CA3189480A1 - Compositions and methods for treating cancer with chimeric tim receptors in combination with inhibitors of poly (adp-ribose) polymerase - Google Patents
Compositions and methods for treating cancer with chimeric tim receptors in combination with inhibitors of poly (adp-ribose) polymeraseInfo
- Publication number
- CA3189480A1 CA3189480A1 CA3189480A CA3189480A CA3189480A1 CA 3189480 A1 CA3189480 A1 CA 3189480A1 CA 3189480 A CA3189480 A CA 3189480A CA 3189480 A CA3189480 A CA 3189480A CA 3189480 A1 CA3189480 A1 CA 3189480A1
- Authority
- CA
- Canada
- Prior art keywords
- domain
- seq
- acid sequence
- amino acid
- signaling domain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 119
- 239000000203 mixture Substances 0.000 title claims abstract description 17
- 206010028980 Neoplasm Diseases 0.000 title claims description 39
- 201000011510 cancer Diseases 0.000 title claims description 22
- 239000003112 inhibitor Substances 0.000 title claims description 21
- 108010061844 Poly(ADP-ribose) Polymerases Proteins 0.000 title claims description 9
- 102000012338 Poly(ADP-ribose) Polymerases Human genes 0.000 title claims description 9
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 title claims description 9
- 239000012661 PARP inhibitor Substances 0.000 claims abstract description 48
- 229940121906 Poly ADP ribose polymerase inhibitor Drugs 0.000 claims abstract description 48
- 230000011664 signaling Effects 0.000 claims description 440
- 150000001413 amino acids Chemical class 0.000 claims description 423
- 102000005962 receptors Human genes 0.000 claims description 340
- 108020003175 receptors Proteins 0.000 claims description 340
- 210000004027 cell Anatomy 0.000 claims description 322
- 230000004068 intracellular signaling Effects 0.000 claims description 287
- 230000027455 binding Effects 0.000 claims description 178
- 230000000139 costimulatory effect Effects 0.000 claims description 163
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 125
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 125
- 108090000623 proteins and genes Proteins 0.000 claims description 116
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 99
- 102000040430 polynucleotide Human genes 0.000 claims description 76
- 108091033319 polynucleotide Proteins 0.000 claims description 76
- 239000002157 polynucleotide Substances 0.000 claims description 76
- 239000013598 vector Substances 0.000 claims description 75
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 65
- 108020001507 fusion proteins Proteins 0.000 claims description 58
- 102000037865 fusion proteins Human genes 0.000 claims description 58
- 102000004169 proteins and genes Human genes 0.000 claims description 55
- PCHKPVIQAHNQLW-CQSZACIVSA-N niraparib Chemical compound N1=C2C(C(=O)N)=CC=CC2=CN1C(C=C1)=CC=C1[C@@H]1CCCNC1 PCHKPVIQAHNQLW-CQSZACIVSA-N 0.000 claims description 53
- 229950011068 niraparib Drugs 0.000 claims description 49
- 101000809875 Homo sapiens TYRO protein tyrosine kinase-binding protein Proteins 0.000 claims description 47
- 102100038717 TYRO protein tyrosine kinase-binding protein Human genes 0.000 claims description 46
- 125000006850 spacer group Chemical group 0.000 claims description 40
- 101000800483 Homo sapiens Toll-like receptor 8 Proteins 0.000 claims description 35
- 102100033110 Toll-like receptor 8 Human genes 0.000 claims description 35
- 102100027207 CD27 antigen Human genes 0.000 claims description 32
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 32
- 238000006467 substitution reaction Methods 0.000 claims description 29
- 239000002253 acid Substances 0.000 claims description 27
- 210000002865 immune cell Anatomy 0.000 claims description 26
- 230000003834 intracellular effect Effects 0.000 claims description 26
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 claims description 25
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 25
- 102100024333 Toll-like receptor 2 Human genes 0.000 claims description 25
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 25
- 101100369802 Caenorhabditis elegans tim-1 gene Proteins 0.000 claims description 23
- 108060003951 Immunoglobulin Proteins 0.000 claims description 23
- 102000003714 TNF receptor-associated factor 6 Human genes 0.000 claims description 23
- 108090000009 TNF receptor-associated factor 6 Proteins 0.000 claims description 23
- 102000018358 immunoglobulin Human genes 0.000 claims description 23
- 101000934346 Homo sapiens T-cell surface antigen CD2 Proteins 0.000 claims description 22
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 claims description 22
- 102100025390 Integrin beta-2 Human genes 0.000 claims description 20
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 claims description 18
- 108090000925 TNF receptor-associated factor 2 Proteins 0.000 claims description 18
- 102100034779 TRAF family member-associated NF-kappa-B activator Human genes 0.000 claims description 18
- 108010064548 Lymphocyte Function-Associated Antigen-1 Proteins 0.000 claims description 17
- 102100040678 Programmed cell death protein 1 Human genes 0.000 claims description 17
- 101710089372 Programmed cell death protein 1 Proteins 0.000 claims description 17
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 16
- 101000914496 Homo sapiens T-cell antigen CD7 Proteins 0.000 claims description 16
- 102100027208 T-cell antigen CD7 Human genes 0.000 claims description 16
- 230000004913 activation Effects 0.000 claims description 16
- 101001109503 Homo sapiens NKG2-C type II integral membrane protein Proteins 0.000 claims description 15
- 102100022683 NKG2-C type II integral membrane protein Human genes 0.000 claims description 15
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims description 14
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 claims description 14
- 238000002560 therapeutic procedure Methods 0.000 claims description 14
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 claims description 13
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 claims description 13
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 claims description 12
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 12
- HMABYWSNWIZPAG-UHFFFAOYSA-N rucaparib Chemical compound C1=CC(CNC)=CC=C1C(N1)=C2CCNC(=O)C3=C2C1=CC(F)=C3 HMABYWSNWIZPAG-UHFFFAOYSA-N 0.000 claims description 12
- 229950004707 rucaparib Drugs 0.000 claims description 12
- 101150013553 CD40 gene Proteins 0.000 claims description 11
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 11
- 150000007513 acids Chemical class 0.000 claims description 11
- 102100038078 CD276 antigen Human genes 0.000 claims description 10
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 10
- 206010033128 Ovarian cancer Diseases 0.000 claims description 9
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 9
- HWGQMRYQVZSGDQ-HZPDHXFCSA-N chembl3137320 Chemical group CN1N=CN=C1[C@H]([C@H](N1)C=2C=CC(F)=CC=2)C2=NNC(=O)C3=C2C1=CC(F)=C3 HWGQMRYQVZSGDQ-HZPDHXFCSA-N 0.000 claims description 9
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 claims description 8
- 230000001413 cellular effect Effects 0.000 claims description 8
- 229960000572 olaparib Drugs 0.000 claims description 8
- 238000009169 immunotherapy Methods 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 230000037361 pathway Effects 0.000 claims description 7
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 6
- 108700010039 chimeric receptor Proteins 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 6
- 108091008036 Immune checkpoint proteins Proteins 0.000 claims description 5
- 229950004550 talazoparib Drugs 0.000 claims description 5
- 229940124597 therapeutic agent Drugs 0.000 claims description 5
- CTLOSZHDGZLOQE-UHFFFAOYSA-N 14-methoxy-9-[(4-methylpiperazin-1-yl)methyl]-9,19-diazapentacyclo[10.7.0.02,6.07,11.013,18]nonadeca-1(12),2(6),7(11),13(18),14,16-hexaene-8,10-dione Chemical compound O=C1C2=C3C=4C(OC)=CC=CC=4NC3=C3CCCC3=C2C(=O)N1CN1CCN(C)CC1 CTLOSZHDGZLOQE-UHFFFAOYSA-N 0.000 claims description 4
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 208000026310 Breast neoplasm Diseases 0.000 claims description 4
- 102000037982 Immune checkpoint proteins Human genes 0.000 claims description 4
- 206010060862 Prostate cancer Diseases 0.000 claims description 4
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 4
- 102000002689 Toll-like receptor Human genes 0.000 claims description 4
- 108020000411 Toll-like receptor Proteins 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000003102 growth factor Substances 0.000 claims description 4
- 229940126546 immune checkpoint molecule Drugs 0.000 claims description 4
- HAVFFEMDLROBGI-UHFFFAOYSA-N m8926c7ilx Chemical compound C1CC(O)CCN1CC1=CC=C(OC=2C3=C(C(NN=C33)=O)C=CC=2)C3=C1 HAVFFEMDLROBGI-UHFFFAOYSA-N 0.000 claims description 4
- JNAHVYVRKWKWKQ-CYBMUJFWSA-N veliparib Chemical compound N=1C2=CC=CC(C(N)=O)=C2NC=1[C@@]1(C)CCCN1 JNAHVYVRKWKWKQ-CYBMUJFWSA-N 0.000 claims description 4
- DENYZIUJOTUUNY-MRXNPFEDSA-N (2R)-14-fluoro-2-methyl-6,9,10,19-tetrazapentacyclo[14.2.1.02,6.08,18.012,17]nonadeca-1(18),8,12(17),13,15-pentaen-11-one Chemical compound FC=1C=C2C=3C=4C(CN5[C@@](C4NC3C1)(CCC5)C)=NNC2=O DENYZIUJOTUUNY-MRXNPFEDSA-N 0.000 claims description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 3
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims description 3
- 238000002512 chemotherapy Methods 0.000 claims description 3
- 201000005202 lung cancer Diseases 0.000 claims description 3
- 208000020816 lung neoplasm Diseases 0.000 claims description 3
- 229950011257 veliparib Drugs 0.000 claims description 3
- 206010009944 Colon cancer Diseases 0.000 claims description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- 244000309459 oncolytic virus Species 0.000 claims description 2
- 229950007072 pamiparib Drugs 0.000 claims description 2
- 150000003384 small molecules Chemical class 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 160
- VEEGZPWAAPPXRB-BJMVGYQFSA-N (3e)-3-(1h-imidazol-5-ylmethylidene)-1h-indol-2-one Chemical compound O=C1NC2=CC=CC=C2\C1=C/C1=CN=CN1 VEEGZPWAAPPXRB-BJMVGYQFSA-N 0.000 claims 1
- KKVYYGGCHJGEFJ-UHFFFAOYSA-N 1-n-(4-chlorophenyl)-6-methyl-5-n-[3-(7h-purin-6-yl)pyridin-2-yl]isoquinoline-1,5-diamine Chemical compound N=1C=CC2=C(NC=3C(=CC=CN=3)C=3C=4N=CNC=4N=CN=3)C(C)=CC=C2C=1NC1=CC=C(Cl)C=C1 KKVYYGGCHJGEFJ-UHFFFAOYSA-N 0.000 claims 1
- 229940122531 Anaplastic lymphoma kinase inhibitor Drugs 0.000 claims 1
- 239000012664 BCL-2-inhibitor Substances 0.000 claims 1
- 102000052609 BRCA2 Human genes 0.000 claims 1
- 108700020462 BRCA2 Proteins 0.000 claims 1
- 229940124291 BTK inhibitor Drugs 0.000 claims 1
- 229940123711 Bcl2 inhibitor Drugs 0.000 claims 1
- 229940118364 Bcr-Abl inhibitor Drugs 0.000 claims 1
- 101001042041 Bos taurus Isocitrate dehydrogenase [NAD] subunit beta, mitochondrial Proteins 0.000 claims 1
- 101150008921 Brca2 gene Proteins 0.000 claims 1
- 229940124204 C-kit inhibitor Drugs 0.000 claims 1
- 229940124297 CDK 4/6 inhibitor Drugs 0.000 claims 1
- 229940124783 FAK inhibitor Drugs 0.000 claims 1
- 229940122242 GTPase inhibitor Drugs 0.000 claims 1
- 229940119324 H-Ras inhibitor Drugs 0.000 claims 1
- 102100035108 High affinity nerve growth factor receptor Human genes 0.000 claims 1
- 101000960234 Homo sapiens Isocitrate dehydrogenase [NADP] cytoplasmic Proteins 0.000 claims 1
- 101001126417 Homo sapiens Platelet-derived growth factor receptor alpha Proteins 0.000 claims 1
- 101000686031 Homo sapiens Proto-oncogene tyrosine-protein kinase ROS Proteins 0.000 claims 1
- 101000932478 Homo sapiens Receptor-type tyrosine-protein kinase FLT3 Proteins 0.000 claims 1
- 102100039905 Isocitrate dehydrogenase [NADP] cytoplasmic Human genes 0.000 claims 1
- 229940123830 K-Ras inhibitor Drugs 0.000 claims 1
- 229940124647 MEK inhibitor Drugs 0.000 claims 1
- 241000642478 Mucia Species 0.000 claims 1
- 101100381978 Mus musculus Braf gene Proteins 0.000 claims 1
- 229940124160 Myc inhibitor Drugs 0.000 claims 1
- 108700019961 Neoplasm Genes Proteins 0.000 claims 1
- 102000048850 Neoplasm Genes Human genes 0.000 claims 1
- 239000012828 PI3K inhibitor Substances 0.000 claims 1
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 claims 1
- 102100030485 Platelet-derived growth factor receptor alpha Human genes 0.000 claims 1
- 102100023347 Proto-oncogene tyrosine-protein kinase ROS Human genes 0.000 claims 1
- 102100020718 Receptor-type tyrosine-protein kinase FLT3 Human genes 0.000 claims 1
- 229940121742 Serine/threonine kinase inhibitor Drugs 0.000 claims 1
- 229940122954 Transcription factor inhibitor Drugs 0.000 claims 1
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 claims 1
- 102100022596 Tyrosine-protein kinase ABL1 Human genes 0.000 claims 1
- 229940124674 VEGF-R inhibitor Drugs 0.000 claims 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims 1
- 102100025093 Zinc fingers and homeoboxes protein 2 Human genes 0.000 claims 1
- 229940121369 angiogenesis inhibitor Drugs 0.000 claims 1
- 239000004037 angiogenesis inhibitor Substances 0.000 claims 1
- 239000003429 antifungal agent Substances 0.000 claims 1
- 229940121375 antifungal agent Drugs 0.000 claims 1
- 239000003443 antiviral agent Substances 0.000 claims 1
- 230000003115 biocidal effect Effects 0.000 claims 1
- 229940121647 egfr inhibitor Drugs 0.000 claims 1
- 229940125829 fibroblast growth factor receptor inhibitor Drugs 0.000 claims 1
- 238000001794 hormone therapy Methods 0.000 claims 1
- 210000005260 human cell Anatomy 0.000 claims 1
- 229940124302 mTOR inhibitor Drugs 0.000 claims 1
- 239000003628 mammalian target of rapamycin inhibitor Substances 0.000 claims 1
- 239000002829 mitogen activated protein kinase inhibitor Substances 0.000 claims 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims 1
- FAQDUNYVKQKNLD-UHFFFAOYSA-N olaparib Chemical compound FC1=CC=C(CC2=C3[CH]C=CC=C3C(=O)N=N2)C=C1C(=O)N(CC1)CCN1C(=O)C1CC1 FAQDUNYVKQKNLD-UHFFFAOYSA-N 0.000 claims 1
- 201000002628 peritoneum cancer Diseases 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 229940043441 phosphoinositide 3-kinase inhibitor Drugs 0.000 claims 1
- 238000001126 phototherapy Methods 0.000 claims 1
- 230000005855 radiation Effects 0.000 claims 1
- 229940124617 receptor tyrosine kinase inhibitor Drugs 0.000 claims 1
- 102200145384 rs34127778 Human genes 0.000 claims 1
- 208000022679 triple-negative breast carcinoma Diseases 0.000 claims 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims 1
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 claims 1
- 239000005483 tyrosine kinase inhibitor Substances 0.000 claims 1
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 claims 1
- 238000002604 ultrasonography Methods 0.000 claims 1
- 238000002648 combination therapy Methods 0.000 abstract 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 66
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 65
- 108010076504 Protein Sorting Signals Proteins 0.000 description 65
- 230000014509 gene expression Effects 0.000 description 62
- 239000000427 antigen Substances 0.000 description 61
- 108091007433 antigens Proteins 0.000 description 59
- 102000036639 antigens Human genes 0.000 description 59
- 108010063954 Mucins Proteins 0.000 description 47
- 102000015728 Mucins Human genes 0.000 description 47
- 210000003719 b-lymphocyte Anatomy 0.000 description 45
- 101000669511 Homo sapiens T-cell immunoglobulin and mucin domain-containing protein 4 Proteins 0.000 description 44
- 150000007523 nucleic acids Chemical class 0.000 description 38
- 102000004196 processed proteins & peptides Human genes 0.000 description 36
- 102000039446 nucleic acids Human genes 0.000 description 35
- 108020004707 nucleic acids Proteins 0.000 description 35
- -1 Veliperib Chemical compound 0.000 description 34
- 230000003213 activating effect Effects 0.000 description 32
- 241000282414 Homo sapiens Species 0.000 description 31
- 239000012636 effector Substances 0.000 description 30
- 102100039367 T-cell immunoglobulin and mucin domain-containing protein 4 Human genes 0.000 description 28
- 229920001184 polypeptide Polymers 0.000 description 28
- 241000894007 species Species 0.000 description 27
- 230000004048 modification Effects 0.000 description 26
- 238000012986 modification Methods 0.000 description 26
- 239000002245 particle Substances 0.000 description 25
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 24
- 230000000694 effects Effects 0.000 description 24
- 239000013603 viral vector Substances 0.000 description 24
- 238000007792 addition Methods 0.000 description 19
- 230000001472 cytotoxic effect Effects 0.000 description 19
- 210000001519 tissue Anatomy 0.000 description 19
- 241000700159 Rattus Species 0.000 description 18
- 238000012217 deletion Methods 0.000 description 18
- 230000037430 deletion Effects 0.000 description 18
- 230000006907 apoptotic process Effects 0.000 description 17
- 230000006870 function Effects 0.000 description 17
- 102000045158 human TIMD4 Human genes 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 16
- 230000001939 inductive effect Effects 0.000 description 16
- 230000019491 signal transduction Effects 0.000 description 16
- 241000283690 Bos taurus Species 0.000 description 15
- 241000282472 Canis lupus familiaris Species 0.000 description 15
- 241000282326 Felis catus Species 0.000 description 15
- 201000010099 disease Diseases 0.000 description 15
- 241000283973 Oryctolagus cuniculus Species 0.000 description 14
- 241001494479 Pecora Species 0.000 description 14
- 241000288906 Primates Species 0.000 description 14
- 230000001640 apoptogenic effect Effects 0.000 description 14
- 241000700198 Cavia Species 0.000 description 13
- 102000004127 Cytokines Human genes 0.000 description 13
- 108090000695 Cytokines Proteins 0.000 description 13
- 241000283086 Equidae Species 0.000 description 13
- 241000282412 Homo Species 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 13
- 241000282887 Suidae Species 0.000 description 13
- 230000004044 response Effects 0.000 description 13
- 210000004881 tumor cell Anatomy 0.000 description 13
- 241000283707 Capra Species 0.000 description 12
- 230000000242 pagocytic effect Effects 0.000 description 12
- 230000003612 virological effect Effects 0.000 description 12
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 description 11
- 102000017578 LAG3 Human genes 0.000 description 11
- 108700019146 Transgenes Proteins 0.000 description 11
- 210000000170 cell membrane Anatomy 0.000 description 11
- 231100000433 cytotoxic Toxicity 0.000 description 11
- 230000001105 regulatory effect Effects 0.000 description 11
- 230000009261 transgenic effect Effects 0.000 description 11
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 10
- 108091008874 T cell receptors Proteins 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 239000002243 precursor Substances 0.000 description 10
- 230000035755 proliferation Effects 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 208000035475 disorder Diseases 0.000 description 9
- 210000000822 natural killer cell Anatomy 0.000 description 9
- 230000001338 necrotic effect Effects 0.000 description 9
- 239000002773 nucleotide Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 8
- 101000669510 Mus musculus T-cell immunoglobulin and mucin domain-containing protein 4 Proteins 0.000 description 8
- 206010057249 Phagocytosis Diseases 0.000 description 8
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 description 8
- 241000700605 Viruses Species 0.000 description 8
- 230000004069 differentiation Effects 0.000 description 8
- FDLYAMZZIXQODN-UHFFFAOYSA-N olaparib Chemical compound FC1=CC=C(CC=2C3=CC=CC=C3C(=O)NN=2)C=C1C(=O)N(CC1)CCN1C(=O)C1CC1 FDLYAMZZIXQODN-UHFFFAOYSA-N 0.000 description 8
- 230000008782 phagocytosis Effects 0.000 description 8
- 230000009258 tissue cross reactivity Effects 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 241000214054 Equine rhinitis A virus Species 0.000 description 7
- 241000124008 Mammalia Species 0.000 description 7
- 108010052285 Membrane Proteins Proteins 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 7
- 238000000684 flow cytometry Methods 0.000 description 7
- 230000036541 health Effects 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 210000001539 phagocyte Anatomy 0.000 description 7
- 210000000680 phagosome Anatomy 0.000 description 7
- 230000028327 secretion Effects 0.000 description 7
- 238000010361 transduction Methods 0.000 description 7
- 230000026683 transduction Effects 0.000 description 7
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 6
- 108010042407 Endonucleases Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 6
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 6
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 6
- 102100033467 L-selectin Human genes 0.000 description 6
- 241000713666 Lentivirus Species 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 102100029215 Signaling lymphocytic activation molecule Human genes 0.000 description 6
- 230000030741 antigen processing and presentation Effects 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 230000016396 cytokine production Effects 0.000 description 6
- 210000004443 dendritic cell Anatomy 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 230000035990 intercellular signaling Effects 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 102100025466 Carcinoembryonic antigen-related cell adhesion molecule 3 Human genes 0.000 description 5
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 5
- 102000004961 Furin Human genes 0.000 description 5
- 108090001126 Furin Proteins 0.000 description 5
- 102100021186 Granulysin Human genes 0.000 description 5
- 101710168479 Granulysin Proteins 0.000 description 5
- 108060005986 Granzyme Proteins 0.000 description 5
- 102000001398 Granzyme Human genes 0.000 description 5
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 description 5
- 101000914337 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 3 Proteins 0.000 description 5
- 101001068133 Homo sapiens Hepatitis A virus cellular receptor 2 Proteins 0.000 description 5
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 5
- 101150069255 KLRC1 gene Proteins 0.000 description 5
- 101100404845 Macaca mulatta NKG2A gene Proteins 0.000 description 5
- 102100022682 NKG2-A/NKG2-B type II integral membrane protein Human genes 0.000 description 5
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 5
- 230000005867 T cell response Effects 0.000 description 5
- 102100025244 T-cell surface glycoprotein CD5 Human genes 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 210000000612 antigen-presenting cell Anatomy 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 238000010362 genome editing Methods 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000005304 joining Methods 0.000 description 5
- 239000002523 lectin Substances 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 210000004698 lymphocyte Anatomy 0.000 description 5
- 229930192851 perforin Natural products 0.000 description 5
- 230000002688 persistence Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 102220077395 rs797044870 Human genes 0.000 description 5
- 230000007781 signaling event Effects 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 230000002123 temporal effect Effects 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 102100025218 B-cell differentiation antigen CD72 Human genes 0.000 description 4
- 102100038077 CD226 antigen Human genes 0.000 description 4
- 102100035793 CD83 antigen Human genes 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 4
- 102100029360 Hematopoietic cell signal transducer Human genes 0.000 description 4
- 102100034459 Hepatitis A virus cellular receptor 1 Human genes 0.000 description 4
- 101000934359 Homo sapiens B-cell differentiation antigen CD72 Proteins 0.000 description 4
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 4
- 101000990188 Homo sapiens Hematopoietic cell signal transducer Proteins 0.000 description 4
- 101000599852 Homo sapiens Intercellular adhesion molecule 1 Proteins 0.000 description 4
- 101001047640 Homo sapiens Linker for activation of T-cells family member 1 Proteins 0.000 description 4
- 101000702132 Homo sapiens Protein spinster homolog 1 Proteins 0.000 description 4
- 102100024032 Linker for activation of T-cells family member 1 Human genes 0.000 description 4
- 102100034709 Lymphocyte cytosolic protein 2 Human genes 0.000 description 4
- 101710195102 Lymphocyte cytosolic protein 2 Proteins 0.000 description 4
- 102000018697 Membrane Proteins Human genes 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 description 4
- 101710163270 Nuclease Proteins 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 description 4
- 230000006044 T cell activation Effects 0.000 description 4
- 230000006052 T cell proliferation Effects 0.000 description 4
- 102000011408 Tripartite Motif Proteins Human genes 0.000 description 4
- 108010023649 Tripartite Motif Proteins Proteins 0.000 description 4
- 101001038499 Yarrowia lipolytica (strain CLIB 122 / E 150) Lysine acetyltransferase Proteins 0.000 description 4
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 4
- 238000011374 additional therapy Methods 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 210000003162 effector t lymphocyte Anatomy 0.000 description 4
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 4
- 239000003623 enhancer Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000001415 gene therapy Methods 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 230000001506 immunosuppresive effect Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 210000001165 lymph node Anatomy 0.000 description 4
- 210000002540 macrophage Anatomy 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 210000001806 memory b lymphocyte Anatomy 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000001177 retroviral effect Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 241001430294 unidentified retrovirus Species 0.000 description 4
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 3
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 3
- 108090000342 C-Type Lectins Proteins 0.000 description 3
- 102000003930 C-Type Lectins Human genes 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 3
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 3
- 206010057248 Cell death Diseases 0.000 description 3
- 108091033380 Coding strand Proteins 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 102100030886 Complement receptor type 1 Human genes 0.000 description 3
- 241000702421 Dependoparvovirus Species 0.000 description 3
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 102000004533 Endonucleases Human genes 0.000 description 3
- 241001663880 Gammaretrovirus Species 0.000 description 3
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 3
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 3
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 3
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 3
- 101000727061 Homo sapiens Complement receptor type 1 Proteins 0.000 description 3
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 3
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 3
- 101000935040 Homo sapiens Integrin beta-2 Proteins 0.000 description 3
- 101000777628 Homo sapiens Leukocyte antigen CD37 Proteins 0.000 description 3
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 3
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 description 3
- 101000971513 Homo sapiens Natural killer cells antigen CD94 Proteins 0.000 description 3
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 3
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 3
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 3
- 102100031586 Leukocyte antigen CD37 Human genes 0.000 description 3
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 3
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 3
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 3
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 description 3
- 102100021462 Natural killer cells antigen CD94 Human genes 0.000 description 3
- 206010028851 Necrosis Diseases 0.000 description 3
- 108010038807 Oligopeptides Proteins 0.000 description 3
- 102000015636 Oligopeptides Human genes 0.000 description 3
- 240000007019 Oxalis corniculata Species 0.000 description 3
- 102000004503 Perforin Human genes 0.000 description 3
- 108010056995 Perforin Proteins 0.000 description 3
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 3
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 3
- 241000700584 Simplexvirus Species 0.000 description 3
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 3
- 101710149792 Triosephosphate isomerase, chloroplastic Proteins 0.000 description 3
- 101710195516 Triosephosphate isomerase, glycosomal Proteins 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 102100040247 Tumor necrosis factor Human genes 0.000 description 3
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 description 3
- 230000002424 anti-apoptotic effect Effects 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000005779 cell damage Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000036755 cellular response Effects 0.000 description 3
- 230000004186 co-expression Effects 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000003325 follicular Effects 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 210000003826 marginal zone b cell Anatomy 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 210000003071 memory t lymphocyte Anatomy 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000009149 molecular binding Effects 0.000 description 3
- 210000000066 myeloid cell Anatomy 0.000 description 3
- 210000000581 natural killer T-cell Anatomy 0.000 description 3
- 230000017074 necrotic cell death Effects 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 210000004180 plasmocyte Anatomy 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 210000001948 pro-b lymphocyte Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 210000003289 regulatory T cell Anatomy 0.000 description 3
- 210000002707 regulatory b cell Anatomy 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 102100034540 Adenomatous polyposis coli protein Human genes 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 2
- 102100023962 Bifunctional arginine demethylase and lysyl-hydroxylase JMJD6 Human genes 0.000 description 2
- 102100032937 CD40 ligand Human genes 0.000 description 2
- 102100037904 CD9 antigen Human genes 0.000 description 2
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000713730 Equine infectious anemia virus Species 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 108010042634 F2A4-K-NS peptide Proteins 0.000 description 2
- 241000713800 Feline immunodeficiency virus Species 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 206010066476 Haematological malignancy Diseases 0.000 description 2
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 2
- 101000924577 Homo sapiens Adenomatous polyposis coli protein Proteins 0.000 description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 2
- 101000868215 Homo sapiens CD40 ligand Proteins 0.000 description 2
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 2
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 2
- 101000818543 Homo sapiens Tyrosine-protein kinase ZAP-70 Proteins 0.000 description 2
- 241000713340 Human immunodeficiency virus 2 Species 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 108091054437 MHC class I family Proteins 0.000 description 2
- 102000043129 MHC class I family Human genes 0.000 description 2
- 241000714177 Murine leukemia virus Species 0.000 description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- 241001672814 Porcine teschovirus 1 Species 0.000 description 2
- 108010074687 Signaling Lymphocytic Activation Molecule Family Member 1 Proteins 0.000 description 2
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 230000024932 T cell mediated immunity Effects 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- 108010073062 Transcription Activator-Like Effectors Proteins 0.000 description 2
- 102100021125 Tyrosine-protein kinase ZAP-70 Human genes 0.000 description 2
- 101150117115 V gene Proteins 0.000 description 2
- 230000001594 aberrant effect Effects 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000006472 autoimmune response Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000004637 cellular stress Effects 0.000 description 2
- 230000004940 costimulation Effects 0.000 description 2
- 230000003436 cytoskeletal effect Effects 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- FGXWKSZFVQUSTL-UHFFFAOYSA-N domperidone Chemical compound C12=CC=CC=C2NC(=O)N1CCCN(CC1)CCC1N1C2=CC=C(Cl)C=C2NC1=O FGXWKSZFVQUSTL-UHFFFAOYSA-N 0.000 description 2
- 230000000925 erythroid effect Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 210000003918 fraction a Anatomy 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 239000000833 heterodimer Substances 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 230000005918 in vitro anti-tumor Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 102000006495 integrins Human genes 0.000 description 2
- 108010044426 integrins Proteins 0.000 description 2
- 210000001821 langerhans cell Anatomy 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000003738 lymphoid progenitor cell Anatomy 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 210000003643 myeloid progenitor cell Anatomy 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 108010077613 phosphatidylserine receptor Proteins 0.000 description 2
- 210000003720 plasmablast Anatomy 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 230000008707 rearrangement Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 229960004641 rituximab Drugs 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 210000000225 synapse Anatomy 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 230000001960 triggered effect Effects 0.000 description 2
- 108010087967 type I signal peptidase Proteins 0.000 description 2
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- RLGNNNSZZAWLAY-UHFFFAOYSA-N 2-(2,3-dimethoxy-4-methylsulfanylphenyl)ethanamine Chemical compound COC1=C(CCN)C=CC(SC)=C1OC RLGNNNSZZAWLAY-UHFFFAOYSA-N 0.000 description 1
- LEELWLKZRKDMAS-UHFFFAOYSA-N 2-(2,4-dimethoxy-3-methylsulfanylphenyl)ethanamine Chemical compound COC1=CC=C(CCN)C(OC)=C1SC LEELWLKZRKDMAS-UHFFFAOYSA-N 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical group N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 108020004491 Antisense DNA Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 102100035687 Bile salt-activated lipase Human genes 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 101710149863 C-C chemokine receptor type 4 Proteins 0.000 description 1
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 102100032976 CCR4-NOT transcription complex subunit 6 Human genes 0.000 description 1
- 102100024263 CD160 antigen Human genes 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 102100026550 Caspase-9 Human genes 0.000 description 1
- 108090000566 Caspase-9 Proteins 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- VYZAMTAEIAYCRO-BJUDXGSMSA-N Chromium-51 Chemical compound [51Cr] VYZAMTAEIAYCRO-BJUDXGSMSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 1
- 206010010144 Completed suicide Diseases 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 101150097493 D gene Proteins 0.000 description 1
- 101150057441 D16 gene Proteins 0.000 description 1
- 239000012623 DNA damaging agent Substances 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 229910052693 Europium Inorganic materials 0.000 description 1
- 241000714165 Feline leukemia virus Species 0.000 description 1
- 241000714174 Feline sarcoma virus Species 0.000 description 1
- 102100031351 Galectin-9 Human genes 0.000 description 1
- 101100229077 Gallus gallus GAL9 gene Proteins 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 108010007712 Hepatitis A Virus Cellular Receptor 1 Proteins 0.000 description 1
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 1
- 101000761938 Homo sapiens CD160 antigen Proteins 0.000 description 1
- 101100273713 Homo sapiens CD2 gene Proteins 0.000 description 1
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101100133483 Homo sapiens NLK gene Proteins 0.000 description 1
- 101001113440 Homo sapiens Poly [ADP-ribose] polymerase 2 Proteins 0.000 description 1
- 101000884271 Homo sapiens Signal transducer CD24 Proteins 0.000 description 1
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 description 1
- 101000763579 Homo sapiens Toll-like receptor 1 Proteins 0.000 description 1
- 101000831496 Homo sapiens Toll-like receptor 3 Proteins 0.000 description 1
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 1
- 101000669460 Homo sapiens Toll-like receptor 5 Proteins 0.000 description 1
- 101000669406 Homo sapiens Toll-like receptor 6 Proteins 0.000 description 1
- 101000669402 Homo sapiens Toll-like receptor 7 Proteins 0.000 description 1
- 101001102797 Homo sapiens Transmembrane protein PVRIG Proteins 0.000 description 1
- 101000666896 Homo sapiens V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 101150017040 I gene Proteins 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 101150008942 J gene Proteins 0.000 description 1
- 102000002698 KIR Receptors Human genes 0.000 description 1
- 108010043610 KIR Receptors Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 102100020943 Leukocyte-associated immunoglobulin-like receptor 1 Human genes 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 108700005089 MHC Class I Genes Proteins 0.000 description 1
- 108700005092 MHC Class II Genes Proteins 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 101100226902 Mus musculus Fcrlb gene Proteins 0.000 description 1
- 101150065403 NECTIN2 gene Proteins 0.000 description 1
- 102100035488 Nectin-2 Human genes 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- 241000713747 Ovine lentivirus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 235000014676 Phragmites communis Nutrition 0.000 description 1
- 208000002151 Pleural effusion Diseases 0.000 description 1
- 108010064218 Poly (ADP-Ribose) Polymerase-1 Proteins 0.000 description 1
- 102100023712 Poly [ADP-ribose] polymerase 1 Human genes 0.000 description 1
- 102100023652 Poly [ADP-ribose] polymerase 2 Human genes 0.000 description 1
- 108091026813 Poly(ADPribose) Proteins 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 241000712909 Reticuloendotheliosis virus Species 0.000 description 1
- 241000712907 Retroviridae Species 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 101100510333 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PKC1 gene Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102100034447 Serine/threonine-protein kinase NLK Human genes 0.000 description 1
- 102100038081 Signal transducer CD24 Human genes 0.000 description 1
- 241000713311 Simian immunodeficiency virus Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 230000020385 T cell costimulation Effects 0.000 description 1
- 101710174757 T-cell immunoglobulin and mucin domain-containing protein 4 Proteins 0.000 description 1
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 241001420369 Thosea Species 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 1
- 102100027010 Toll-like receptor 1 Human genes 0.000 description 1
- 102100024324 Toll-like receptor 3 Human genes 0.000 description 1
- 102100039360 Toll-like receptor 4 Human genes 0.000 description 1
- 102100039357 Toll-like receptor 5 Human genes 0.000 description 1
- 102100039387 Toll-like receptor 6 Human genes 0.000 description 1
- 102100039390 Toll-like receptor 7 Human genes 0.000 description 1
- 102100033117 Toll-like receptor 9 Human genes 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 102100039630 Transmembrane protein PVRIG Human genes 0.000 description 1
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 1
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 102100038929 V-set domain-containing T-cell activation inhibitor 1 Human genes 0.000 description 1
- 102100038282 V-type immunoglobulin domain-containing suppressor of T-cell activation Human genes 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 241000713325 Visna/maedi virus Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000001668 ameliorated effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000003816 antisense DNA Substances 0.000 description 1
- 230000025194 apoptotic cell clearance Effects 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 230000008236 biological pathway Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 201000008274 breast adenocarcinoma Diseases 0.000 description 1
- 208000003362 bronchogenic carcinoma Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 229960005395 cetuximab Drugs 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000002074 deregulated effect Effects 0.000 description 1
- PGUYAANYCROBRT-UHFFFAOYSA-N dihydroxy-selanyl-selanylidene-lambda5-phosphane Chemical compound OP(O)([SeH])=[Se] PGUYAANYCROBRT-UHFFFAOYSA-N 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 210000003515 double negative t cell Anatomy 0.000 description 1
- 229960003722 doxycycline Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 1
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- IJJVMEJXYNJXOJ-UHFFFAOYSA-N fluquinconazole Chemical compound C=1C=C(Cl)C=C(Cl)C=1N1C(=O)C2=CC(F)=CC=C2N=C1N1C=NC=N1 IJJVMEJXYNJXOJ-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 229960002963 ganciclovir Drugs 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 108091022928 glucosylglycerol-phosphate synthase Proteins 0.000 description 1
- 150000002337 glycosamines Chemical group 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 102000045108 human EGFR Human genes 0.000 description 1
- 102000045892 human TYROBP Human genes 0.000 description 1
- 102000027596 immune receptors Human genes 0.000 description 1
- 108091008915 immune receptors Proteins 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006882 induction of apoptosis Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 108010025001 leukocyte-associated immunoglobulin-like receptor 1 Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 208000025113 myeloid leukemia Diseases 0.000 description 1
- DAZSWUUAFHBCGE-KRWDZBQOSA-N n-[(2s)-3-methyl-1-oxo-1-pyrrolidin-1-ylbutan-2-yl]-3-phenylpropanamide Chemical compound N([C@@H](C(C)C)C(=O)N1CCCC1)C(=O)CCC1=CC=CC=C1 DAZSWUUAFHBCGE-KRWDZBQOSA-N 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000007030 peptide scission Effects 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 201000005825 prostate adenocarcinoma Diseases 0.000 description 1
- 230000009873 pyroptotic effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000007420 radioactive assay Methods 0.000 description 1
- 210000003370 receptor cell Anatomy 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- JRPHGDYSKGJTKZ-UHFFFAOYSA-K selenophosphate Chemical compound [O-]P([O-])([O-])=[Se] JRPHGDYSKGJTKZ-UHFFFAOYSA-K 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 108091005475 signaling receptors Proteins 0.000 description 1
- 102000035025 signaling receptors Human genes 0.000 description 1
- 239000002924 silencing RNA Substances 0.000 description 1
- 230000037432 silent mutation Effects 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000011885 synergistic combination Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 101150016462 tut-2 gene Proteins 0.000 description 1
- 230000004222 uncontrolled growth Effects 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0012—Lipids; Lipoproteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
- A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/50—Pyridazines; Hydrogenated pyridazines
- A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/50—Pyridazines; Hydrogenated pyridazines
- A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/80—Vaccine for a specifically defined cancer
- A61K2039/892—Reproductive system [uterus, ovaries, cervix, testes]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Mycology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present disclosure relates to combination therapy compositions and methods comprising chimeric Tim receptors, host cells modified to include chimeric Tim receptor molecules, and PARP inhibitors.
Description
COMPOSITIONS AND METHODS FOR TREATING CANCER WITH CHIMERIC
TIM RECEPTORS IN COMBINATION WITH INHIBITORS OF POLY (ADP-RIBOSE) POLY1V1ERASE
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200265_415W0_ST25.txt. The text file is 425 KB, was created on August 13, 2021, and is being submitted electronically via EFS-Web.
BACKGROUND
Upon exposure to antigen, naïve antigen-specific CD8+ T cells undergo activation that promotes their clonal expansion, differentiation, and development into functional, effector T cells that can kill cells expressing the cognate antigen (e.g., tumor cells). Following antigen clearance, the majority of effector T cells undergo apoptosis, and a subset of the surviving effector T cells differentiate into memory T
cells that can confer long-tenn protection against antigen re-exposure. However, prolonged antigen exposure may result in T cell exhaustion, enabling the persistence of tumor cells. T cell exhaustion refers to a dysfunctional state acquired by T cells experiencing persistent TCR stimulation characterized by upregulated expression of immune checkpoint molecules (e.g., PD-1, CTLA-4, Tim-3), impaired effector function, poor proliferation, and metabolic defects. Engineered T cells expressing chimeric antigen receptors (CARs) can also develop exhaustion.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIGS. IA-1B show that the Kuramochi model of ovarian cancer is moderately sensitive to PARP inhibition. FIG. IA shows the results of Kuramochi cells expressing an mCherry-NLS plasmid incubated with the PARP inhibitor Niraparib. FIG. 1B shows the results of Kuramochi cells expressing an mCherry-NLS
plasmid incubated with the PARP inhibitors Niraparib, Olaparib, Talazoparib, Veliperib, and Rucaparib.
FIGS. 2A-2C show Kuramochi cells challenged with Niraparib bind Tim4, indicating increased levels of surface PS in response to PARP
inhibition. FIG.
2A shows staining of Niraparib treated Kuramochi cells by Tim4-Fc chimera and anti-mouse IgG2a antibody conjugated to Alexa488. FIG. 2B shows staining for IgG
control. FIG. 2C shows increase in Kuramochi cell number in response to 1.56-12.5 iiM Niraparib.
FIG. 3 shows that Niraparib has negligible impact on CD4/CD8 T cell health, cell size, or expansion.
FIG. 4 shows the results of Kuramochi cells were incubated with chimeric Tim receptor Construct 13A, control 1' cells, and Niraparib.
FIG. 5 shows that combination of chimeric Tim4 receptor (CTX140) and niraparib treatment reduces Kuramochi cell numbers in vitro. Kuramochi cells were pre-incubated with 1.56 M niraparib. CTX156 (tEGFR) is a transduction control.
FIG. 6 shows that combination of chimeric Tim4 receptor (CTX137) and niraparib treatment reduces Kuramochi cell numbers in vitro. Kuramochi cells were pre-incubated with 25 M. niraparib and given maintenance dose of 0.52 LIM
nariparib. CTX156 (tEGFR) is a transduction control.
FIG. 7 shows that combination of chimeric Tim4 receptor (CTX137) with nirparib treatment at different doses (6.25 M, I 2.5 M, or 25 M pre-treatment +
0.52 M maintenance dose) and different effectortarget cell ratios reduces Kuramochi cell numbers in vitro. CTX156 (tEGFR) is a transduction control.
FIGS. 8A-8B show that pCTX133, a TLR-2 containing Chimeric Tim4 Receptor Enhances Potency of Niraparib in an Ovarian Cancer Model. FIG. 8A:
Flow cytometry measurement of surface PtdSer. Kuramochi cells were treated with 1.56 or 25 M Niraparib or with equivalent volume of DMSO (control). 48 hours later samples were trypsinized and stained using a Tim4-Fc followed by a fluoreseently-labeled secondary antibody to the Tim4-Fc. 'FIG. 8B: Kuramochi cells pre-treated for --
TIM RECEPTORS IN COMBINATION WITH INHIBITORS OF POLY (ADP-RIBOSE) POLY1V1ERASE
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200265_415W0_ST25.txt. The text file is 425 KB, was created on August 13, 2021, and is being submitted electronically via EFS-Web.
BACKGROUND
Upon exposure to antigen, naïve antigen-specific CD8+ T cells undergo activation that promotes their clonal expansion, differentiation, and development into functional, effector T cells that can kill cells expressing the cognate antigen (e.g., tumor cells). Following antigen clearance, the majority of effector T cells undergo apoptosis, and a subset of the surviving effector T cells differentiate into memory T
cells that can confer long-tenn protection against antigen re-exposure. However, prolonged antigen exposure may result in T cell exhaustion, enabling the persistence of tumor cells. T cell exhaustion refers to a dysfunctional state acquired by T cells experiencing persistent TCR stimulation characterized by upregulated expression of immune checkpoint molecules (e.g., PD-1, CTLA-4, Tim-3), impaired effector function, poor proliferation, and metabolic defects. Engineered T cells expressing chimeric antigen receptors (CARs) can also develop exhaustion.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIGS. IA-1B show that the Kuramochi model of ovarian cancer is moderately sensitive to PARP inhibition. FIG. IA shows the results of Kuramochi cells expressing an mCherry-NLS plasmid incubated with the PARP inhibitor Niraparib. FIG. 1B shows the results of Kuramochi cells expressing an mCherry-NLS
plasmid incubated with the PARP inhibitors Niraparib, Olaparib, Talazoparib, Veliperib, and Rucaparib.
FIGS. 2A-2C show Kuramochi cells challenged with Niraparib bind Tim4, indicating increased levels of surface PS in response to PARP
inhibition. FIG.
2A shows staining of Niraparib treated Kuramochi cells by Tim4-Fc chimera and anti-mouse IgG2a antibody conjugated to Alexa488. FIG. 2B shows staining for IgG
control. FIG. 2C shows increase in Kuramochi cell number in response to 1.56-12.5 iiM Niraparib.
FIG. 3 shows that Niraparib has negligible impact on CD4/CD8 T cell health, cell size, or expansion.
FIG. 4 shows the results of Kuramochi cells were incubated with chimeric Tim receptor Construct 13A, control 1' cells, and Niraparib.
FIG. 5 shows that combination of chimeric Tim4 receptor (CTX140) and niraparib treatment reduces Kuramochi cell numbers in vitro. Kuramochi cells were pre-incubated with 1.56 M niraparib. CTX156 (tEGFR) is a transduction control.
FIG. 6 shows that combination of chimeric Tim4 receptor (CTX137) and niraparib treatment reduces Kuramochi cell numbers in vitro. Kuramochi cells were pre-incubated with 25 M. niraparib and given maintenance dose of 0.52 LIM
nariparib. CTX156 (tEGFR) is a transduction control.
FIG. 7 shows that combination of chimeric Tim4 receptor (CTX137) with nirparib treatment at different doses (6.25 M, I 2.5 M, or 25 M pre-treatment +
0.52 M maintenance dose) and different effectortarget cell ratios reduces Kuramochi cell numbers in vitro. CTX156 (tEGFR) is a transduction control.
FIGS. 8A-8B show that pCTX133, a TLR-2 containing Chimeric Tim4 Receptor Enhances Potency of Niraparib in an Ovarian Cancer Model. FIG. 8A:
Flow cytometry measurement of surface PtdSer. Kuramochi cells were treated with 1.56 or 25 M Niraparib or with equivalent volume of DMSO (control). 48 hours later samples were trypsinized and stained using a Tim4-Fc followed by a fluoreseently-labeled secondary antibody to the Tim4-Fc. 'FIG. 8B: Kuramochi cells pre-treated for --
2 hours with 1.56 IVI Niraparib were co-cultured with pCTX133 and Untransduced T cells from donor 32 at a 2:1 T cell:Kuramochi ratio and a final Niraparib concentration of 1.56 M. Samples treated with Niraparib pCTX133 exhibited substantially fewer tumor cells in culture -3 days later when compared to samples treated with Niraparib alone, or Niraparib + untransduced T cells. All data were collected via IncuCyte.
FIGS. 9A-9B: CER pCTX247 (S:EQ ID NO:257) in vitro anti-tumor activity in two Ovarian cancer models. FIG. 9A: A2780-mCh-NLS cells pre-treated for -20 hours with 25 M Niraparib were cocultured with CER 247 and MOCK 236 CD4/8 T cells from donor 21 at a 2:1 T cell:A2780 ratio and a final Niraparib concentration of 6.25 pM. Samples treated with Niraparib + CER 247 exhibited substantially fewer tumor cells in culture -5 days later when compared to samples treated with Niraparib alone, CER. 247 alone, or Niraparib + MOCK 236. FIG.
9B:
Kuramochi-mCh-NLS cells pre-treated for -20 hours with 25 laM Niraparib were cocultured with CER 247 and MOCK 236 CD4/8 T cells from donor 21 at a 2:1 T
cell : A2780 ratio and a final Niraparib concentration of 1.56 M. Samples treated with Niraparib + CER 247 exhibited substantially fewer tumor cells in culture -5 days later when compared to samples treated with Niraparib alone, CER 247 alone, or Niraparib MOCK 236. All data were collected via IncuCyte.
FIG. 10: CER pC'FX797 (SEQ ID NO:258) in vitro antitumor activity in the A2780 Ovarian cancer model. A2780-mCh-NLS cells pre-treated for -20 hours with 0.52 M .Niraparib were co-cultured with CER. 797 and MOCK 236 CD4/8 T
cells from donor 32 at a 2:1 T cell :A2780 ratio and a final Niraparib concentration of 0.52 p.M. Samples treated with Niraparib + CER 247 exhibited substantially fewer tumor cells in culture -5 days later when compared to samples treated with Niraparib alone, CER 247 alone, or Niraparib + MOCK 236. All data were collected via IncuCyte.
FIGS. 11A-11B: Phosphatidylserine is present on the cell surface in response to Niraparib in two Ovarian cancer models. FIG. 11A: A2780-rnCh-NLS
cells treated with 1.56 and 25 M. Niraparib exhibit a significantly higher fraction of PS+ cells and an increase in Tim4-Fe WI compared to samples treated with equivalent
FIGS. 9A-9B: CER pCTX247 (S:EQ ID NO:257) in vitro anti-tumor activity in two Ovarian cancer models. FIG. 9A: A2780-mCh-NLS cells pre-treated for -20 hours with 25 M Niraparib were cocultured with CER 247 and MOCK 236 CD4/8 T cells from donor 21 at a 2:1 T cell:A2780 ratio and a final Niraparib concentration of 6.25 pM. Samples treated with Niraparib + CER 247 exhibited substantially fewer tumor cells in culture -5 days later when compared to samples treated with Niraparib alone, CER. 247 alone, or Niraparib + MOCK 236. FIG.
9B:
Kuramochi-mCh-NLS cells pre-treated for -20 hours with 25 laM Niraparib were cocultured with CER 247 and MOCK 236 CD4/8 T cells from donor 21 at a 2:1 T
cell : A2780 ratio and a final Niraparib concentration of 1.56 M. Samples treated with Niraparib + CER 247 exhibited substantially fewer tumor cells in culture -5 days later when compared to samples treated with Niraparib alone, CER 247 alone, or Niraparib MOCK 236. All data were collected via IncuCyte.
FIG. 10: CER pC'FX797 (SEQ ID NO:258) in vitro antitumor activity in the A2780 Ovarian cancer model. A2780-mCh-NLS cells pre-treated for -20 hours with 0.52 M .Niraparib were co-cultured with CER. 797 and MOCK 236 CD4/8 T
cells from donor 32 at a 2:1 T cell :A2780 ratio and a final Niraparib concentration of 0.52 p.M. Samples treated with Niraparib + CER 247 exhibited substantially fewer tumor cells in culture -5 days later when compared to samples treated with Niraparib alone, CER 247 alone, or Niraparib + MOCK 236. All data were collected via IncuCyte.
FIGS. 11A-11B: Phosphatidylserine is present on the cell surface in response to Niraparib in two Ovarian cancer models. FIG. 11A: A2780-rnCh-NLS
cells treated with 1.56 and 25 M. Niraparib exhibit a significantly higher fraction of PS+ cells and an increase in Tim4-Fe WI compared to samples treated with equivalent
3 volumes of DMSO. Niraparib also has an anti-tumor effect beginning at a concentration of at least 1.56 KM Niraparib. FIG. 11B: Kuramochi-mCh-NLS cells treated with laM Niraparib exhibit a significantly higher fraction of PS+ cells and an increase in Tim4-FcIVIFI compared to samples treated with equivalent volumes of DMSO.
Niraparib also has an anti-tumor effect beginning at a concentration of at least 25 pM
Niraparib. All samples were analyzed via Flow Cytometry.
FIG. 12: CERs exhibit synergy with additional PARP inhibitors (Rucaparib and Olaparib, in addition to Niraparib). A2780-mCh-NLS cells pre-treated for ¨20 hours with 0.52 pM Niraparib, Rucaparib, or Olaparib were co-cultured with CER 247 and MOCK 236 CD4/8 T cells from donor 32 at a 2:1 T cell :A2780 ratio and a final Niraparib concentration of 0.52 KM. Samples treated with Niraparib +
CER 247, Rucaparib + CER247, or Olaparib + CER 247 exhibited substantially fewer tumor cells in culture ¨5 days later when compared to samples treated with Niraparib, Rucaparib, or Olaparib alone, CER 247 alone, or Niraparib + MOCK 236. All data were collected via IncuCyte.
DETAILED DESCRIPTION
In one aspect, the present disclosure provides methods for the treatment of cancer using chimeric T-cell immunoglobulin mucin protein (Tim) receptors, also referred to as chimeric engulfment receptors (CERs), in combination with an inhibitor of Poly (ADP-ribose) polymerase (PARP). In another aspect, the present disclosure provides a pharmaceutical compositions or combination comprising a Chimeric Tim receptor and a PARP inhibitor. Chimeric Tim receptors useful in compositions and methods of the present disclosure confer engulfment, cytotoxicity, and/or antigen presentation activity to chimeric Tim receptor-modified host cells (e.g., T
cells), with the cytotoxic activity being induced upon binding of the chimeric Tim receptor to its target antigen, phosphatidylserine. DNA damaging agents such as PARP
inhibitors may synergize with chimeric Tim receptors by promoting cell damage and externalization of phosphatidylscrinc, which induces effector function of chimeric Tim
Niraparib also has an anti-tumor effect beginning at a concentration of at least 25 pM
Niraparib. All samples were analyzed via Flow Cytometry.
FIG. 12: CERs exhibit synergy with additional PARP inhibitors (Rucaparib and Olaparib, in addition to Niraparib). A2780-mCh-NLS cells pre-treated for ¨20 hours with 0.52 pM Niraparib, Rucaparib, or Olaparib were co-cultured with CER 247 and MOCK 236 CD4/8 T cells from donor 32 at a 2:1 T cell :A2780 ratio and a final Niraparib concentration of 0.52 KM. Samples treated with Niraparib +
CER 247, Rucaparib + CER247, or Olaparib + CER 247 exhibited substantially fewer tumor cells in culture ¨5 days later when compared to samples treated with Niraparib, Rucaparib, or Olaparib alone, CER 247 alone, or Niraparib + MOCK 236. All data were collected via IncuCyte.
DETAILED DESCRIPTION
In one aspect, the present disclosure provides methods for the treatment of cancer using chimeric T-cell immunoglobulin mucin protein (Tim) receptors, also referred to as chimeric engulfment receptors (CERs), in combination with an inhibitor of Poly (ADP-ribose) polymerase (PARP). In another aspect, the present disclosure provides a pharmaceutical compositions or combination comprising a Chimeric Tim receptor and a PARP inhibitor. Chimeric Tim receptors useful in compositions and methods of the present disclosure confer engulfment, cytotoxicity, and/or antigen presentation activity to chimeric Tim receptor-modified host cells (e.g., T
cells), with the cytotoxic activity being induced upon binding of the chimeric Tim receptor to its target antigen, phosphatidylserine. DNA damaging agents such as PARP
inhibitors may synergize with chimeric Tim receptors by promoting cell damage and externalization of phosphatidylscrinc, which induces effector function of chimeric Tim
4
5 receptors, such as engulfment, cytotoxicity, costimulatory activity, antigen presentation, or a combination thereof.
Chimeric Tim receptors described herein comprise a single chain chimeric protein, the single chain chimeric protein comprising: (a) an extracellular domain comprising a binding domain comprising: (i) a Tim I IgV domain or a Tim4 I8V domain; and (ii) a Timl mucin domain or a Tim4 mucin domain; (b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) a transrnembrane domain positioned between and connecting the extracellular domain and the intracellular signaling domain.
In certain embodiments, the extracellular domain of the chimeric Tim receptors described herein optionally includes an extracellular spacer domain positioned between and connecting the binding domain and transmembrane domain.
In certain embodiments, chimeric Tim receptors may also be capable of costimulating T cells via a different signaling pathway than the "classical" T
cell costimulation pathways (e.g., CD28). For example, in addition to binding phosphatidylserine, Tim4 is also a ligand for Tim I, which is expressed on the surface of activated T cells. Timl is also capable of binding to phosphatidylserine.
Tim44nduced 'rim I signaling has been found to costimulate T cell proliferation and survival (Ham Meyers etal., 2005, Nat. Immunol. 6:455). Thus, in certain embodiments, cytotoxic chimeric Tim receptors may reduce or inhibit T cell exhaustion, or restore exhausted T
cells by providing costimulatory signals via at least one signaling pathway.
In certain embodiments, cytotoxic chimeric Tim receptors provide cosh mulatoty signals via at least two distinct signaling pathways (e.g., via the selected costimulatory signaling domain in the cytotoxic chimeric Tim receptor and Tim I).
In certain embodiments, when expressed in a host cell, the chimeric Tim receptors of the present disclosure also confer engulfment activity to the host cell. For example, in certain such embodiments, binding of the chimeric Tim receptor expressed in a host cell to a phosphatidylserinc target may induce both cytolytic and engulfment responses by the host cell. In particular embodiments of the modified host cells described herein, the host cell does not naturally exhibit an engulfment phenotype prior to modification with the chimeric Tim receptor.
In embodiments, administration of host cells modified with chimeric Tim receptors of the present disclosure can be used in methods for eliminating target cells bearing surface exposed phosphatidylserine, e.g., for the treatment of cancer. In non-nal, healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. H:owever, certain cellular events, such as damage, apoptosis, necrosis, and stress, activates a "scramblase" that quickly exposes phosphatidylserine on the cell surface, where it can bind to receptors such as Ti m4 or Tim 1. Endogenous tumor-specific effector T cells can induce exposure of phosphatidylserine on the outer membrane of targeted tumor cells during cytolysis. Furthermore, certain cancer therapies (e.g., chemotherapy, radiotherapy, CAR-T cells, etc.) can induce exposure of phosphatidylserine on targeted tumor cells or cells in the tumor microenvironment by inducing apoptosis, cellular stress, cellular damage, etc. Engineered host cells expressing the presently disclosed chimeric Tim receptors may clear damaged, stressed, apoptotic, or necrotic tumor cells bearing surface exposed phosphatidylserine by inducing apoptosis in the tumor cells bearing surface exposed phosphatidylseiine. En certain embodiments, host cells expressing chimeric Tim receptors disclosed herein clear damaged, stressed, apoptotic, or necrotic tumor cells bearing surface exposed phosphatidylserine by inducing apoptosis and by engulfment. In another aspect, host cells modified with chimeric Tim receptors of the present disclosure can be used to enhance the effect of a PARP inhibitor that induces cellular stress, damage, necrosis, or apoptosis. For example, administration of a PARP inhibitor may increase levels of surface phosphatidylserine, thus resulting in a synergistic combination. Cells expressing a chimeric Tim receptor as presently described can bind to the phosphatidylserine moieties exposed on the outer leaflet of damaged or dying cells resulting from administration of the PARP inhibitor and induce cytolysis or both cytolysis and engulfment of the targeted cells.
The combination of engineered host cells comprising chimeric Tim receptors and a PARP inhibitor according to the present description may be
Chimeric Tim receptors described herein comprise a single chain chimeric protein, the single chain chimeric protein comprising: (a) an extracellular domain comprising a binding domain comprising: (i) a Tim I IgV domain or a Tim4 I8V domain; and (ii) a Timl mucin domain or a Tim4 mucin domain; (b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) a transrnembrane domain positioned between and connecting the extracellular domain and the intracellular signaling domain.
In certain embodiments, the extracellular domain of the chimeric Tim receptors described herein optionally includes an extracellular spacer domain positioned between and connecting the binding domain and transmembrane domain.
In certain embodiments, chimeric Tim receptors may also be capable of costimulating T cells via a different signaling pathway than the "classical" T
cell costimulation pathways (e.g., CD28). For example, in addition to binding phosphatidylserine, Tim4 is also a ligand for Tim I, which is expressed on the surface of activated T cells. Timl is also capable of binding to phosphatidylserine.
Tim44nduced 'rim I signaling has been found to costimulate T cell proliferation and survival (Ham Meyers etal., 2005, Nat. Immunol. 6:455). Thus, in certain embodiments, cytotoxic chimeric Tim receptors may reduce or inhibit T cell exhaustion, or restore exhausted T
cells by providing costimulatory signals via at least one signaling pathway.
In certain embodiments, cytotoxic chimeric Tim receptors provide cosh mulatoty signals via at least two distinct signaling pathways (e.g., via the selected costimulatory signaling domain in the cytotoxic chimeric Tim receptor and Tim I).
In certain embodiments, when expressed in a host cell, the chimeric Tim receptors of the present disclosure also confer engulfment activity to the host cell. For example, in certain such embodiments, binding of the chimeric Tim receptor expressed in a host cell to a phosphatidylserinc target may induce both cytolytic and engulfment responses by the host cell. In particular embodiments of the modified host cells described herein, the host cell does not naturally exhibit an engulfment phenotype prior to modification with the chimeric Tim receptor.
In embodiments, administration of host cells modified with chimeric Tim receptors of the present disclosure can be used in methods for eliminating target cells bearing surface exposed phosphatidylserine, e.g., for the treatment of cancer. In non-nal, healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. H:owever, certain cellular events, such as damage, apoptosis, necrosis, and stress, activates a "scramblase" that quickly exposes phosphatidylserine on the cell surface, where it can bind to receptors such as Ti m4 or Tim 1. Endogenous tumor-specific effector T cells can induce exposure of phosphatidylserine on the outer membrane of targeted tumor cells during cytolysis. Furthermore, certain cancer therapies (e.g., chemotherapy, radiotherapy, CAR-T cells, etc.) can induce exposure of phosphatidylserine on targeted tumor cells or cells in the tumor microenvironment by inducing apoptosis, cellular stress, cellular damage, etc. Engineered host cells expressing the presently disclosed chimeric Tim receptors may clear damaged, stressed, apoptotic, or necrotic tumor cells bearing surface exposed phosphatidylserine by inducing apoptosis in the tumor cells bearing surface exposed phosphatidylseiine. En certain embodiments, host cells expressing chimeric Tim receptors disclosed herein clear damaged, stressed, apoptotic, or necrotic tumor cells bearing surface exposed phosphatidylserine by inducing apoptosis and by engulfment. In another aspect, host cells modified with chimeric Tim receptors of the present disclosure can be used to enhance the effect of a PARP inhibitor that induces cellular stress, damage, necrosis, or apoptosis. For example, administration of a PARP inhibitor may increase levels of surface phosphatidylserine, thus resulting in a synergistic combination. Cells expressing a chimeric Tim receptor as presently described can bind to the phosphatidylserine moieties exposed on the outer leaflet of damaged or dying cells resulting from administration of the PARP inhibitor and induce cytolysis or both cytolysis and engulfment of the targeted cells.
The combination of engineered host cells comprising chimeric Tim receptors and a PARP inhibitor according to the present description may be
6 administered to a subject alone, or in further combination with one or more additional therapeutic agents, including for example CAR-T cells, TCRs, antibodies, radiation therapy, chemotherapies, small molecules, oncolytic viruses, el ectropulse therapy, etc.
Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.
In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components.
The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "have"
and "coinpiise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.
Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein. The term "antibody" is used in the broadest sense and includes polyclonal and monoclonal antibodies. An "antibody" may refer to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as an antigen-binding portion (or antigen-binding domain) of an intact antibody that has or retains the capacity to bind a target molecule. An antibody may be naturally occurring, recombinantly produced, genetically engineered, or modified forms of immunoglobulins, for example intrabodies, peptibodies, nanobodies, single domain antibodies, SMIPs, multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodics, tctrabodics, tandem di-scFV, tandem tri-scFv, ADAPTIR). A
monoclonal antibody or antigen-binding portion thereof may be non-human, chimeric, humanized,
Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.
In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components.
The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "have"
and "coinpiise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.
Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein. The term "antibody" is used in the broadest sense and includes polyclonal and monoclonal antibodies. An "antibody" may refer to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as an antigen-binding portion (or antigen-binding domain) of an intact antibody that has or retains the capacity to bind a target molecule. An antibody may be naturally occurring, recombinantly produced, genetically engineered, or modified forms of immunoglobulins, for example intrabodies, peptibodies, nanobodies, single domain antibodies, SMIPs, multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodics, tctrabodics, tandem di-scFV, tandem tri-scFv, ADAPTIR). A
monoclonal antibody or antigen-binding portion thereof may be non-human, chimeric, humanized,
7 or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). "Antigen-binding portion" or "antigen-binding domain" of an intact antibody is meant to encompass an "antibody fragment," which indicates a portion of an intact antibody and refers to the antigenic determining variable regions or complementary determining regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab.)2, and Fv fragments, Fab'-S1-1, F(ab')2, diabodi es, linear antibodies, sel7v antibodies, VII, and multispecific antibodies formed from antibody fragments. A "Fab" (fragment antigen binding) is a portion of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond. An antibody may be of any class or subclass, including IgG and subclasses thereof (IgGt, IgCr2, IgG3, IgG4), IgM, IgE, IgA, and IgD.
The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to antigen.
The variable domains of the heavy chain and light chain (VH and VIõ
respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al.
Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL
domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL
domain from an antibody that binds the antigen to screen a library of complementary VL or VII
domains, respectively. See, e.g., Portolano et al., J. 1m munol. 150:880-887 (1993);
Clarkson et al., Nature 352:624-628 (1991).
The terms "complementarity determining region" and "CDR," which are synonymous with "hyper-variable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LC DR3).
The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to antigen.
The variable domains of the heavy chain and light chain (VH and VIõ
respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al.
Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL
domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL
domain from an antibody that binds the antigen to screen a library of complementary VL or VII
domains, respectively. See, e.g., Portolano et al., J. 1m munol. 150:880-887 (1993);
Clarkson et al., Nature 352:624-628 (1991).
The terms "complementarity determining region" and "CDR," which are synonymous with "hyper-variable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LC DR3).
8 As used herein, the terms "binding domain", "binding region", and "binding moiety" refer to a molecule, such as a peptide, oligopeptide, polypeptide, or protein that possesses the ability to specifically and non-covalently bind, associate, unite, recognize, or combine with a target molecule (e.g., phosphatidylserine). A
binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or other target of interest. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody or functional binding domain or antigen-binding portion thereof.
Exemplary binding domains include single chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), receptor ectodomains (e.g., Tim4), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for the specific ability to bind to a biological molecule.
"T cell receptor" (TCR) refers to a molecule found on the surface of T
cells (also referred to as T lymphocytes) that is generally responsible for recognizing antigens bound to major histocompatibility complex (1µ41-1C) molecules. The TCR is generally composed of a disulfide-linked heterodimer of the highly variable a and 13 chains (also known as Taket and TCR13, respectively) in most T cells. In a small subset of T cells, the TCR is made up of a heterodimer of y and 8 chains (also known as TCRy and TCR8, respectively). Each chain of the Tat is a member of the immunoglobulin superfamily and possesses one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end (see .laneway el al., Immunohiology: The Immune System in Health and Disease, 3rdEd., Current Biology Publications, p. 4:33, 1997). TCRs of the present disclosure may be from various animal species, including human, mouse, rat, cat, dog, goat, horse, or other mammals. TCRs may be cell-bound (i.e., have a transmembrane region or domain) or in soluble form. TCRs include recombinantly produced, genetically engineered, fusion, or modified forms of TCRs, including for example, scTCRs, soluble TCRs, TCR fusion constructs (TRuCm; see, U.S. Patent Publication No. 2017/0166622).
binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or other target of interest. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody or functional binding domain or antigen-binding portion thereof.
Exemplary binding domains include single chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), receptor ectodomains (e.g., Tim4), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for the specific ability to bind to a biological molecule.
"T cell receptor" (TCR) refers to a molecule found on the surface of T
cells (also referred to as T lymphocytes) that is generally responsible for recognizing antigens bound to major histocompatibility complex (1µ41-1C) molecules. The TCR is generally composed of a disulfide-linked heterodimer of the highly variable a and 13 chains (also known as Taket and TCR13, respectively) in most T cells. In a small subset of T cells, the TCR is made up of a heterodimer of y and 8 chains (also known as TCRy and TCR8, respectively). Each chain of the Tat is a member of the immunoglobulin superfamily and possesses one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end (see .laneway el al., Immunohiology: The Immune System in Health and Disease, 3rdEd., Current Biology Publications, p. 4:33, 1997). TCRs of the present disclosure may be from various animal species, including human, mouse, rat, cat, dog, goat, horse, or other mammals. TCRs may be cell-bound (i.e., have a transmembrane region or domain) or in soluble form. TCRs include recombinantly produced, genetically engineered, fusion, or modified forms of TCRs, including for example, scTCRs, soluble TCRs, TCR fusion constructs (TRuCm; see, U.S. Patent Publication No. 2017/0166622).
9 The term "variable region" or "variable domain" of a TCR a-chain (Va) and 13-chain (Vf3), or Vy and VS for yo TCRs, are involved in binding of the TCR to antigen. The Va and V of a native TCR generally have similar structures, with each variable domain comprising four conserved FRs and three CDRs. The Va domain is encoded by two separate DNA segments, the variable gene segment (V gene) and the joining gene segment (I gene); the Vo domain is encoded by three separate DNA
segments, the variable gene segment (V gene), the diversity gene segment (D
gene), and the joining gene segment (J gene). A single Va or V domain may be sufficient to confer antigen-binding specificity. "Major histocompatibility complex molecule"
(MI-IC molecule) refers to a glycoprotein that delivers a peptide antigen to a cell surface. MHC class I molecules are heterodimers composed of a membrane spanning a chain (with three a domains) and a non-covalentl y associated 132 microglobulin. MHC
class II molecules are composed of two transmembrane glycoproteins, a and 13, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where peptide:MHC
complex is recognized by CD8' T cells. WIC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4- T cells. An MHC molecule may be from various animal species, including human, mouse, rat, or other mammals.
"Chimeric antigen receptor" (CAR) refers to a chimeric protein comprising two or more distinct domains and can function as a receptor when expressed on the surface of a cell. CARs are generally composed of an extracellular domain comprising a binding domain that binds a target antigen, an optional extracellular spacer domain, a transmembrane domain, and an intracellular signaling domain (e.g., an immunoreceptor tyrosine-based activation motif (UAW-containing T cell activating motif, and optionally an intracellular costimulatory domain). In certain embodiments, an intracellular signaling domain of a CAR has an ITAM-containing T cell activating domain (e.g. CD3C) and an intracellular costimulatory domain (e.g., CD28). In certain embodiments, a CAR is synthesized as a single polypeptide chain or is encoded by a nucleic acid molecule as a single chain polypepfide.
A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain affinities, such as Western blot, ELISA, and BIACORE analysis (see also, e.g., Scatchard etal., Ann. N.Y. Acad. Sci. 51:660, 1949; and U.S.
Patent Nos.
5,283,173, 5,468,614, or the equivalent). As used herein, "specifically binds"
refers to an association or union of a binding domain, or a fusion protein thereof, to a target molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 105 Mt, while not significantly associating or uniting with any other molecules or components in a sample.
The terms "antigen" and "Ag" refer to a molecule that is capable of inducing an immune response. The immune response that is induced may involve antibody production, the activation of specific immunologically-competent cells, or both. Macromolecules, including proteins, glycoproteins, and glycolipids, can serve as an antigen. Antigens can be derived from recombinant or genomic DNA. As contemplated herein, an antigen need not be encoded (i) solely by a full length nucleotide sequence of a gene or (ii) by a "gene" at all. An antigen can be generated or synthesized, or an antigen can be derived from a biological sample. Such a biological sample can include, but is not limited, to a tissue sample, a tumor sample, a cell, or a biological fluid.
The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence or protein determinant within an antigen that is specifically bound by a cognate immune binding molecule, such as an antibody or fragment thereof (e.g., scFv), T cell receptor (TCR), chimeric Tim receptor, or other binding molecule, domain or protein. :Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics. An epitope may be a linear epitope or a conformational cpitopc.
As used herein, the term "PARP" (Poly (ADP-ribose) polymerase) refers to a family of proteins, each of which have two ribose moieties and two phosphates per unit polymer. PARP1 and PARP2 are enzymes involved in a DNA. repair pathway.
As used herein, the term "Tim4" (T-cell immunoglobulin and mucin domain containing protein 4), also known as "1imD4", refers to a phosphatidylserine receptor that is typically expressed on antigen presenting cells, such as macrophages and dendritic cells. 1'irn4 mediates the phagocytosis of apoptotic, necrotic, damaged, injured, or stressed cells, which present phosphatidylserine (PtdSer) on the exofacial (outer) leaflet of the cell membrane. Tim4 is also capable of binding to Timl expressed on the surface of T cells and inducing proliferation and survival. In certain embodiments, Tim4 refers to human Tim4. An exemplary human Tim4 protein comprises an amino acid sequence of SEQ ID NO: 1.
As used herein, the term "Tim4 binding domain" refers to the N-terminal immunoglobulin-fold domain of Tim4 that possesses a metal ion¨dependent pocket that selectively binds PtdSer. An exemplary human Tim4 binding domain comprises an amino acid sequence of SEQ ID NO:2, and an exemplary mouse Tim4 binding domain comprises an amino acid sequence of SEQ ID NO:24.
A Tim4 binding domain includes a variable immunoglobulin (1gV) like domain (referred to herein as an "IgV domain") and a Mucin like domain ("referred to herein as a "mucin domain"). An exemplary human Tim4 IgV domain comprises an amino acid sequence of SEQ ID NO:34, and an exemplary human Tim4 mucin domain comprises an amino acid sequence of SEQ ID NO:35. In certain embodiments, the Tim4 binding domain does not include a signal peptide. An exemplary human Tim4 signal peptide has the amino acid sequences of SEQ ID NO:11. An exemplary mouse Tim4 signal peptide has the amino acid sequences of SEQ ID NO:25.
As used herein, the term "Tim 1" (T-cell immunoglobulin and mucin domain containing protein 1), refers to a phosphatidylserine receptor that is expressed on the surface of T cells. Tim I, as noted above is also capable of binding to Tim4 expressed on the surface of antigen presenting cells. In certain embodiments, Timl refers to human Tim 1. An exemplary human Timl protein comprises an amino acid sequence of SEQ ID NO:36.
As used herein, the term "Timl binding domain" refers to the N-terminal immunoglobulin-fold domain of Timl that selectively binds PtdSer. An exemplary human Tim I binding domain comprises an amino acid sequence of SEQ ID NO:37.
A Timl binding domain includes an IgV domain and a mucin domain.
An exemplary human Timl IgV domain comprises an amino acid sequence of SEQ ID
NO:38, and an exemplary human Tim I rnucin domain comprises an amino acid sequence of SEQ ID NO:39. In certain embodiments, the Timl binding domain does not include a signal peptide. An exemplary human Timl signal peptide has the amino acid sequences of SEQ ID NO:40.
As used herein, an "effector domain" is an intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response in a cell expressing the effector domain when receiving the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response.
As used herein, a "costimulatory signaling domain" refers to an intracellular signaling domain, or functional portion thereof, of a costimulatory molecule, which, when activated in conjunction with a primary or classic (e.g., ITAM-driven) activation signal (provided by, for example, a CD3C, intracellular signaling domain), promotes or enhances a T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or combinations thereof Costimulatory signaling domains include, for example, CD27, CD28, CD4OL, GITR, NI(G2C, CARD!, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMI71), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, SLP76, TRIM, or any combination thereof.
As used herein, an "immunoreceptor tyrosine-based activation motif (ITAM) activating domain" refers to an intracellular signaling domain or functional portion thereof which is naturally or endogenously present on an immune cell receptor or a cell surface marker and contains at least one immunoreceptor tyrosine-based activation motif (ITAM). ITAM refers to a conserved motif of YXXL/I-X6.8-YXXL/I.
In certain embodiments an ITAM signaling domain contains one, two, three, four, or more ITAMs. An ITAM signaling domain may initiate T cell activation signaling following antigen binding or ligand engagement. ITAM-signaling domains include, for example, intracellular signaling domains of CD3y, CD38, CD3e, CD3C, CD79a, and CD66d.
"Junction amino acids" or "junction amino acid residues" refer to one or more (e.g., about 2-20) amino acid residues between two adjacent motifs, regions or domains of a polypeptide. Junction amino acids may result from the construct design of a chimeric protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a chimeric protein).
"Nucleic acid molecule" and "polynucleotide" can be in the form of RNA or DNA., which includes c:DN A, genomic DNA, and synthetic DNA. A nucleic acid molecule may be composed of naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. A nucleic acid molecule may be double stranded or single stranded, and if single stranded, may be the coding strand or non-coding (anti-sense strand). A
coding molecule may have a coding sequence identical to a coding sequence known in the art or may have a different coding sequence, which, as the result of the redundancy or degeneracy of the genetic code, or by splicing, can encode the same polypeptide.
"Encoding" refers to the inherent property of specific polynucleotide sequences, such as DNA, cDNA, and mRNA sequences, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a polynucleotide encodes a protein if transcription and translation of mitNA
corresponding to that polynucleotide produces the protein in a cell or other biological system. Both a coding strand and a non-coding strand can be referred to as encoding a protein or other product of the polynucleotide. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
"Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
As used herein, the term "mature polypeptide" or "mature protein" refers to a protein or polypeptide that is secreted or localized in the cell membrane or inside certain cell organelles (e.g., the endoplasmic reticulum, golgi, or endosome) and does not include an N-terminal signal peptide.
A "signal peptide", also referred to as "signal sequence", "leader sequence", "leader peptide", "localization signal" or "localization sequence", is a short peptide (usually 15-30 amino acids in length) present at the N-terminus of newly synthesized proteins that are destined for the secretory pathway. A signal peptide typically comprises a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a cleavage site for a signal peptidase. In eukaryotes, a signal peptide prompts translocation of the newly synthesized protein to the endoplasmic reticulum where it is cleaved by the signal peptidase, creating a mature protein that then proceeds to its appropriate destination.
The term "chimeric" refers to any nucleic acid molecule or protein that is not endogenous and comprises sequences joined or linked together that are not normally found joined or linked together in nature. For example, a chimeric nucleic acid molecule may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from the same source but arranged in a manner different than that found in nature.
As used herein, the term "endogenous" or "native" refers to a gene, protein, cornpound, molecule, or activity that is normally present in a host or host cell, including naturally occurring variants of the gene, protein, compound, molecule, or activity.
As used herein, "homologous" or "homolog" refers to a molecule or activity from a host cell that is related by ancestry to a second gene or activity, e.g., from the same host cell, from a different host cell, from a different organism, from a different strain, from a different species. For example, a heterologous molecule or heterologous gene. encoding the molecule may be homologous to a native host cell molecule or gene that encodes the molecule, respectively, and may optionally have an altered structure, sequence, expression level or any combination thereof.
As used herein, "heterologous" nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but can be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell. The source of the heterologous nucleic acid molecule, construct or sequence can be from a different genus or species. In some embodiments, the heterologous nucleic acid molecules are not naturally occurring. In certain embodiments, a heterologous nucleic acid molecule is added (i.e., not endogenous or native) into a host cell or host genorne by, for example, conjugation, transformation, transfection, transduction, electroporation, or the like, wherein the added molecule can integrate into the host cell genome or exist as extra-chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can be present in multiple copies. In addition, "heterologous" refers to a non-native enzyme, protein or other activity encoded by a non-endogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.
As used herein, the term "engineered," "recombinant," "modified" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule, or vector that has been modified by introduction of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been genetically engineered by human intervention that is, modified by introduction of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, where such alterations or modifications can be introduced by genetic engineering.
Human-generated genetic alterations can include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins, chimeric receptors, or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell's genetic material. Exemplary modifications include those in coding regions or functional fragments thereof heterologous or homologous polypeptides from a reference or parent molecule. Additional exemplary modifications include, for example, modifications in non-coding regulatory regions in which the modifications alter expression of a gene or operon.
As used herein, the term "transgene" refers to a gene or polynucleotide encoding a protein of interest (e.g., chimeric Tim receptor) whose expression is desired in a host cell and that has been transferred by genetic engineering techniques into a cell.
A transgene may encode proteins of therapeutic interest as well as proteins that are reporters, tags, markers, suicide proteins, etc. A transgene may be from a natural source, modification of a natural gene, or a recombinant or synthetic molecule. In certain embodiments, a transgene is a component of a vector.
The term "overexpressed" or "overexpression" of an antigen refers to an abnormally high level of antigen expression in a cell. Overexpressed antigen or overexpression of antigen is often associated with a disease state, such as in hematological malignancies and cells forming a solid tumor within a specific tissue or organ of a subject. Solid tumors or hematological malignancies characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.
The "percent identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. The comparison of sequences and determination of percent identity between two or more sequences can be accomplished using a mathematical algorithm, such as BLAST and Gapped BLAST programs at their default parameters (e.g.. Altschul etal., J. Mot Biol. 2/5:403, 1990; see also BLASTN
at www.nthi.nlm.nih.gov/BLAST).
A "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).
The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements that are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one that expresses the gene product in a tissue specific manner.
A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
The phrase "under transcriptional control" or "operatively linked" as used herein means that a promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, for example, plasmids, cosmids, viruses, or phage. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells. An "expression vector"
is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
In certain embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gamma retrovirus vectors, and lentivirus vectors.
"Retroviruses" are viruses having an RNA genome. "Gamma retrovirus" refers to a genus of the retroviridae family. Examples of gamma retroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses. "Lentivirus" refers to a genus of retrovimses that are capable of infecting dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (B1V), and simian immunodeficiency virus (Sly).
In other embodiments, the vector is a non-viral vector. Examples of non-viral vectors include lipid-based DNA vectors, modified mR_NA (modRNA), self-amplifying mRNA, closed-ended linear duplex (CELiD) DNA, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty). Where a non-viral delivery system is used, the delivery vehicle can be aliposome. Lipid formulations can be used to introduce nucleic acids into a host cell in vitro, ex vivo, or in vivo The nucleic acid may be encapsulated in the interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, contained or complexed with a micelle, or otherwise associated with a lipid.
As used herein, the term "engulfment" refers to a receptor-mediated process wherein endogenous or exogenous cells or particles greater than 100 nm in diameter are internalized by a phagocyte or host cell of the present disclosure Engulfment is typically composed of multiple steps: (1) tethering of the target cell or particle via binding of an engulfment receptor to a pro-engulfment marker or antigenic marker directly or indirectly (via a bridging molecule) on a target cell or particle; and (2) internalization or engulfment of the whole target cell or particle, or a portion thereof. In certain embodiments, internalization may occur via cytoskeletal rearrangement of a phagocyte or host cell to form a phagosome, a membrane-bound compartment containing the internalized target. Engulfment may further include maturation of the phagosome, wherein the phagosome becomes increasingly acidic and fuses with lysosomes (to form a phagolysosorne), whereupon the engulfed target is degraded (e.g., "phagocytosis"). Alternatively, phagosome-lysosome fusion may not be observed in engulfment. In yet another embodiment, a phagosome may regurgitate or discharge its contents to the extracellular environment before complete degradation. In some embodiments, engulfment refers to phagocytosis. In some embodiments, engulfment includes tethering of the target cell or particle by the phagocyte of host cell of the present disclosure, but not internalization. In some embodiments, engulfment includes tethering of the target cell or particle by the phagocyte of host cell of the present disclosure and internalization of part of the target cell or particle.
As used herein, the term "phagocytosis" refers to an engulfment process of cells or large particles (2_;.-- 0.5 gm) wherein tethering of a target cell or particle, engulfment of the target cell or particle, and degradation of the internalized target cell or particle occurs. In certain embodiments, phagocytosis comprises formation of a phagosome that encompasses the internalized target cell or particle and phagosome fusion with a I ysosome to form a phagolysosome, wherein the contents therein are degraded. In certain embodiments, during phagocytosis, following binding of a chimeric Tim receptor expressed on a host cell of the present disclosure to a phosphatidylserine expressed by a target cell or particle, a phagocytic synapse is formed; an actin-rich phagocytic cup is generated at the phagocytic synapse;
phagocytic arms are extended around the target cell or particle through cytoskeletal rearrangements; and ultimately, the target cell or particle is pulled into the phagocyte or host cell through force generated by motor proteins. As used herein, "phagocytosis"
includes the process of "efferocytosis", which specifically refers to the phagocytosis of apoptotic or necrotic cells in a non-inflammatory manner.
The term "immune system cell" or "immune cell" means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow.
Hematopoietic stem cells give rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a T6 T cell, a regulatory T cell, a natural killer cell, and a dendritic cell.
Macrophages and dendritic cells may also be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MEW) receptor on the surface of the .APC complexed with a peptide interacts with a TCR on the surface of a T cell.
The term "T cells" refers to cells of T cell lineage. "Cells of T cell lineage" refer to cells that show at least one phenotypic characteristic of a T cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for T cells (e.g. , CD3+, CD4, CD8-), or a physiological, morphological, functional, or immunological feature specific for a T cell. For example, cells of the T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25+ immature and inactivated T cells;
cells that have undergone CD4 or CD8 linage commitment; thyrnocyte progenitor cells that are CD4-CD8+ double positive; single positive CD4 + or CD8+; TCRap or TCR y8; or mature and functional or activated T cells. The term "T cells" encompasses naïve T
cells (CD45 RA+, CCR7+, CD62L+, CD27+, C045R0-), central memory T cells (CD45R0+, CD62L', CD8+), effector memory T cells (CD45RA+, CD45R0-, CCR7-, CD621,-, CD27-), mucosal-associated invariant T (MATT) cells, Tregs, natural killer T
cells, and tissue resident T cells.
The term "B cells" refers to cells of the B cell lineage. "Cells of B cell lineage" refer to cells that show at least one phenotypic characteristic of a B cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for B cells (e.g. , CD19+, CD72+, CD24+, CD20.4), or a physiological, morphological, functional, or immunological feature specific for a B cell. For example, cells of the B cell lineage may be progenitor or precursor cells committed to the B cell lineage (e.g., pre-pro-B cells, pro-B cells, and pre-B cells); immature and inactivated B cells or mature and functional or activated B
cells. Thus, "B cells" encompass naïve B cells, plasma cells, regulatory B
cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmahlast cells, and memory B cells (e.g., CD27+, The term "cytotoxic activity," also referred to as "cytolyfic activity,"
with respect to a cell (e.g., a T cell or NK cell) expressing an immune receptor (e.g., a TCR) or a chimeric Tim receptor according to the present disclosure on its surface, means that upon antigen-specific signaling (e.g., via the TCR, chimeric Tim receptor), the cell induces a target cell to undergo apoptosis. In some embodiments, a cytotoxic cell may induce apoptosis in a target cell via the release of cytotoxins, such as perforin, granzyme, and granulysin, from granules. Perforins insert into the target cell membrane and form pores that allow water and salts to rapidly enter the target cell.
Granzymes are serine proteases that induce apoptosis in the target cell. Granulysin is also capable of forming pores in the target cell membrane and is a proinflamtnatory molecule.
In some embodiments, a cytotoxic cell may induce apoptosis in a target cell via interaction of Fas ligand, which is upregulated on T cell following antigen-specific signaling, with Fas molecules expressed on the target cell. Fas is an apoptosis-signaling receptor molecule on the surface of a number of different cells.
The term "exhaustion" with respect to immune cells refers to a state of immune cell dysfunction defined by poor effector function (e.g., reduced cytokine production, reduced cytotoxic activity), reduced proliferative capacity, increased expression of immune checkpoint molecules, and a transcriptional state distinct from that of functional effector or memory cells. In certain embodiments, an exhausted immune cell becomes unresponsive to the presence of its target antigen. Immune cell exhaustion may result from chronic exposure to a target antigen (e.g., as may result from chronic infection) or when it enters an immunosuppressive environment (e.g., a tumor inicroenvironment). In certain embodiments, immune cell exhaustion refers to T
cell exhaustion, NK cell exhaustion, or both. In certain embodiments, exhausted T cells exhibit; (a) increased expression of PD-I, LAG3, TIM3, or any combination thereof; (b) decreased production of IFN-y, IL-2, TNT-a, or any combination thereof; or both (a) and (b). In certain embodiments, exhausted NK cells exhibit; (a) increased expression of PD-1, NKG2A, TIM3, or any combination thereof; (b) decreased production of IFN-y, TN. F-a, or both; or both (a) and (b).
A "disease" is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein, if the disease is not ameliorated, then the subject's health continues to deteriorate. In contrast, a "disorder" or "undesirable condition" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder or undesirable condition. Left untreated, a disorder or undesirable condition does not necessarily result in a further decrease in the subject's state of health.
The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. The aberrant cells may form solid tumors or constitute a hematological malignancy. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.
Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.
The term "subject," "patient" and "individual" are used interchangeably herein and are intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenie species thereof.
"Adoptive cellular immunotherapy" or "adoptive immunotherapy" refers to the administration of naturally occurring or genetically engineered disease antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient).
"Autologous" refers to any material (e.g., a graft of organ, tissue, cells) derived from the same subject to which it is later to be re-introduced.
"Allogeneic" refers to a graft derived from a different subject of the same species.
A "therapeutically effective amount" or "effective amount" of a chimeric protein or cell expressing a chimeric protein of this disclosure (e.g., a chimeric Tim receptor or a cell expressing a chimeric Tim receptor) refers to that amount of protein or cells sufficient to result in amelioration of one or more symptoms of the disease, disorder, or undesired condition being treated. When referring to an individual active ingredient or a cell expressing a single active ingredient, administered alone, a therapeutically effective dose refers to the effects of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective dose refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.
"Treat" or "treatment" or "ameliorate" refers to medical management of a disease, disorder, or undesired condition of a subject. in general, an appropriate dose or treatment regimen comprising a host cell expressing a chimeric protein of this disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease, disorder, or undesired condition; decreased occurrence of symptoms; improved quality of life;
longer disease-free status; diminishment of extent of disease, disorder, or undesired condition; stabilization of disease state; delay of disease progression;
remission;
survival; prolonged survival; or any combination thereof.
The term "anti-tumor effect" refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by prevention of a hematological malignancy or tumor formation.
"Autoimmune disease" refers to a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriately excessive response to a self-antigen. An autoimmune response may involve self-reactive B-cells that produce autoantibodies, self-reactive T-cells, or both.
An "autoantibody" as used herein is an antibody produced by a subject that binds to a self-antigen also produced by the subject.
Additional definitions are provided throughout the present disclosure.
As noted above, methods of the disclosure are useful for treating cancer in a subject. Therapeutic agents of the disclosure (i.e., a chimeric Tim receptor and a PARP inhibitor), when administered together or sequentially, treat a cancer in a subject (e.g., (i) arrest the cancer's development; (ii) cause regression of the cancer; or (iii) relieve the symptoms resulting from the cancer).
In embodiments, the methods of treatment disclosed herein comprise administering an effective amount of a chimeric Tim receptor and a PARP
inhibitor to the subject, thereby treating their cancer.
Chimeric Tim Receptors The present disclosure provides a chimeric Tim receptor comprising a single chain chimeric protein, the single chain chimeric protein comprising:
an cxtraccllular domain comprising a Tim binding domain; an intracellular signaling domain comprising a first costintulatory signaling domain; and a transmembrane domain positioned between and connecting the extracellular domain and intracellular signaling domain. In certain embodiments, the extracellular domain of the chimeric Tim receptors described herein optionally includes an extracellular spacer domain positioned between and connecting the binding domain and transmembrane domain.
When expressed in a host cell, chimeric Tim receptors of the present disclosure can confer a phosphatidylserine-specific, cytotoxic phenotype to the modified host cell (e.g., the host cell becomes cytotoxic to a stressed, damaged, injured, apoptotic, or necrotic cell expressing phosphatidylserine on its surface). In certain embodiments, the chimeric Tim receptors induce apoptosis in targeted cells via release of granzymes, perforin, granulysin, or any combination thereof. In further embodiments, cells expressing a chimeric Tim receptor according to the present description exhibit an engulfment phenotype specific to phosphatidylserine presenting cells.
The intracellular signaling domain can include one or more effector domains that are capable of transmitting functional signals to a cell in response to binding of the extracellular domain of the chimeric Tim receptor and phosphatidylserine. Signaling by the intracellular signaling domain(s) is triggered by binding of the extracellular domain to phosphatidylserine. The signals transduced by the intracellular signaling domain promote effector function of the chimeric Tim receptor containing cell. Examples of effector function include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, expansion, engulfment of a target cell or particle expressing phosphatidylserine on its surface, antigen presentation, or any combination thereof In certain embodiments, the intracellular signaling domain comprises a first intracellular signaling domain. In further embodiments, the intracellular signaling domain comprises a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a first intracellular signaling domain, a second intracellular signaling domain, and a third intracellular signaling domain. Chimeric Tim receptors according to the present disclosure can be used in a variety of therapeutic methods where clearance of apoptotic, necrotic, damaged, or stressed cells is beneficial, while providing costimulation that enhances cellular immune response, reduces immune cell exhaustion, or both.
Component parts of the fusion proteins of the present disclosure are further described in detail herein.
Extracellular Domain A Tim4 binding domain suitable for use in a chimeric Tim4 receptor of the present disclosure may be any polypeptide or peptide derived from a Tim4 molecule that specifically binds phosphatidylserine. In certain embodiments, the Tim4 binding domain is derived from human Tim4. An exemplary human Tim4 molecule is provided in Uniprot. Ref. Q96H15 (SEQ ID NO:1). An exemplary human Tim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:2 or amino acids 314 of SEQ ID NO:2. An exemplary mouse Tim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:24 or amino acids 23-279 of SEQ ID
NO:24. In certain embodiments, the Tim4 binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%õ 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID NO:24 or amino acids 23-279 of SEQ ID
NO:24. In certain embodiments, the Tim4 binding domain comprises an amino acid sequence haying at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of S:EQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID
NO:24 or amino acids 23-279 of SEQ ID NO:24.
In certain embodiments, the extracellular domain optionally comprises an extracellular, non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable proper cell/cell contact, binding, and activation. When included in a chimeric receptor as described herein, an extracellular spacer domain is generally located between the extracellular binding domain and the transmembranc domain of the chimeric Tim4 receptor. The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest et immunother.
28:203-11, 2005; PCT Publication No. WO 2014/031687). In certain embodiments, an extracellular spacer domain is an immunoglobulin hinge region (e.g., IgGl, IgG2, IgG3, IgG4, igA, 1g1)). An immunoglobulin hinge region may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region.
An altered IgG4 hinge region is described in PCT Publication No. WO
2014/031687, which hinge region is incorporated herein by reference in its entirety. In a particular embodiment, an extracellular spacer domain comprises a modified IgG4 hinge region having an amino acid sequence of ESK.YGPPCPPCP (SEQ ID NO:3). Other examples of hinge regions that may be used in the chimeric Tim4 receptors described herein include the hinge region from the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof In some embodiments, an extracellular spacer domain comprises a CD28 hinge region having an amino acid sequence of SEQ ID NO:32. In further embodiments, an extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from. a CH1 domain, a CH2 domain, a CH3 domain, or combinations thereof (see, e.g., PCT Publication W02014/031.687, which spacers are incorporated herein by reference in their entirety). In yet further embodiments, an extracellular spacer domain may comprise a stalk region of a type H C-lectin (the extracellular domain located between the C-type lectin domain and the transmembrane domain). Type II C-Iectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.
In certain embodiments, an (rX tracellular domain comprises polynucleoti de sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof. in certain embodiments, an extracellular domain is mudne, human, or chitneric.
The intracellular signaling domain of a chimeric Tim4 receptor as described herein is an intmccHular effector domain and is capable of transmitting functional signals to a cell in response to binding of the extracellular domain of the chimeric Tim4 receptor and phosphatidylserine. The signals transduced by the intracellular signaling domain promote effector function of the chimeric Tim4 receptor containing cell. Examples of effector function include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, expansion, engulfment of a target cell or particle expressing phosphatidylserine on its surface, or any combination thereof.
In certain embodiments, an intracellular signaling domain comprises a costimulatory signaling domain. The costimulatory signaling domain may be any portion of a costimulatory signaling molecule that retains sufficient signaling activity.
In some embodiments, a full length or full length intracellular component of a costimulatory signaling molecule is used. In some embodiments, a truncated portion of a costimulatory signaling molecule or intracellular component of a costimulatory signaling molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, a costimulatory signaling domain is a variant of a whole or truncated portion of a costimulatory signaling molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).
In certain embodiments, the costimulatory signaling domain comprises a CD27, CD28, CD4OL, GYM, NKG2C, CARD], CD2, CD7, CD27, CD30, C040, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAIVIF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (FIVEM), PD-1, CD273 (PD-L2), CD274 (PD-L I), B7-H3 (CD276), ICOS (CD278), DAP10, LAT, LFA-1 (CD I 1 a/CD 1 8 ), LIGHT, NKG2C, SLP76, TRIM, or ZAP70 signaling domain. In particular embodiments, the costimulatory signaling domain comprises an 0X40, CD2, CD27, CD28, LFA-1 (CD1 la/CD18), 1COS (CD278), or 4-1BB (CD137) signaling domain. An exemplary CD28 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:118 or 119. An exemplary 0X40 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID
NO:120.
An exemplary CD2 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:121. An exemplary 4-1BB costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:122. An exemplary CD27 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:123. An exemplary ICAM-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:124. An exemplary LF A-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:125. An exemplary ICOS costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:126. An exemplary CD30 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:127. An exemplary CD40 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:128. An exemplary PD-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:129. An exemplary CD7 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:130. An exemplary LIGHT costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:131. An exemplary NKG2C costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO: 132. An exemplary B7-H3 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:133. In certain embodiments, the costimulatory signaling domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOS:118-133. In certain embodiments, the costimulatory signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,
segments, the variable gene segment (V gene), the diversity gene segment (D
gene), and the joining gene segment (J gene). A single Va or V domain may be sufficient to confer antigen-binding specificity. "Major histocompatibility complex molecule"
(MI-IC molecule) refers to a glycoprotein that delivers a peptide antigen to a cell surface. MHC class I molecules are heterodimers composed of a membrane spanning a chain (with three a domains) and a non-covalentl y associated 132 microglobulin. MHC
class II molecules are composed of two transmembrane glycoproteins, a and 13, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where peptide:MHC
complex is recognized by CD8' T cells. WIC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4- T cells. An MHC molecule may be from various animal species, including human, mouse, rat, or other mammals.
"Chimeric antigen receptor" (CAR) refers to a chimeric protein comprising two or more distinct domains and can function as a receptor when expressed on the surface of a cell. CARs are generally composed of an extracellular domain comprising a binding domain that binds a target antigen, an optional extracellular spacer domain, a transmembrane domain, and an intracellular signaling domain (e.g., an immunoreceptor tyrosine-based activation motif (UAW-containing T cell activating motif, and optionally an intracellular costimulatory domain). In certain embodiments, an intracellular signaling domain of a CAR has an ITAM-containing T cell activating domain (e.g. CD3C) and an intracellular costimulatory domain (e.g., CD28). In certain embodiments, a CAR is synthesized as a single polypeptide chain or is encoded by a nucleic acid molecule as a single chain polypepfide.
A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain affinities, such as Western blot, ELISA, and BIACORE analysis (see also, e.g., Scatchard etal., Ann. N.Y. Acad. Sci. 51:660, 1949; and U.S.
Patent Nos.
5,283,173, 5,468,614, or the equivalent). As used herein, "specifically binds"
refers to an association or union of a binding domain, or a fusion protein thereof, to a target molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 105 Mt, while not significantly associating or uniting with any other molecules or components in a sample.
The terms "antigen" and "Ag" refer to a molecule that is capable of inducing an immune response. The immune response that is induced may involve antibody production, the activation of specific immunologically-competent cells, or both. Macromolecules, including proteins, glycoproteins, and glycolipids, can serve as an antigen. Antigens can be derived from recombinant or genomic DNA. As contemplated herein, an antigen need not be encoded (i) solely by a full length nucleotide sequence of a gene or (ii) by a "gene" at all. An antigen can be generated or synthesized, or an antigen can be derived from a biological sample. Such a biological sample can include, but is not limited, to a tissue sample, a tumor sample, a cell, or a biological fluid.
The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence or protein determinant within an antigen that is specifically bound by a cognate immune binding molecule, such as an antibody or fragment thereof (e.g., scFv), T cell receptor (TCR), chimeric Tim receptor, or other binding molecule, domain or protein. :Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics. An epitope may be a linear epitope or a conformational cpitopc.
As used herein, the term "PARP" (Poly (ADP-ribose) polymerase) refers to a family of proteins, each of which have two ribose moieties and two phosphates per unit polymer. PARP1 and PARP2 are enzymes involved in a DNA. repair pathway.
As used herein, the term "Tim4" (T-cell immunoglobulin and mucin domain containing protein 4), also known as "1imD4", refers to a phosphatidylserine receptor that is typically expressed on antigen presenting cells, such as macrophages and dendritic cells. 1'irn4 mediates the phagocytosis of apoptotic, necrotic, damaged, injured, or stressed cells, which present phosphatidylserine (PtdSer) on the exofacial (outer) leaflet of the cell membrane. Tim4 is also capable of binding to Timl expressed on the surface of T cells and inducing proliferation and survival. In certain embodiments, Tim4 refers to human Tim4. An exemplary human Tim4 protein comprises an amino acid sequence of SEQ ID NO: 1.
As used herein, the term "Tim4 binding domain" refers to the N-terminal immunoglobulin-fold domain of Tim4 that possesses a metal ion¨dependent pocket that selectively binds PtdSer. An exemplary human Tim4 binding domain comprises an amino acid sequence of SEQ ID NO:2, and an exemplary mouse Tim4 binding domain comprises an amino acid sequence of SEQ ID NO:24.
A Tim4 binding domain includes a variable immunoglobulin (1gV) like domain (referred to herein as an "IgV domain") and a Mucin like domain ("referred to herein as a "mucin domain"). An exemplary human Tim4 IgV domain comprises an amino acid sequence of SEQ ID NO:34, and an exemplary human Tim4 mucin domain comprises an amino acid sequence of SEQ ID NO:35. In certain embodiments, the Tim4 binding domain does not include a signal peptide. An exemplary human Tim4 signal peptide has the amino acid sequences of SEQ ID NO:11. An exemplary mouse Tim4 signal peptide has the amino acid sequences of SEQ ID NO:25.
As used herein, the term "Tim 1" (T-cell immunoglobulin and mucin domain containing protein 1), refers to a phosphatidylserine receptor that is expressed on the surface of T cells. Tim I, as noted above is also capable of binding to Tim4 expressed on the surface of antigen presenting cells. In certain embodiments, Timl refers to human Tim 1. An exemplary human Timl protein comprises an amino acid sequence of SEQ ID NO:36.
As used herein, the term "Timl binding domain" refers to the N-terminal immunoglobulin-fold domain of Timl that selectively binds PtdSer. An exemplary human Tim I binding domain comprises an amino acid sequence of SEQ ID NO:37.
A Timl binding domain includes an IgV domain and a mucin domain.
An exemplary human Timl IgV domain comprises an amino acid sequence of SEQ ID
NO:38, and an exemplary human Tim I rnucin domain comprises an amino acid sequence of SEQ ID NO:39. In certain embodiments, the Timl binding domain does not include a signal peptide. An exemplary human Timl signal peptide has the amino acid sequences of SEQ ID NO:40.
As used herein, an "effector domain" is an intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response in a cell expressing the effector domain when receiving the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response.
As used herein, a "costimulatory signaling domain" refers to an intracellular signaling domain, or functional portion thereof, of a costimulatory molecule, which, when activated in conjunction with a primary or classic (e.g., ITAM-driven) activation signal (provided by, for example, a CD3C, intracellular signaling domain), promotes or enhances a T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or combinations thereof Costimulatory signaling domains include, for example, CD27, CD28, CD4OL, GITR, NI(G2C, CARD!, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMI71), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, SLP76, TRIM, or any combination thereof.
As used herein, an "immunoreceptor tyrosine-based activation motif (ITAM) activating domain" refers to an intracellular signaling domain or functional portion thereof which is naturally or endogenously present on an immune cell receptor or a cell surface marker and contains at least one immunoreceptor tyrosine-based activation motif (ITAM). ITAM refers to a conserved motif of YXXL/I-X6.8-YXXL/I.
In certain embodiments an ITAM signaling domain contains one, two, three, four, or more ITAMs. An ITAM signaling domain may initiate T cell activation signaling following antigen binding or ligand engagement. ITAM-signaling domains include, for example, intracellular signaling domains of CD3y, CD38, CD3e, CD3C, CD79a, and CD66d.
"Junction amino acids" or "junction amino acid residues" refer to one or more (e.g., about 2-20) amino acid residues between two adjacent motifs, regions or domains of a polypeptide. Junction amino acids may result from the construct design of a chimeric protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a chimeric protein).
"Nucleic acid molecule" and "polynucleotide" can be in the form of RNA or DNA., which includes c:DN A, genomic DNA, and synthetic DNA. A nucleic acid molecule may be composed of naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. A nucleic acid molecule may be double stranded or single stranded, and if single stranded, may be the coding strand or non-coding (anti-sense strand). A
coding molecule may have a coding sequence identical to a coding sequence known in the art or may have a different coding sequence, which, as the result of the redundancy or degeneracy of the genetic code, or by splicing, can encode the same polypeptide.
"Encoding" refers to the inherent property of specific polynucleotide sequences, such as DNA, cDNA, and mRNA sequences, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a polynucleotide encodes a protein if transcription and translation of mitNA
corresponding to that polynucleotide produces the protein in a cell or other biological system. Both a coding strand and a non-coding strand can be referred to as encoding a protein or other product of the polynucleotide. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
"Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
As used herein, the term "mature polypeptide" or "mature protein" refers to a protein or polypeptide that is secreted or localized in the cell membrane or inside certain cell organelles (e.g., the endoplasmic reticulum, golgi, or endosome) and does not include an N-terminal signal peptide.
A "signal peptide", also referred to as "signal sequence", "leader sequence", "leader peptide", "localization signal" or "localization sequence", is a short peptide (usually 15-30 amino acids in length) present at the N-terminus of newly synthesized proteins that are destined for the secretory pathway. A signal peptide typically comprises a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a cleavage site for a signal peptidase. In eukaryotes, a signal peptide prompts translocation of the newly synthesized protein to the endoplasmic reticulum where it is cleaved by the signal peptidase, creating a mature protein that then proceeds to its appropriate destination.
The term "chimeric" refers to any nucleic acid molecule or protein that is not endogenous and comprises sequences joined or linked together that are not normally found joined or linked together in nature. For example, a chimeric nucleic acid molecule may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from the same source but arranged in a manner different than that found in nature.
As used herein, the term "endogenous" or "native" refers to a gene, protein, cornpound, molecule, or activity that is normally present in a host or host cell, including naturally occurring variants of the gene, protein, compound, molecule, or activity.
As used herein, "homologous" or "homolog" refers to a molecule or activity from a host cell that is related by ancestry to a second gene or activity, e.g., from the same host cell, from a different host cell, from a different organism, from a different strain, from a different species. For example, a heterologous molecule or heterologous gene. encoding the molecule may be homologous to a native host cell molecule or gene that encodes the molecule, respectively, and may optionally have an altered structure, sequence, expression level or any combination thereof.
As used herein, "heterologous" nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but can be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell. The source of the heterologous nucleic acid molecule, construct or sequence can be from a different genus or species. In some embodiments, the heterologous nucleic acid molecules are not naturally occurring. In certain embodiments, a heterologous nucleic acid molecule is added (i.e., not endogenous or native) into a host cell or host genorne by, for example, conjugation, transformation, transfection, transduction, electroporation, or the like, wherein the added molecule can integrate into the host cell genome or exist as extra-chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can be present in multiple copies. In addition, "heterologous" refers to a non-native enzyme, protein or other activity encoded by a non-endogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.
As used herein, the term "engineered," "recombinant," "modified" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule, or vector that has been modified by introduction of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been genetically engineered by human intervention that is, modified by introduction of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, where such alterations or modifications can be introduced by genetic engineering.
Human-generated genetic alterations can include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins, chimeric receptors, or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell's genetic material. Exemplary modifications include those in coding regions or functional fragments thereof heterologous or homologous polypeptides from a reference or parent molecule. Additional exemplary modifications include, for example, modifications in non-coding regulatory regions in which the modifications alter expression of a gene or operon.
As used herein, the term "transgene" refers to a gene or polynucleotide encoding a protein of interest (e.g., chimeric Tim receptor) whose expression is desired in a host cell and that has been transferred by genetic engineering techniques into a cell.
A transgene may encode proteins of therapeutic interest as well as proteins that are reporters, tags, markers, suicide proteins, etc. A transgene may be from a natural source, modification of a natural gene, or a recombinant or synthetic molecule. In certain embodiments, a transgene is a component of a vector.
The term "overexpressed" or "overexpression" of an antigen refers to an abnormally high level of antigen expression in a cell. Overexpressed antigen or overexpression of antigen is often associated with a disease state, such as in hematological malignancies and cells forming a solid tumor within a specific tissue or organ of a subject. Solid tumors or hematological malignancies characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.
The "percent identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. The comparison of sequences and determination of percent identity between two or more sequences can be accomplished using a mathematical algorithm, such as BLAST and Gapped BLAST programs at their default parameters (e.g.. Altschul etal., J. Mot Biol. 2/5:403, 1990; see also BLASTN
at www.nthi.nlm.nih.gov/BLAST).
A "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).
The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements that are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one that expresses the gene product in a tissue specific manner.
A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
The phrase "under transcriptional control" or "operatively linked" as used herein means that a promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, for example, plasmids, cosmids, viruses, or phage. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells. An "expression vector"
is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
In certain embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gamma retrovirus vectors, and lentivirus vectors.
"Retroviruses" are viruses having an RNA genome. "Gamma retrovirus" refers to a genus of the retroviridae family. Examples of gamma retroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses. "Lentivirus" refers to a genus of retrovimses that are capable of infecting dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (B1V), and simian immunodeficiency virus (Sly).
In other embodiments, the vector is a non-viral vector. Examples of non-viral vectors include lipid-based DNA vectors, modified mR_NA (modRNA), self-amplifying mRNA, closed-ended linear duplex (CELiD) DNA, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty). Where a non-viral delivery system is used, the delivery vehicle can be aliposome. Lipid formulations can be used to introduce nucleic acids into a host cell in vitro, ex vivo, or in vivo The nucleic acid may be encapsulated in the interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, contained or complexed with a micelle, or otherwise associated with a lipid.
As used herein, the term "engulfment" refers to a receptor-mediated process wherein endogenous or exogenous cells or particles greater than 100 nm in diameter are internalized by a phagocyte or host cell of the present disclosure Engulfment is typically composed of multiple steps: (1) tethering of the target cell or particle via binding of an engulfment receptor to a pro-engulfment marker or antigenic marker directly or indirectly (via a bridging molecule) on a target cell or particle; and (2) internalization or engulfment of the whole target cell or particle, or a portion thereof. In certain embodiments, internalization may occur via cytoskeletal rearrangement of a phagocyte or host cell to form a phagosome, a membrane-bound compartment containing the internalized target. Engulfment may further include maturation of the phagosome, wherein the phagosome becomes increasingly acidic and fuses with lysosomes (to form a phagolysosorne), whereupon the engulfed target is degraded (e.g., "phagocytosis"). Alternatively, phagosome-lysosome fusion may not be observed in engulfment. In yet another embodiment, a phagosome may regurgitate or discharge its contents to the extracellular environment before complete degradation. In some embodiments, engulfment refers to phagocytosis. In some embodiments, engulfment includes tethering of the target cell or particle by the phagocyte of host cell of the present disclosure, but not internalization. In some embodiments, engulfment includes tethering of the target cell or particle by the phagocyte of host cell of the present disclosure and internalization of part of the target cell or particle.
As used herein, the term "phagocytosis" refers to an engulfment process of cells or large particles (2_;.-- 0.5 gm) wherein tethering of a target cell or particle, engulfment of the target cell or particle, and degradation of the internalized target cell or particle occurs. In certain embodiments, phagocytosis comprises formation of a phagosome that encompasses the internalized target cell or particle and phagosome fusion with a I ysosome to form a phagolysosome, wherein the contents therein are degraded. In certain embodiments, during phagocytosis, following binding of a chimeric Tim receptor expressed on a host cell of the present disclosure to a phosphatidylserine expressed by a target cell or particle, a phagocytic synapse is formed; an actin-rich phagocytic cup is generated at the phagocytic synapse;
phagocytic arms are extended around the target cell or particle through cytoskeletal rearrangements; and ultimately, the target cell or particle is pulled into the phagocyte or host cell through force generated by motor proteins. As used herein, "phagocytosis"
includes the process of "efferocytosis", which specifically refers to the phagocytosis of apoptotic or necrotic cells in a non-inflammatory manner.
The term "immune system cell" or "immune cell" means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow.
Hematopoietic stem cells give rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a T6 T cell, a regulatory T cell, a natural killer cell, and a dendritic cell.
Macrophages and dendritic cells may also be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MEW) receptor on the surface of the .APC complexed with a peptide interacts with a TCR on the surface of a T cell.
The term "T cells" refers to cells of T cell lineage. "Cells of T cell lineage" refer to cells that show at least one phenotypic characteristic of a T cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for T cells (e.g. , CD3+, CD4, CD8-), or a physiological, morphological, functional, or immunological feature specific for a T cell. For example, cells of the T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25+ immature and inactivated T cells;
cells that have undergone CD4 or CD8 linage commitment; thyrnocyte progenitor cells that are CD4-CD8+ double positive; single positive CD4 + or CD8+; TCRap or TCR y8; or mature and functional or activated T cells. The term "T cells" encompasses naïve T
cells (CD45 RA+, CCR7+, CD62L+, CD27+, C045R0-), central memory T cells (CD45R0+, CD62L', CD8+), effector memory T cells (CD45RA+, CD45R0-, CCR7-, CD621,-, CD27-), mucosal-associated invariant T (MATT) cells, Tregs, natural killer T
cells, and tissue resident T cells.
The term "B cells" refers to cells of the B cell lineage. "Cells of B cell lineage" refer to cells that show at least one phenotypic characteristic of a B cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for B cells (e.g. , CD19+, CD72+, CD24+, CD20.4), or a physiological, morphological, functional, or immunological feature specific for a B cell. For example, cells of the B cell lineage may be progenitor or precursor cells committed to the B cell lineage (e.g., pre-pro-B cells, pro-B cells, and pre-B cells); immature and inactivated B cells or mature and functional or activated B
cells. Thus, "B cells" encompass naïve B cells, plasma cells, regulatory B
cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmahlast cells, and memory B cells (e.g., CD27+, The term "cytotoxic activity," also referred to as "cytolyfic activity,"
with respect to a cell (e.g., a T cell or NK cell) expressing an immune receptor (e.g., a TCR) or a chimeric Tim receptor according to the present disclosure on its surface, means that upon antigen-specific signaling (e.g., via the TCR, chimeric Tim receptor), the cell induces a target cell to undergo apoptosis. In some embodiments, a cytotoxic cell may induce apoptosis in a target cell via the release of cytotoxins, such as perforin, granzyme, and granulysin, from granules. Perforins insert into the target cell membrane and form pores that allow water and salts to rapidly enter the target cell.
Granzymes are serine proteases that induce apoptosis in the target cell. Granulysin is also capable of forming pores in the target cell membrane and is a proinflamtnatory molecule.
In some embodiments, a cytotoxic cell may induce apoptosis in a target cell via interaction of Fas ligand, which is upregulated on T cell following antigen-specific signaling, with Fas molecules expressed on the target cell. Fas is an apoptosis-signaling receptor molecule on the surface of a number of different cells.
The term "exhaustion" with respect to immune cells refers to a state of immune cell dysfunction defined by poor effector function (e.g., reduced cytokine production, reduced cytotoxic activity), reduced proliferative capacity, increased expression of immune checkpoint molecules, and a transcriptional state distinct from that of functional effector or memory cells. In certain embodiments, an exhausted immune cell becomes unresponsive to the presence of its target antigen. Immune cell exhaustion may result from chronic exposure to a target antigen (e.g., as may result from chronic infection) or when it enters an immunosuppressive environment (e.g., a tumor inicroenvironment). In certain embodiments, immune cell exhaustion refers to T
cell exhaustion, NK cell exhaustion, or both. In certain embodiments, exhausted T cells exhibit; (a) increased expression of PD-I, LAG3, TIM3, or any combination thereof; (b) decreased production of IFN-y, IL-2, TNT-a, or any combination thereof; or both (a) and (b). In certain embodiments, exhausted NK cells exhibit; (a) increased expression of PD-1, NKG2A, TIM3, or any combination thereof; (b) decreased production of IFN-y, TN. F-a, or both; or both (a) and (b).
A "disease" is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein, if the disease is not ameliorated, then the subject's health continues to deteriorate. In contrast, a "disorder" or "undesirable condition" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder or undesirable condition. Left untreated, a disorder or undesirable condition does not necessarily result in a further decrease in the subject's state of health.
The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. The aberrant cells may form solid tumors or constitute a hematological malignancy. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.
Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.
The term "subject," "patient" and "individual" are used interchangeably herein and are intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenie species thereof.
"Adoptive cellular immunotherapy" or "adoptive immunotherapy" refers to the administration of naturally occurring or genetically engineered disease antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient).
"Autologous" refers to any material (e.g., a graft of organ, tissue, cells) derived from the same subject to which it is later to be re-introduced.
"Allogeneic" refers to a graft derived from a different subject of the same species.
A "therapeutically effective amount" or "effective amount" of a chimeric protein or cell expressing a chimeric protein of this disclosure (e.g., a chimeric Tim receptor or a cell expressing a chimeric Tim receptor) refers to that amount of protein or cells sufficient to result in amelioration of one or more symptoms of the disease, disorder, or undesired condition being treated. When referring to an individual active ingredient or a cell expressing a single active ingredient, administered alone, a therapeutically effective dose refers to the effects of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective dose refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.
"Treat" or "treatment" or "ameliorate" refers to medical management of a disease, disorder, or undesired condition of a subject. in general, an appropriate dose or treatment regimen comprising a host cell expressing a chimeric protein of this disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease, disorder, or undesired condition; decreased occurrence of symptoms; improved quality of life;
longer disease-free status; diminishment of extent of disease, disorder, or undesired condition; stabilization of disease state; delay of disease progression;
remission;
survival; prolonged survival; or any combination thereof.
The term "anti-tumor effect" refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by prevention of a hematological malignancy or tumor formation.
"Autoimmune disease" refers to a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriately excessive response to a self-antigen. An autoimmune response may involve self-reactive B-cells that produce autoantibodies, self-reactive T-cells, or both.
An "autoantibody" as used herein is an antibody produced by a subject that binds to a self-antigen also produced by the subject.
Additional definitions are provided throughout the present disclosure.
As noted above, methods of the disclosure are useful for treating cancer in a subject. Therapeutic agents of the disclosure (i.e., a chimeric Tim receptor and a PARP inhibitor), when administered together or sequentially, treat a cancer in a subject (e.g., (i) arrest the cancer's development; (ii) cause regression of the cancer; or (iii) relieve the symptoms resulting from the cancer).
In embodiments, the methods of treatment disclosed herein comprise administering an effective amount of a chimeric Tim receptor and a PARP
inhibitor to the subject, thereby treating their cancer.
Chimeric Tim Receptors The present disclosure provides a chimeric Tim receptor comprising a single chain chimeric protein, the single chain chimeric protein comprising:
an cxtraccllular domain comprising a Tim binding domain; an intracellular signaling domain comprising a first costintulatory signaling domain; and a transmembrane domain positioned between and connecting the extracellular domain and intracellular signaling domain. In certain embodiments, the extracellular domain of the chimeric Tim receptors described herein optionally includes an extracellular spacer domain positioned between and connecting the binding domain and transmembrane domain.
When expressed in a host cell, chimeric Tim receptors of the present disclosure can confer a phosphatidylserine-specific, cytotoxic phenotype to the modified host cell (e.g., the host cell becomes cytotoxic to a stressed, damaged, injured, apoptotic, or necrotic cell expressing phosphatidylserine on its surface). In certain embodiments, the chimeric Tim receptors induce apoptosis in targeted cells via release of granzymes, perforin, granulysin, or any combination thereof. In further embodiments, cells expressing a chimeric Tim receptor according to the present description exhibit an engulfment phenotype specific to phosphatidylserine presenting cells.
The intracellular signaling domain can include one or more effector domains that are capable of transmitting functional signals to a cell in response to binding of the extracellular domain of the chimeric Tim receptor and phosphatidylserine. Signaling by the intracellular signaling domain(s) is triggered by binding of the extracellular domain to phosphatidylserine. The signals transduced by the intracellular signaling domain promote effector function of the chimeric Tim receptor containing cell. Examples of effector function include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, expansion, engulfment of a target cell or particle expressing phosphatidylserine on its surface, antigen presentation, or any combination thereof In certain embodiments, the intracellular signaling domain comprises a first intracellular signaling domain. In further embodiments, the intracellular signaling domain comprises a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a first intracellular signaling domain, a second intracellular signaling domain, and a third intracellular signaling domain. Chimeric Tim receptors according to the present disclosure can be used in a variety of therapeutic methods where clearance of apoptotic, necrotic, damaged, or stressed cells is beneficial, while providing costimulation that enhances cellular immune response, reduces immune cell exhaustion, or both.
Component parts of the fusion proteins of the present disclosure are further described in detail herein.
Extracellular Domain A Tim4 binding domain suitable for use in a chimeric Tim4 receptor of the present disclosure may be any polypeptide or peptide derived from a Tim4 molecule that specifically binds phosphatidylserine. In certain embodiments, the Tim4 binding domain is derived from human Tim4. An exemplary human Tim4 molecule is provided in Uniprot. Ref. Q96H15 (SEQ ID NO:1). An exemplary human Tim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:2 or amino acids 314 of SEQ ID NO:2. An exemplary mouse Tim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:24 or amino acids 23-279 of SEQ ID
NO:24. In certain embodiments, the Tim4 binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%õ 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID NO:24 or amino acids 23-279 of SEQ ID
NO:24. In certain embodiments, the Tim4 binding domain comprises an amino acid sequence haying at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of S:EQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID
NO:24 or amino acids 23-279 of SEQ ID NO:24.
In certain embodiments, the extracellular domain optionally comprises an extracellular, non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable proper cell/cell contact, binding, and activation. When included in a chimeric receptor as described herein, an extracellular spacer domain is generally located between the extracellular binding domain and the transmembranc domain of the chimeric Tim4 receptor. The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest et immunother.
28:203-11, 2005; PCT Publication No. WO 2014/031687). In certain embodiments, an extracellular spacer domain is an immunoglobulin hinge region (e.g., IgGl, IgG2, IgG3, IgG4, igA, 1g1)). An immunoglobulin hinge region may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region.
An altered IgG4 hinge region is described in PCT Publication No. WO
2014/031687, which hinge region is incorporated herein by reference in its entirety. In a particular embodiment, an extracellular spacer domain comprises a modified IgG4 hinge region having an amino acid sequence of ESK.YGPPCPPCP (SEQ ID NO:3). Other examples of hinge regions that may be used in the chimeric Tim4 receptors described herein include the hinge region from the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof In some embodiments, an extracellular spacer domain comprises a CD28 hinge region having an amino acid sequence of SEQ ID NO:32. In further embodiments, an extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from. a CH1 domain, a CH2 domain, a CH3 domain, or combinations thereof (see, e.g., PCT Publication W02014/031.687, which spacers are incorporated herein by reference in their entirety). In yet further embodiments, an extracellular spacer domain may comprise a stalk region of a type H C-lectin (the extracellular domain located between the C-type lectin domain and the transmembrane domain). Type II C-Iectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.
In certain embodiments, an (rX tracellular domain comprises polynucleoti de sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof. in certain embodiments, an extracellular domain is mudne, human, or chitneric.
The intracellular signaling domain of a chimeric Tim4 receptor as described herein is an intmccHular effector domain and is capable of transmitting functional signals to a cell in response to binding of the extracellular domain of the chimeric Tim4 receptor and phosphatidylserine. The signals transduced by the intracellular signaling domain promote effector function of the chimeric Tim4 receptor containing cell. Examples of effector function include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, expansion, engulfment of a target cell or particle expressing phosphatidylserine on its surface, or any combination thereof.
In certain embodiments, an intracellular signaling domain comprises a costimulatory signaling domain. The costimulatory signaling domain may be any portion of a costimulatory signaling molecule that retains sufficient signaling activity.
In some embodiments, a full length or full length intracellular component of a costimulatory signaling molecule is used. In some embodiments, a truncated portion of a costimulatory signaling molecule or intracellular component of a costimulatory signaling molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, a costimulatory signaling domain is a variant of a whole or truncated portion of a costimulatory signaling molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).
In certain embodiments, the costimulatory signaling domain comprises a CD27, CD28, CD4OL, GYM, NKG2C, CARD], CD2, CD7, CD27, CD30, C040, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAIVIF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (FIVEM), PD-1, CD273 (PD-L2), CD274 (PD-L I), B7-H3 (CD276), ICOS (CD278), DAP10, LAT, LFA-1 (CD I 1 a/CD 1 8 ), LIGHT, NKG2C, SLP76, TRIM, or ZAP70 signaling domain. In particular embodiments, the costimulatory signaling domain comprises an 0X40, CD2, CD27, CD28, LFA-1 (CD1 la/CD18), 1COS (CD278), or 4-1BB (CD137) signaling domain. An exemplary CD28 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:118 or 119. An exemplary 0X40 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID
NO:120.
An exemplary CD2 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:121. An exemplary 4-1BB costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:122. An exemplary CD27 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:123. An exemplary ICAM-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:124. An exemplary LF A-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:125. An exemplary ICOS costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:126. An exemplary CD30 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:127. An exemplary CD40 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:128. An exemplary PD-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:129. An exemplary CD7 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:130. An exemplary LIGHT costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:131. An exemplary NKG2C costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO: 132. An exemplary B7-H3 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:133. In certain embodiments, the costimulatory signaling domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOS:118-133. In certain embodiments, the costimulatory signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of any one of SEQ ID
NOS:118-133. in certain embodiments, the intracellular signaling comprises a second costimulatory signaling domain. In preferred embodiments, the first costimulatory signaling domain and second costimulatory signaling domain are different.
In certain embodiments, the intracellular signaling domain further comprises an ITAM-containing activating domain. The ITAM-containing activating domain may recapitulate TCR signaling independently of endogenous TCR
complexes.
In certain embodiments, signaling via the ITAM-containing activating domain leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. The ITAM-containing activating domain may be any portion of an ITAM-containing activating domain molecule that retains sufficient signaling activity. in some embodiments, a full length or full length intracellular component of an ITAM-containing activating domain molecule is used. In some embodiments, a truncated portion of an ITAM-containing activating domain molecule or intracellular component of an ITAM-containing activating domain molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, an. ITAM-containing activating domain is a variant of a whole or truncated portion of an ITAM-containing activating domain molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional valiant).
Examples of ITAM-containing activating domains that may be used in the chimeric Tim4 receptors of the present disclosure include those derived from CD3c CD3y, CD35, CD3e, CD5, CD22, CD79a, CD278 (ICOS), and CD66d. In specific embodiments, the ITAM-containing activating domain is a CD3 signaling domain.
An exemplary CD3C, signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:134 or 135. An exemplary CD3y signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:136. An exemplary CD35 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:137. An exemplary CD3e signaling domain comprises or consists of an amino acid sequence of SEQ
ID
NO: .138. An exemplary CD5 signaling domain comprises or consists of an amino acid sequence of SEQ ID .NO:139. An exemplary CD22 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:140. An exemplary CD79a signaling domain comprises or consists of an amino acid sequence of SEQ ID
NO:141.
An exemplary CD66d signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:143. In certain embodiments, the ITAM-containing activating domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%
identity to any one of SEQ ID NOS:134-141, and 143. In certain embodiments, the ITAM containing activating domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) of an amino acid sequence to any one of SEQ ID NOS:134-141, and143.
In another embodiment, an intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3C signaling domain. In another embodiment, an intracellular signaling domain comprises a 4-1BB costimulatory signaling domain and a CD3C signaling domain. In yet another embodiment, an intracellular signaling domain comprises a CD27 costimulatory signaling domain and a CD3C signaling domain. In another embodiment, an intracellular signaling domain comprises a ICOS costimulatory signaling domain and a CD3C signaling domain.
In another embodiment, an intracellular signaling domain comprises a LFA-1 costimulatory signaling domain and a CD3C signaling domain. In another embodiment, an intracellular signaling domain comprises an 0X40 costimulatory signaling domain and a CD3C signaling domain. In yet another embodiment, an intracellular signaling domain comprises a CD2 costimulatory signaling domain and a CD3C signaling domain. In still another embodiment, an intracellular signaling domain comprises an ICAM-1 costimulatory signaling domain and a CD3C signaling domain.
Intracellular signaling domains may be derived from a mammalian species, including humans, primates, cows, horses, goats., sheep, dogs, cats., mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
The transmembrane domain of a chimeric Tim4 receptor connects and is positioned between the extracellular domain and the intracellular signaling domain.
The transmembrane domain is a hydrophobic alpha helix that transverses the host cell membrane. The transmembrane domain may be directly fused to the binding domain or to the extracellular spacer domain if present. In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g., receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, or the like). In onc embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, a chimeric Tim4 receptor may comprise a Tim4 binding domain and a Tim4 transmembrane domain. In another example, a chimeric Tim4 receptor may comprise a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules; the transmembrane domain and the intracellular signaling domain are derived from different molecules; or the transmembrane domain, extracellular domain, and intracellular signaling domain are all derived from different molecules. Examples of transmembrane domains that may be used in chimeric Tim4 receptors of the present disclosure include transmembrane domains from Tim4, Tim1, CD3c, CD37, CD35, CD3e, CD28, CD45, CD4, CD5, CD8, CD9, C D16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3. An exemplary Tim4 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:144 or 23. An exemplary Timl transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:8. An exemplary CD28 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:145. An exemplary 4-1BB transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:146. An exemplary 0X40 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:147. An exemplary CD27 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:148. An exemplary ICOS transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:149. An exemplary CD2 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:150. An exemplary LFA-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:151. An exemplary CD30 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:152. An exemplary CD40 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:153. An exemplary PD-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:154. An exemplary CD7 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:155. An exemplary LIGHT transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:156. An exemplary N'KG2C transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:157. An exemplary B7-H3 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:158. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOS:8, 23 and 144-158. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletion, additions, substitutions) to an amino acid sequence of any one of SEQ ID
NOS:8, 23 and 144-158.
Transmembrane domains may derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
Exemplary components, configurations and chimeric Tim4 receptor sequences of the present disclosure are provided in Table 1.
Table!.
Name Amino Acid Sequence SEQ ID
NO:
_ Tim4 binding MSKEPLILWLMIEFWWLYLTPVTSETVVTEVLGHRVT 2 domain LPCLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGM
RVISRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCR
IEVPGWFNDVKINVRLNLQRASTTTHRTATTTTRRTTT
TSPTTTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAV
FTTANTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESET
VLPSDSWSSVESTSADTVLLTSKESKVWDLPSTSIIVSM
WKTSDSVSSPQPGA.SDTAVPEQNICITKTGQMDGIPMS
MKNEMPISQ
Modified IgG4 ESKYGPPCPPCP
hinge Wildtype RSKRSIkLMSDYMNMTP1212.PGPTRKIIYQPYAPPREW A 118 costimulatory signaling domain costimulatory , AYRS
signaling domain with substitutions (positions reference to full length protein) costimulatory LAK1 signaling domain costimulatory QNPATSQHPPPPPGHRSQAPSHRPPPPGHR.VQHQPQKR
signaling PPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSS
domain 4-1.BB KRGRKKLINIFKQ.PFMRPVQTTQEE.DGCSCRFPEEEEG
costimulatory GCEL
signaling domain CD27 QRRKYR.SNKGESPVEPAEPCITYSCPREEEGSTIPIQEDY 123 costimulatory RKPEPACSP
signaling domain costimulatory signaling domain costimulatory MNPKFAES
signaling domain ICOS CWLTKKK.YSSSVHDPNGEYMFMRAVNTAKK SRLTDV 126 costimulatory TL
signaling domain CD30 HRRACRKR.R.Q.KLiii.,CYPVQTSQPKILELVDSRPRRSST 127 costimulatory QLRSGASVIEPVAEE.RGLM.SQ.PLMETCHSVGAAYLES
signaling LPLQDASPAGGPSSPRDLPEPRVSTEHTNN. KIEKIYLMK
domain ADTVIV GT VIC AELPECiRGIAGP AEPELEEELEADHTPH
YPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK
costimulatory TLHGCQPVTQEDGKESRISVQERQ
signaling domain costimulatory WREKTPEPPVPC'VPEQTEYATIVFPSGMGTSSPARR.GS
signaling ADGPRSAQPLRPEDGHCSWPL
domain CD7 RTQIKKLCSWRDKNSAA.0 VVYEDMSHSRCNTLSSPNQ 130 costimulatory YQ
signaling domain LIGHT MEESVVRP SVFVVDGQTDIP FTRLGRSHRRQ SC S VAR
costimulatory signaling domain costimulatory FQVELNLQNPSLNHQGIDKIYDCQGLLPPPEK
signaling domain costimulatory SKEDDGQEIA.
signaling domain wi dtype CD3 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD 134 activating K RRGRDPEMGCTK PQRRKNPQEGINNELQK DK M A EAY
domain SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALEIMQ
ALP:PR
mutated human RVKISRSADAPAYQQGQNQLYNELNLGRREEYDVLD 135 CD3c, ITAM- KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
containing EIGMK GERRRGKCiHDGLYQGL ST A TKDINDALHMQA
activating LPPR
domain containing QGNQLRRN
activating domain CD38 ITAM- Cil-IETGRLSGA ADTQALLANDQVYQPI,RDRDDAQ VS1-I 137 containing LGGNWARNK
activating domain C'D3 e ITAM- KNRK AK AKPVTRGAGAGGRQRGQ NKERPPPVP NPDY 138 containing EPIRKGQRDINSGLNQRRI
activating domain C'D5 ITAM- j KKLVK KFRQKK QRQW1GP17GMNQNM STHRNIf FATVR 139 containing SHAENPTA.SEIVDNEYSQPPRNSHLSAYPALEGALHR.SS
activating MQPDNSSDSDYDLHGAQRL
domain CD22 ITAM- 1 1<1,QRRWKR TQSQQGI,QFNSS'GQSFINRNKKVRR API ,S 140 containing EGPHSLGCYNPMMEDGISYTTLRFPEMN1PRTGDAESS
activating EMQRPPPDCDDTVTYSALHKRQ V GDYEN V1PDFP EDE
domain GIHYSELIQFGVGERPQAQEN'VDYVILKII
CD 79a ITAM- RKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYED 141 con tai fling :IS RGLQGTYQD V GSLNIGD VQLEKP
activating domain con tai ning activating domain CD66d ITAM- AKTGRTSIQRDLKEQQPQALAPGRGPSHSSAFSMSPLS 143 containing TAQAPLPNPRIAA SIYEELLKHDTNIYCRMDHKAEVAS
activating domain Tim4 LLM. HAPSLGFVLF ALFVAFL
transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain ICOS FWLPIGCAAFVVVCILGCILI
transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain PD-1 VGVVCrGLE.GSINLINVVVIAVI
transmembrane domain transmembrane domain LIGHT VGLGLLLLLMGAGLAVQGWFL
transmembrane domain NKG2C LTAEVLGIICIVLMATVLK.TIVL
transmembrane domain transmembrane domain Tim4 binding MSKGLLLLWLVTELWWLYLTPAASEDTIIGFLGQPVTL 159 domain-CD28 PCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTR
transmembrane IISRK.STKYTLLGKVQFGEVSLTISNTNRGDSGVYCCRI
costimulatory VTITTPELLPTTymyrS'VLPTTTPPQMATTAFSTAv-rT
signaling CPSTTPGSFSQETTKGSAFTTESETLPASMISQRSMMTI
domain, amino STDIAVLRPTGSNPGILPSTSQLTTQKTTLTTSESLQKTT
acids 1-22 are KSHQINSRQTFWVLVVVGGVLACYSLLVTVAFILFWVR
signal peptide SICRSRGGIISDYIvLNMTPRRPGPTRKHYQPYAPPRDFAA
YRS
Ti m 4 binding M SK CILLLLW L V TEL W W LYLTPAASEDTIIGFLGQPVTL 160 domain-Tim4 PCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTR
transmembrane IISRKSTKYTLLGKVQFGEVSLTISNTN. RGDSGVYCCRI
-CD28 E'VPGVvr.FNDVICKNVRI,ELRRAT`FIKK.PrrtTRPTTTPY
costimulatory VTTTTPELLPTTVMTTSVLPTTTPPQTLATTAFSTAVTT
signaling CPSTTPGSFSQETTKGSAF'TTESETLPASNHSQRSM.MT1 domain. amino STDIAVLRPTGSNPGILPSTSQLTTQKTTLTTSESLQKTT
acids 1-.22 are KSHQINSRQT1LIIACCVGFVLMVLLFLAFLRSKRSRGG
signal peptide HSDYMNIVITPRRPGPTRICHYQPYAPPRDFAAYR.S
Ti m4 binding MSKGLILLWINTELWWINUTPA A SEDTTIGFLGQPVTL 161 domain-Tim4 PCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTR
transmembrane IISRKSTKYTLLGKVQFGEVSLTISNINRGDSGVYCCRI
costimulatory V'TTTTPELLPTTVMTTSVLPTTTPPQTLATTAFSTAVTT
signaling CPSTTPGSFSQETTKGSAFTTESETI,PASNIISQRSMMTI
domain-CD3 t STDIAVLRPTGSNTPGILPSTSQLTTQKTTLTTSESLQKTT
TTAM KSHQINSRATTILIIACCVGFVLMVLLFLAFIKRGIKKKLL
containing YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR.VKF
activating SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG
domain, amino RD.PEMGGKPRRKNPQEGLYNELQKDKMAEAYSE1GM
acids 1-22 are KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
signal peptide MSKEPLILWLMIEFWWLYLTPVTS
Construct 13A
ETV'VTEVLGHRVTLPCLYSSWSHNSNSMCWGKDQCP
TIM4 binding YSCICKEALIRTDGIVERVTSRKSAKYR.I,QGTIPRGDVSLT
domain-CD28 1LNPSESDSGVYCCRIEVPGWFNDVKINVRLNLQRAST
TM¨ CD28 õ TTH R RTATTTTRTT T
TFSPTIRQM'TTTPAALPTTVV'I"TP
costim- CD.5(, DLTTGTPLQMTTIAVFTTANTCLSLTPSTLPEEATGLLT
(amino acids 1-PEPSICEGRILTAESETVLPSDSWSSVESTSADTVLLTSKE
24 signal SKVWDLPSTSTIVSMW.KTSDSVSSPQPGA.SDTAVPEQN
peptide) KTTKTGQIVIDGIPMSMKNEMPISQ
FW VLVVVGGVLA.0 Y S LLVTVAFIIFW V
RSKRSRLLHS'DYMWMTPRRPGPTRKHYOPYAPPRDFAAYR
SSRVIUSRSADAF'AYQQGQNQLYNELNLGRREEYDVL
DKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEA
YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM
signal peptide Tim4 signal MSKEPLILWLMIEFWWLYtTPVTS
NOS:118-133. in certain embodiments, the intracellular signaling comprises a second costimulatory signaling domain. In preferred embodiments, the first costimulatory signaling domain and second costimulatory signaling domain are different.
In certain embodiments, the intracellular signaling domain further comprises an ITAM-containing activating domain. The ITAM-containing activating domain may recapitulate TCR signaling independently of endogenous TCR
complexes.
In certain embodiments, signaling via the ITAM-containing activating domain leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. The ITAM-containing activating domain may be any portion of an ITAM-containing activating domain molecule that retains sufficient signaling activity. in some embodiments, a full length or full length intracellular component of an ITAM-containing activating domain molecule is used. In some embodiments, a truncated portion of an ITAM-containing activating domain molecule or intracellular component of an ITAM-containing activating domain molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, an. ITAM-containing activating domain is a variant of a whole or truncated portion of an ITAM-containing activating domain molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional valiant).
Examples of ITAM-containing activating domains that may be used in the chimeric Tim4 receptors of the present disclosure include those derived from CD3c CD3y, CD35, CD3e, CD5, CD22, CD79a, CD278 (ICOS), and CD66d. In specific embodiments, the ITAM-containing activating domain is a CD3 signaling domain.
An exemplary CD3C, signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:134 or 135. An exemplary CD3y signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:136. An exemplary CD35 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:137. An exemplary CD3e signaling domain comprises or consists of an amino acid sequence of SEQ
ID
NO: .138. An exemplary CD5 signaling domain comprises or consists of an amino acid sequence of SEQ ID .NO:139. An exemplary CD22 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:140. An exemplary CD79a signaling domain comprises or consists of an amino acid sequence of SEQ ID
NO:141.
An exemplary CD66d signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:143. In certain embodiments, the ITAM-containing activating domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%
identity to any one of SEQ ID NOS:134-141, and 143. In certain embodiments, the ITAM containing activating domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) of an amino acid sequence to any one of SEQ ID NOS:134-141, and143.
In another embodiment, an intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3C signaling domain. In another embodiment, an intracellular signaling domain comprises a 4-1BB costimulatory signaling domain and a CD3C signaling domain. In yet another embodiment, an intracellular signaling domain comprises a CD27 costimulatory signaling domain and a CD3C signaling domain. In another embodiment, an intracellular signaling domain comprises a ICOS costimulatory signaling domain and a CD3C signaling domain.
In another embodiment, an intracellular signaling domain comprises a LFA-1 costimulatory signaling domain and a CD3C signaling domain. In another embodiment, an intracellular signaling domain comprises an 0X40 costimulatory signaling domain and a CD3C signaling domain. In yet another embodiment, an intracellular signaling domain comprises a CD2 costimulatory signaling domain and a CD3C signaling domain. In still another embodiment, an intracellular signaling domain comprises an ICAM-1 costimulatory signaling domain and a CD3C signaling domain.
Intracellular signaling domains may be derived from a mammalian species, including humans, primates, cows, horses, goats., sheep, dogs, cats., mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
The transmembrane domain of a chimeric Tim4 receptor connects and is positioned between the extracellular domain and the intracellular signaling domain.
The transmembrane domain is a hydrophobic alpha helix that transverses the host cell membrane. The transmembrane domain may be directly fused to the binding domain or to the extracellular spacer domain if present. In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g., receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, or the like). In onc embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, a chimeric Tim4 receptor may comprise a Tim4 binding domain and a Tim4 transmembrane domain. In another example, a chimeric Tim4 receptor may comprise a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules; the transmembrane domain and the intracellular signaling domain are derived from different molecules; or the transmembrane domain, extracellular domain, and intracellular signaling domain are all derived from different molecules. Examples of transmembrane domains that may be used in chimeric Tim4 receptors of the present disclosure include transmembrane domains from Tim4, Tim1, CD3c, CD37, CD35, CD3e, CD28, CD45, CD4, CD5, CD8, CD9, C D16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3. An exemplary Tim4 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:144 or 23. An exemplary Timl transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:8. An exemplary CD28 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:145. An exemplary 4-1BB transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:146. An exemplary 0X40 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:147. An exemplary CD27 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:148. An exemplary ICOS transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:149. An exemplary CD2 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:150. An exemplary LFA-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:151. An exemplary CD30 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:152. An exemplary CD40 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:153. An exemplary PD-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:154. An exemplary CD7 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:155. An exemplary LIGHT transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:156. An exemplary N'KG2C transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:157. An exemplary B7-H3 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:158. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOS:8, 23 and 144-158. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletion, additions, substitutions) to an amino acid sequence of any one of SEQ ID
NOS:8, 23 and 144-158.
Transmembrane domains may derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
Exemplary components, configurations and chimeric Tim4 receptor sequences of the present disclosure are provided in Table 1.
Table!.
Name Amino Acid Sequence SEQ ID
NO:
_ Tim4 binding MSKEPLILWLMIEFWWLYLTPVTSETVVTEVLGHRVT 2 domain LPCLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGM
RVISRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCR
IEVPGWFNDVKINVRLNLQRASTTTHRTATTTTRRTTT
TSPTTTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAV
FTTANTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESET
VLPSDSWSSVESTSADTVLLTSKESKVWDLPSTSIIVSM
WKTSDSVSSPQPGA.SDTAVPEQNICITKTGQMDGIPMS
MKNEMPISQ
Modified IgG4 ESKYGPPCPPCP
hinge Wildtype RSKRSIkLMSDYMNMTP1212.PGPTRKIIYQPYAPPREW A 118 costimulatory signaling domain costimulatory , AYRS
signaling domain with substitutions (positions reference to full length protein) costimulatory LAK1 signaling domain costimulatory QNPATSQHPPPPPGHRSQAPSHRPPPPGHR.VQHQPQKR
signaling PPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSS
domain 4-1.BB KRGRKKLINIFKQ.PFMRPVQTTQEE.DGCSCRFPEEEEG
costimulatory GCEL
signaling domain CD27 QRRKYR.SNKGESPVEPAEPCITYSCPREEEGSTIPIQEDY 123 costimulatory RKPEPACSP
signaling domain costimulatory signaling domain costimulatory MNPKFAES
signaling domain ICOS CWLTKKK.YSSSVHDPNGEYMFMRAVNTAKK SRLTDV 126 costimulatory TL
signaling domain CD30 HRRACRKR.R.Q.KLiii.,CYPVQTSQPKILELVDSRPRRSST 127 costimulatory QLRSGASVIEPVAEE.RGLM.SQ.PLMETCHSVGAAYLES
signaling LPLQDASPAGGPSSPRDLPEPRVSTEHTNN. KIEKIYLMK
domain ADTVIV GT VIC AELPECiRGIAGP AEPELEEELEADHTPH
YPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK
costimulatory TLHGCQPVTQEDGKESRISVQERQ
signaling domain costimulatory WREKTPEPPVPC'VPEQTEYATIVFPSGMGTSSPARR.GS
signaling ADGPRSAQPLRPEDGHCSWPL
domain CD7 RTQIKKLCSWRDKNSAA.0 VVYEDMSHSRCNTLSSPNQ 130 costimulatory YQ
signaling domain LIGHT MEESVVRP SVFVVDGQTDIP FTRLGRSHRRQ SC S VAR
costimulatory signaling domain costimulatory FQVELNLQNPSLNHQGIDKIYDCQGLLPPPEK
signaling domain costimulatory SKEDDGQEIA.
signaling domain wi dtype CD3 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD 134 activating K RRGRDPEMGCTK PQRRKNPQEGINNELQK DK M A EAY
domain SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALEIMQ
ALP:PR
mutated human RVKISRSADAPAYQQGQNQLYNELNLGRREEYDVLD 135 CD3c, ITAM- KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
containing EIGMK GERRRGKCiHDGLYQGL ST A TKDINDALHMQA
activating LPPR
domain containing QGNQLRRN
activating domain CD38 ITAM- Cil-IETGRLSGA ADTQALLANDQVYQPI,RDRDDAQ VS1-I 137 containing LGGNWARNK
activating domain C'D3 e ITAM- KNRK AK AKPVTRGAGAGGRQRGQ NKERPPPVP NPDY 138 containing EPIRKGQRDINSGLNQRRI
activating domain C'D5 ITAM- j KKLVK KFRQKK QRQW1GP17GMNQNM STHRNIf FATVR 139 containing SHAENPTA.SEIVDNEYSQPPRNSHLSAYPALEGALHR.SS
activating MQPDNSSDSDYDLHGAQRL
domain CD22 ITAM- 1 1<1,QRRWKR TQSQQGI,QFNSS'GQSFINRNKKVRR API ,S 140 containing EGPHSLGCYNPMMEDGISYTTLRFPEMN1PRTGDAESS
activating EMQRPPPDCDDTVTYSALHKRQ V GDYEN V1PDFP EDE
domain GIHYSELIQFGVGERPQAQEN'VDYVILKII
CD 79a ITAM- RKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYED 141 con tai fling :IS RGLQGTYQD V GSLNIGD VQLEKP
activating domain con tai ning activating domain CD66d ITAM- AKTGRTSIQRDLKEQQPQALAPGRGPSHSSAFSMSPLS 143 containing TAQAPLPNPRIAA SIYEELLKHDTNIYCRMDHKAEVAS
activating domain Tim4 LLM. HAPSLGFVLF ALFVAFL
transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain ICOS FWLPIGCAAFVVVCILGCILI
transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain PD-1 VGVVCrGLE.GSINLINVVVIAVI
transmembrane domain transmembrane domain LIGHT VGLGLLLLLMGAGLAVQGWFL
transmembrane domain NKG2C LTAEVLGIICIVLMATVLK.TIVL
transmembrane domain transmembrane domain Tim4 binding MSKGLLLLWLVTELWWLYLTPAASEDTIIGFLGQPVTL 159 domain-CD28 PCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTR
transmembrane IISRK.STKYTLLGKVQFGEVSLTISNTNRGDSGVYCCRI
costimulatory VTITTPELLPTTymyrS'VLPTTTPPQMATTAFSTAv-rT
signaling CPSTTPGSFSQETTKGSAFTTESETLPASMISQRSMMTI
domain, amino STDIAVLRPTGSNPGILPSTSQLTTQKTTLTTSESLQKTT
acids 1-22 are KSHQINSRQTFWVLVVVGGVLACYSLLVTVAFILFWVR
signal peptide SICRSRGGIISDYIvLNMTPRRPGPTRKHYQPYAPPRDFAA
YRS
Ti m 4 binding M SK CILLLLW L V TEL W W LYLTPAASEDTIIGFLGQPVTL 160 domain-Tim4 PCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTR
transmembrane IISRKSTKYTLLGKVQFGEVSLTISNTN. RGDSGVYCCRI
-CD28 E'VPGVvr.FNDVICKNVRI,ELRRAT`FIKK.PrrtTRPTTTPY
costimulatory VTTTTPELLPTTVMTTSVLPTTTPPQTLATTAFSTAVTT
signaling CPSTTPGSFSQETTKGSAF'TTESETLPASNHSQRSM.MT1 domain. amino STDIAVLRPTGSNPGILPSTSQLTTQKTTLTTSESLQKTT
acids 1-.22 are KSHQINSRQT1LIIACCVGFVLMVLLFLAFLRSKRSRGG
signal peptide HSDYMNIVITPRRPGPTRICHYQPYAPPRDFAAYR.S
Ti m4 binding MSKGLILLWINTELWWINUTPA A SEDTTIGFLGQPVTL 161 domain-Tim4 PCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTR
transmembrane IISRKSTKYTLLGKVQFGEVSLTISNINRGDSGVYCCRI
costimulatory V'TTTTPELLPTTVMTTSVLPTTTPPQTLATTAFSTAVTT
signaling CPSTTPGSFSQETTKGSAFTTESETI,PASNIISQRSMMTI
domain-CD3 t STDIAVLRPTGSNTPGILPSTSQLTTQKTTLTTSESLQKTT
TTAM KSHQINSRATTILIIACCVGFVLMVLLFLAFIKRGIKKKLL
containing YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR.VKF
activating SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG
domain, amino RD.PEMGGKPRRKNPQEGLYNELQKDKMAEAYSE1GM
acids 1-22 are KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
signal peptide MSKEPLILWLMIEFWWLYLTPVTS
Construct 13A
ETV'VTEVLGHRVTLPCLYSSWSHNSNSMCWGKDQCP
TIM4 binding YSCICKEALIRTDGIVERVTSRKSAKYR.I,QGTIPRGDVSLT
domain-CD28 1LNPSESDSGVYCCRIEVPGWFNDVKINVRLNLQRAST
TM¨ CD28 õ TTH R RTATTTTRTT T
TFSPTIRQM'TTTPAALPTTVV'I"TP
costim- CD.5(, DLTTGTPLQMTTIAVFTTANTCLSLTPSTLPEEATGLLT
(amino acids 1-PEPSICEGRILTAESETVLPSDSWSSVESTSADTVLLTSKE
24 signal SKVWDLPSTSTIVSMW.KTSDSVSSPQPGA.SDTAVPEQN
peptide) KTTKTGQIVIDGIPMSMKNEMPISQ
FW VLVVVGGVLA.0 Y S LLVTVAFIIFW V
RSKRSRLLHS'DYMWMTPRRPGPTRKHYOPYAPPRDFAAYR
SSRVIUSRSADAF'AYQQGQNQLYNELNLGRREEYDVL
DKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEA
YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM
signal peptide Tim4 signal MSKEPLILWLMIEFWWLYtTPVTS
11 peptide Mouse Tim4 ILIIACCVGFVLMVLLFLAFL
transmembrane domain Mouse Tim4 M SK GLLLLWLVTELW Vv' LYLTPAASEDTI1GFLGQPVTL 24 binding PCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTR
domain, amino IISRKSTKYILLGKVQFGEVSLTISNTN. RGDSGVYCCRI
acids 1-22 are EVPGVvr.FNDVIKKNV.RLEI.,RRAT`FIKK.PrrtTRPT'FTPY
signal peptide VTTTTPELLPTTVMTTSVLPTTTPPQTLATTAFSTAVTT
CPSTTPGSFSQETTKGSAF'TTESETLPASNHSQRSMMTI
STDIAVLRPTGSNPGILPSTSQLTTQKTTLTTSESLQKTT
KSHQINSRQT
Mouse Tim4 MSICGLLLLWLVTELWWLYLTPA
signal peptide Further embodiments of the chimeric Tim4 receptors described herein comprise a single chain chimeric protein, the single chain chimeric protein comprising:
an extracellular domain comprising a Tim4 binding domain; an intracellular signaling domain comprising a first costimulatory signaling domain and an HAM-containing activating domain, wherein the ITAIVI-containing activating domain comprises a DAP12 signaling domain; and a transmembrane domain positioned between and connecting the extracellular domain and the intracellular signaling domain.
A Tim4 binding domain suitable for use in a chimeric Tim4 receptor of the present disclosure may be any polypeptide or peptide derived from a Ti m4 molecule that specifically binds phosphatidylserine. In certain embodiments, the Tim4 binding domain is derived from human Tim4. An exemplary human Tim4 molecule is provided in liniprot. Ref. Q96H15 (SEQ ID .N0:1). An exemplary human Tim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:2 or amino acids 314 of SEQ ID NO:2. An exemplary mouse *Iim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:24 or amino acids 23-279 of SEQ ID
NO:24. In certain embodiments, the Tim4 binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID NO:24 or amino acids 23-279 of SEQ ID
NO:24. In certain embodiments, the Tim4 binding domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID
NO:24 or amino acids 23-279 of SEQ ID NO:24.
In certain embodiments, the ex tracellular domain optionally comprises an extracellular, non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable proper cell/cell contact, binding, and activation. When included in a chimeric receptor as described herein, an extracellular spacer domain is generally located between the extracellular binding domain and the transniembrane domain of the chimeric Tim4 receptor. The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest etal., J.
Immunother.
28:203-11, 2005; PCT Publication No. WO 2014/031687). In certain embodiments, an extracellular spacer domain is an immunoglobulin hinge region (e.g., IgGl, IgG2, IgG3, IgG4, IgA, IgD). An immunoglobulin hinge region may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region. An altered IgG4 hinge region is described in PCT Publication No. WO 2014/031687, which hinge region is incorporated herein by reference in its entirety. In a particular embodiment, an extracellular spacer domain comprises a modified IgG4 hinge region having an amino acid sequence of ESKYGPPCPPCP (SEQ ID NO:3). Other examples of hinge regions that may be used in the chimeric Ti m4 receptors described herein include the hinge region from the extracellular regions of type I membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof In some embodiments, an extracellular spacer domain comprises a CD28 hinge region having an amino acid sequence of SEQ ID NO:32. In further embodiments, an extracellular spacer domain comprises all or a portion of an immunoglobulin Fe domain selected from: a CHI
domain, a CH2 domain, a CH3 domain, or combinations thereof (see. e.g., PCT
Publication W02014/031687, which spacers are incorporated herein by reference in their entirety). In yet further embodiments, an extracellular spacer domain may comprise a stalk region of a type II C-lectin (the extraccllular domain located between the C-type lectin domain and the transmembrane domain). Type II C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.
In certain embodiments, an extracellular domain comprises polynucleotide sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof. In certain embodiments, an extracellular domain is murine, human, or chimeric.
In certain embodiments, an intracellular signaling domain comprises a costimulatory signaling domain and an ITAM-containing activating domain, wherein the ITAM-containing activating domain, comprises a DAP12 signaling domain. The costimulatory signaling domain may be any portion of a costimulatory signaling molecule that retains sufficient signaling activity. In some embodiments, a full length or full length intracellular component of a costimulatory signaling molecule is used. In some embodiments, a truncated portion of a costimulatory signaling molecule or intracellular component of a costimulatory signaling molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, costimulatory signaling domain is a variant of a whole or truncated portion of a costimulatory signaling molecule, provided that the variant retains sufficient signal transduction activity e . , is a functional variant).
In certain embodiments, the costimulatory signaling domain comprises a CD27, CD28, CD4OL, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 ([CAM:), CD83, CD134 (0X-40), CD137 (4-IBB), CD150 (SLAM:Fp, CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), PD-1, CD273 (PD-L2), CD274 (PD-L1), B7-H3 (CD276), ICOS (CD278), DAP10, LAT, LFA-1 (CD 1 1a/0318), LIGHT, NKG2C, SLP76, or TRIM: signaling domain. In particular embodiments, the costimulatory signaling domain comprises an 0X40, CD2, CD27, CD28, 1CAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), or 4-1BB (CD137) signaling domain. An exemplary CD28 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO: 164 or 165. An exemplary 0X40 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:166. An exemplary CD2 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:167. An exemplary 4-1BB costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:168. An exemplary CD27 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:169. An exemplary ICAM-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:170. An exemplary LFA-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:171. An exemplary ICOS costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:172. An exemplary CD30 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:173. An exemplary CD40 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID .NO:174. An exemplary PD-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:175. An exemplary CD7 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:176. An exemplary LIGHT
costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:177. An exemplary NKG2C costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:178. An exemplary B7-H3 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:179. In certain embodiments, the costimulatory signaling domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOS:164-179. In certain embodiments, the costimulatory signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of any one of SEQ ID
NOS:164-179. In certain embodiments, the intracellular signaling comprises a second costimulatory signaling domain. In preferred embodiments, the first costimulatory signaling domain and second costimulatory signaling domain arc different.
The DAP12 signaling domain may recapitulate TCR signaling independently of endogenous TCR complexes. In certain embodiments, signaling via the DAP12 signaling domain leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. The DAP12 signaling domain may be any portion of the DAP12 molecule that retains sufficient signaling activity. In some embodiments, a full length or full length intracellular component of the DAP12 molecule is used. In some embodiments, a truncated portion of DAP12 or intracellular component of a DAP12 is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, the DAP12 signaling domain is a variant of a whole or truncated portion of DAP12, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).
An exemplary human DAP12 molecule is provided in Uniprot. Ref.
043914. An exemplary DAP12 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:180. In certain embodiments, the DAP12 signaling domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%õ 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%
identity to SEQ ID NO:180. In certain embodiments, the DAP12 signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
transmembrane domain Mouse Tim4 M SK GLLLLWLVTELW Vv' LYLTPAASEDTI1GFLGQPVTL 24 binding PCHYLSWSQSRNSMCWGKGSCPNSKCNAELLRTDGTR
domain, amino IISRKSTKYILLGKVQFGEVSLTISNTN. RGDSGVYCCRI
acids 1-22 are EVPGVvr.FNDVIKKNV.RLEI.,RRAT`FIKK.PrrtTRPT'FTPY
signal peptide VTTTTPELLPTTVMTTSVLPTTTPPQTLATTAFSTAVTT
CPSTTPGSFSQETTKGSAF'TTESETLPASNHSQRSMMTI
STDIAVLRPTGSNPGILPSTSQLTTQKTTLTTSESLQKTT
KSHQINSRQT
Mouse Tim4 MSICGLLLLWLVTELWWLYLTPA
signal peptide Further embodiments of the chimeric Tim4 receptors described herein comprise a single chain chimeric protein, the single chain chimeric protein comprising:
an extracellular domain comprising a Tim4 binding domain; an intracellular signaling domain comprising a first costimulatory signaling domain and an HAM-containing activating domain, wherein the ITAIVI-containing activating domain comprises a DAP12 signaling domain; and a transmembrane domain positioned between and connecting the extracellular domain and the intracellular signaling domain.
A Tim4 binding domain suitable for use in a chimeric Tim4 receptor of the present disclosure may be any polypeptide or peptide derived from a Ti m4 molecule that specifically binds phosphatidylserine. In certain embodiments, the Tim4 binding domain is derived from human Tim4. An exemplary human Tim4 molecule is provided in liniprot. Ref. Q96H15 (SEQ ID .N0:1). An exemplary human Tim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:2 or amino acids 314 of SEQ ID NO:2. An exemplary mouse *Iim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:24 or amino acids 23-279 of SEQ ID
NO:24. In certain embodiments, the Tim4 binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID NO:24 or amino acids 23-279 of SEQ ID
NO:24. In certain embodiments, the Tim4 binding domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID
NO:24 or amino acids 23-279 of SEQ ID NO:24.
In certain embodiments, the ex tracellular domain optionally comprises an extracellular, non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable proper cell/cell contact, binding, and activation. When included in a chimeric receptor as described herein, an extracellular spacer domain is generally located between the extracellular binding domain and the transniembrane domain of the chimeric Tim4 receptor. The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest etal., J.
Immunother.
28:203-11, 2005; PCT Publication No. WO 2014/031687). In certain embodiments, an extracellular spacer domain is an immunoglobulin hinge region (e.g., IgGl, IgG2, IgG3, IgG4, IgA, IgD). An immunoglobulin hinge region may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region. An altered IgG4 hinge region is described in PCT Publication No. WO 2014/031687, which hinge region is incorporated herein by reference in its entirety. In a particular embodiment, an extracellular spacer domain comprises a modified IgG4 hinge region having an amino acid sequence of ESKYGPPCPPCP (SEQ ID NO:3). Other examples of hinge regions that may be used in the chimeric Ti m4 receptors described herein include the hinge region from the extracellular regions of type I membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof In some embodiments, an extracellular spacer domain comprises a CD28 hinge region having an amino acid sequence of SEQ ID NO:32. In further embodiments, an extracellular spacer domain comprises all or a portion of an immunoglobulin Fe domain selected from: a CHI
domain, a CH2 domain, a CH3 domain, or combinations thereof (see. e.g., PCT
Publication W02014/031687, which spacers are incorporated herein by reference in their entirety). In yet further embodiments, an extracellular spacer domain may comprise a stalk region of a type II C-lectin (the extraccllular domain located between the C-type lectin domain and the transmembrane domain). Type II C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.
In certain embodiments, an extracellular domain comprises polynucleotide sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof. In certain embodiments, an extracellular domain is murine, human, or chimeric.
In certain embodiments, an intracellular signaling domain comprises a costimulatory signaling domain and an ITAM-containing activating domain, wherein the ITAM-containing activating domain, comprises a DAP12 signaling domain. The costimulatory signaling domain may be any portion of a costimulatory signaling molecule that retains sufficient signaling activity. In some embodiments, a full length or full length intracellular component of a costimulatory signaling molecule is used. In some embodiments, a truncated portion of a costimulatory signaling molecule or intracellular component of a costimulatory signaling molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, costimulatory signaling domain is a variant of a whole or truncated portion of a costimulatory signaling molecule, provided that the variant retains sufficient signal transduction activity e . , is a functional variant).
In certain embodiments, the costimulatory signaling domain comprises a CD27, CD28, CD4OL, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 ([CAM:), CD83, CD134 (0X-40), CD137 (4-IBB), CD150 (SLAM:Fp, CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), PD-1, CD273 (PD-L2), CD274 (PD-L1), B7-H3 (CD276), ICOS (CD278), DAP10, LAT, LFA-1 (CD 1 1a/0318), LIGHT, NKG2C, SLP76, or TRIM: signaling domain. In particular embodiments, the costimulatory signaling domain comprises an 0X40, CD2, CD27, CD28, 1CAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), or 4-1BB (CD137) signaling domain. An exemplary CD28 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO: 164 or 165. An exemplary 0X40 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:166. An exemplary CD2 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:167. An exemplary 4-1BB costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:168. An exemplary CD27 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:169. An exemplary ICAM-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:170. An exemplary LFA-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:171. An exemplary ICOS costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:172. An exemplary CD30 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:173. An exemplary CD40 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID .NO:174. An exemplary PD-1 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:175. An exemplary CD7 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:176. An exemplary LIGHT
costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:177. An exemplary NKG2C costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:178. An exemplary B7-H3 costimulatory signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:179. In certain embodiments, the costimulatory signaling domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOS:164-179. In certain embodiments, the costimulatory signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of any one of SEQ ID
NOS:164-179. In certain embodiments, the intracellular signaling comprises a second costimulatory signaling domain. In preferred embodiments, the first costimulatory signaling domain and second costimulatory signaling domain arc different.
The DAP12 signaling domain may recapitulate TCR signaling independently of endogenous TCR complexes. In certain embodiments, signaling via the DAP12 signaling domain leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. The DAP12 signaling domain may be any portion of the DAP12 molecule that retains sufficient signaling activity. In some embodiments, a full length or full length intracellular component of the DAP12 molecule is used. In some embodiments, a truncated portion of DAP12 or intracellular component of a DAP12 is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, the DAP12 signaling domain is a variant of a whole or truncated portion of DAP12, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).
An exemplary human DAP12 molecule is provided in Uniprot. Ref.
043914. An exemplary DAP12 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:180. In certain embodiments, the DAP12 signaling domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%õ 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%
identity to SEQ ID NO:180. In certain embodiments, the DAP12 signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence to SEQ ID NO:180.
In another embodiment, an intracellular signaling domain comprises a CD28 costimulatory signaling domain and a DAP12 signaling domain. In another embodiment, an intracellular signaling domain comprises a 4-1 BB costimulatory signaling domain and a DAP12 signaling domain. In yet another embodiment, an intracellular signaling domain comprises a CD27 costimulatory signaling domain and a DAP12 signaling domain. In another embodiment, an intracellular signaling domain comprises a ICOS costimulatory signaling domain and a DAP12 signaling domain.
In another embodiment, an intracellular signaling domain comprises a I,FA-1 costimulatory signaling domain and a DAP12 signaling domain. In another embodiment, an intracellular signaling domain comprises an 0X40 costimulatory signaling domain and a DAP12 signaling domain. In yet another embodiment, an intracellular signaling domain comprises a CD2 costimulatory signaling domain and a DAP12 signaling domain. In still another embodiment, an intracellular signaling domain comprises an ICAM-1 costimulatory signaling domain and a DAP12 signaling domain.
Intracellular signaling domains may be derived from a mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g., receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, or the like). In one embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, a chimeric 711m4 receptor may comprise a Tim4 binding domain and a Tim4 transmembrane domain. In another example, a chimeric Tim4 receptor may comprise a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules; the transmembrane domain and the intracellular signaling domain are derived from different molecules; or the transmembrane domain, extracellular domain, and intracellular signaling domain are all derived from different molecules. Examples of transmembrane domains that may be used in chimeric Tim4 receptors of the present disclosure include transmembrane domains from Tim4, CD3, CD3y, CD38, CD3e, CD28, CD45, CD4, CD5, CD8, CD9, CD1.6, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3. An exemplary Tim4 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:144 or 23. An exemplary CD28 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:181. An exemplary 4-1 BB transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:182. An exemplary 0X40 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO: 183. An exemplary CD27 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID
NO:184. An exemplary ICOS transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:185. An exemplary CD2 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:186. An exemplary LFA-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:187. An exemplary CD30 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:188. An exemplary CD40 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:189. An exemplary PD-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:190. An exemplary CD7 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:191. An exemplary LIGHT transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:192. An exemplary NKG2C transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:193. An exemplary 1B7-H3 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:194. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOS:23, 144, and 181-194. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletion, additions, substitutions) to an amino acid sequence of any one of SEQ113 NOS:23, 144, and 181-194.
Transmembrane domains may derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
In certain embodiments, a chimeric Tirn4 receptor comprises polynucleoti de sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof. In certain embodiments, a chimeric Tim4 receptor is murine, chimeric, human, or humanized.
It is understood that direct fusion of one domain to another domain of a chimeric Tim4 receptor described herein does not preclude the presence of intervening junction amino acids. Junction amino acids may be natural or non-natural (e.g., resulting from the construct design of a chimeric protein). For example, junction amino acids may result from restriction enzyme sites used for joining one domain to another domain or cloning polynucleotides encoding chimeric Tim4 receptors into vectors.
Exemplary components, configurations and chimeric Tim4 receptor sequences of the present disclosure are provided in Table 2.
Table 2.
SEQ
Name Amino Acid Sequence ID
NO:
human Tim4 MSKEPL1LWLMIEFWWLYLTPVTSETVVTEVLGHRVTLP
binding CLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGMRV 2 domain (amino TSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEV
acids 1-24 PGWFNDVKINVRLNLQRASTT'FHERTATTTTRRTTTTSPT
signal peptide) TTRQMTITPAALPTTvvrTpDLTTGTPLQMTTIAVF17TA
NTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESETVLPSDS
WSSVESTSADTVLLTSKESKVWDLPSTSHVSMWKTSDS
VSSPQPGASDTA.VPEQNKTTKTGQMDGIPMSMKNEMPI
SQ
mouse Tim4 MSKGLLLLWLVTELWWLYLTPAASEDTIIGFLGQPVTLP A
binding CH Y LS W SQ SRN SMC W GKGSCPN SKC N
AELLRTDGTRIIS
domain (amino RKSTKYTLI.,GKVQFGEVSLTISNTNRGDSGVYCCRIEV.P
acids 1-22 GWFNDVKKNVRLELRRATTTKICPTITTRPTTTPYVITTT
signal peptide) PELLPTitv-mTTSVLPTTTPPQTLATTAFS'FAvrrcPS`11-PG
SFSQETTKGSAFTTESETI,PASNITSQRSMMTISTDIAVIR
PTGSNPGILPSTSQLTTQKTTLTTSESLQKTTKSHQINSRQ
.1' Modified IgG4 ESKYGPPCPPCP
hinge Wildtype RSKRSR1_,LIISDYMNMTPRRPGPTRKETYQPY.APPRDFA A 164 costimulatory YRS
signaling domain CD28 RSKRS RGGH S DY?vINM TPRRPGPTRKI 1 YQPY APPRD FA
A
costimulatory YRS
signaling domain with substitutions (positions reference to full length protein) mulatory ALYLLRRDQRLPPDAHK..PPGG(3 SF RTRIQEEQADAHSTL 166 costi signaling MCI
domain CD2 KRK KQRSRRNDEELETRAHR.VA.TEERGRKPHQIPASTPQ
costimulatory NPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPP 167 signaling APSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN
domain 4-1BB ....................... KRGRKKLLYEFKQPFMRPVQTTQEEDGCSCRFPEEEEG-G
________________ costimulatory CEL
signaling domain CD27 QRR.K YR SNK
_____________________________________ GESPVEP AEPCHYSCPREEEG STIPIQEDYR
16 ) costimulatory KPEPAC SP
signaling domain costimulatory signaling domain ________________ costimulatory MNPKFAES
signaling domain ICOS
TCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVT
costimulatory L
signaling domain costimulatory LRSGASVTEPVAEERGLMSQPLIVIETCHSVGAAYLESLPL "
signaling QDA SPA GGPS S PRDLPEPRV STEHTNNKIEK WIMKADTV
domain IV GTVKAELPEGRGLAGPAEPELEEELEADHTPH YPEQE-1.
EPPLGSC SDVMLS VEEEGKEDPLpT A ASGK
costimutatory LHGCQPVTQEDGKESRISVQERQ
signaling domain PD-1 C SRAAR.GT IGARRTGQ PLK EDP S A.VPVF S VDYGE LDFQ
costi mul atory WREKTPEPPVPCYPEQTEVATIVFPSGMGTSSP ARR GS A
signaling DGPRSAQPLRPEDGH C SW PL
domain costimulatory Q
signaling domain LIGHT MEE SVVRP SWVVDGQTDIPFTRLGRSHRRQ SC SVAR
costimul atory signaling domain costimulatory QVELNLQNP SLNHQGIDKIYDC QGLLPPPEK
signaling domain costi m ulatory KEDDGQEI A
signaling domain ___________________________ VYSDLNTQRPYYK
Tim4 LLIVIIIAPSLGFVLF ALFVAFL
transmembrane domain transmembrane domain transmembrane domain __________________________ transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane 1)0 domain transmembrane domain LIGHT VGLGLLLLLMGAGLAVQGWFL
transmembrane domain transmembrane domain transmembrane domain Tim4 binding MSKEPLILWLMIEFWWLYLTPVTSETVVTEVLGIIRATTLP
domain-CD28 CLYS SW SI-LNSNSMCWGKDQCPYSGCKEALIRTDGMRV
hinge-CD28 TSRKSAK YRLQGT IPRGDVSLTILNPSESDSGVYCCRIE V
transmembrane PGVVFNDVKINVRINLQRASTTTEIRTATT.TTRRTTTTSPT
costimulatory NTCLSLTPSTLPEEA'FGLLTPEPSKEGP1LTAESETVLPSDS
signaling WSSVESTSADTVLLTSKESKVWDLPSTSHVSMWKTSDS
domain- VS SPQPGASDTAVPEQNKTTIC17GQMDGIPMSMKNEMPI
DAP12 SQIEVMYPPPYLDNEKSNGTHHVKGKHLCPSPLITGP,S'KPF
signaling W VL V V VGGVLACY SLINTVAFIIFWVIZSKRSRLDISDIM
domain, amino AMTPRIZPGPTRKHYQPYAPPRDFAA YRS'YFLGRINPRGRG
acids 1-24 are AAEAATICKORITETESPYQELQGQRSDVYSDLNTQRPYY
signal peptide K
(CTX140) Ti m4 binding MSKEPLILWLMIEFWWLYL'IPVTSETVVTEVLGIIRVTLP nr_ domain- CLYS SW SI-INSN SMCW GKDQCPYSGCKEALIRTDGMRV 17`"
modified Igth TSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEV
hinge-CD28 PGWFNDVKINVRLNLQR A STTMRTATTITRICITTTSPT
transmembrane TTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAVFTTA
costimulatory WSSVESTSADTVLLTSKESKVWDLPSTSHVSMWKTSDS
signaling VS SPQPGASDTAVPEQNKTTKTGQMDGIPMSMKNEMPI
domain- SQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF
DAP12 WV R,S'KRS'RLLHSDYMNMTPRI?PGP7RKHYOPYAPPRDPAA
signaling YRSYFLG.RLVPRGRGAAEAATRKQ.RITETESPYQELQGQ
domain, amino RSDVYSDLNTQRPYYK
acids 1-24 are signal peptide (CTX143) Tim4 binding MSKEPLILWLM1EFWWLYLTPVTSETVVTEVLGHRVTLP
domain-CD28 CLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGMRV
transmembrane TSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEV
-CD28 PGWFINIDVKINVRLNLQRASTT'FHRTATTTTRRTTTTSPT
costimulatory TTRQMTTTPAALPTTVVTTPDLTTGTVLQMTTIAVFTTA
signaling NTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESETVLPSDS
domain- WSSVESTSADTVLLTSKE:SKVWDLPSTSHVSMWKTSDS
signaling SQFWVLVVVGGVLACYSLLVTVAFIEFWVRS'KR,SRUHSD
domai n, amino YAMMTPRRPGPTRKHYQPYAPPRIVA A YRS'YFLGRINPRG
acids 1:24 are RGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRP
signal peptide , YYK
(CTX137) 1 Tim4 binding MSKEPLILWLMIEFWWLYLTPVTSETVVTEVLGHRVTLP 198 domain ¨ Tim4 CLYSSWSHNSNSMCWGKDQCPYSGCKEAL1RTDGMRV
DAP12; ammo PGWFNDVKINVRLNLQRA STT1'1-1WrATTITRRTT: [1 SP
acids 1-24 are TTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAVFTTA
signal peptide NTCLSLTPSTLPEEATGLLTPEPSKEGPlurAESET'VLPSDS
(CTX256) WSSVESTSADTVULTSKESKVWDLPSTSHVSMWKTSDS
VSSPQPGAS:DTAVPEQNKTTKTGQIVEDGIPMSMKNEMP1 SQII,MIIAPSLGFVLFALEVAFLRSKRSRLLI-ISDYMNMIP
RRPGPTRKHYQPYAPPRDFAAYRSYFLGRLVPRGRGAA
EAAIRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK
In embodiments, a chimeric Tim receptor comprises a single chain chimeric protein, the single chain chimeric protein comprising: (a) an extracellular domain comprising a binding domain comprising: (i) a Timl IgV domain or Tim4 IgV
domain and (ii) a Timl mucin domain or Tim4 mucin domain; (b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) a transmembrane domain positioned between and connecting the extracellular domain and the intracellular signaling domain.
In particular embodiments, the binding domain comprises: (i) a Tim!
IgV domain and a Timl mucin domain; (ii) a Tim4 IgV domain and a Tim4 mucin domain; (iii) a Timl IgV domain and a Tim4 mucin domain; or (iv) a Tim4 IgV
domain and a Timl mucin domain. In certain embodiments, the extracellular domain of the chimeric Tim receptors described herein optionally includes an extracellular spacer domain positioned between and connecting the binding domain and transmembrane domain When expressed in a host cell, chimeric Tim receptors of the present disclosure can confer a phosphatidylserine-specific, cytotoxic phenotype to the modified host cell (e.g., the host cell becomes cytotoxic to a stressed, damaged, injured, apoptotic, or necrotic cell expressing phosphatidylserine on its surface). In certain embodiments, the chimeric Tim receptors induce apoptosis in targeted cells via release of granzymes, perforin, granulysin, or any combination thereof. In further embodiments, cells expressing a chimeric Tim receptor according to the present description exhibit an engulfment phenotype specific to phosphatidylserine presenting cells.
The intracellular signaling domain can include one or more effector (also referred to as "costimulatory signaling") domains that costimulate the modified host cell. Signaling by the intracellular signaling domain(s) is triggered by binding of the extracellular domain to phosphatidylserine. In certain embodiments, the intracellular signaling domain comprises a first intracellular signaling domain. In further embodiments, the intracellular signaling domain comprises a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a first intracellular signaling domain, a second intracellular signaling domain, and a third intracellular signaling domain.
Chimeric Tim receptors according to the present disclosure can be used in a variety of therapeutic methods where clearance of apoptotic, necrotic, damaged, or stressed cells is beneficial, while providing costimulation that enhances cellular immune response, reduces immune cell exhaustion, or both.
As described herein, a chimeric Tim receptor corn prises an extracellular domain comprising a Tim binding domain. The Tim binding domain confers specificity to phosphatidylserine (PtdSer), which is a phospholipid with a negatively charged head-group and a component of the cell membrane. In healthy cells, phosphatidylserine is preferentially found in the inner leaflet of the cell membrane. However, when cells are stressed, damaged or undergo apoptosis or necrosis, phosphatidylserine is exposed on the outer leaflet of the cell membrane. Thus, phosphatidylserine may be used as a marker to distinguish stressed, damaged, apoptotic, necrotic, pyroptotic, or oncotic cells. Binding of phosphatidylserine by the Tim binding domain may block the interaction between the phosphatidylserine and another molecule and, for example, interfere with, reduce or eliminate certain functions of the phosphatidylserine (e.g., signal transduction). In some embodiments, the binding of a phosphatidylserine may induce certain biological pathways or identify the phosphatidylserine molecule or a cell expressing phosphatidylserine for elimination.
A Tim binding domain suitable for use in a chimeric Tim receptor of the present disclosure may be any polypeptide or peptide derived from a Tim I
and/or Tim4 molecule that specifically binds phosphatidylserine. In embodiments, a Tim binding domain comprises an IgV domain from Timl or Tim4, and a mucin domain from Tim I
or Tim4. For example, a Tim binding domain may comprise a Timl IgV domain and a Tim I mucin domain. In another example, a Tim binding domain may comprise a Tim I
IgV domain and a Tim4 mucin domain. In another example, a Tim binding domain may comprise a Tim4 IgV domain and a Tim 1 mucin domain. In another example, a Tim binding domain may comprise a Tim4 IgV domain and a Tim4 mucin domain.
Phosphatidylserine binding is generally regulated by the I8\' domain.
The core phosphatidylserine binding domain is a four amino acid sequence in the 18V
domain (e.g., amino acids 95-98 of SEQ ID NO:34 or amino acids 92-95 of SEQ
NO:38). A Tim4 binding domain binds minimally to cells with low phosphatidylserine density. A 717im I. binding domain binds more strongly to a lower phosphatidylserine density, resulting in a lower threshold for response. A summary of Timl and Tim4 binding to phosphatidylserine is provided in Table 3. By combining Timi IgV
domain and Tim4 mucin domain, or Tim4 IgV domain and Timl rnucin domain, the binding affinity of the binding domain to phosphatidylserine can be modulated.
Further, such a combination in the Tim binding domain also provides a combination of the sensitivity to phosphatidylserine of Tim4 and the stability in protein expression of Tim 1.
Table 3. .
Low PtdSer 1 Moderate PtdSer High PtdSer i Cooperativity Tim-1 Moderate binding Strong binding Strong binding I
Tim-4 Minimal binding Moderate binding Strong binding I.
1 Yes Additionally, an RGD domain (e.g., amino acids 68-70 of SEQ ID
NO:34) in an IgV domain may regulate integrin binding as a co-receptor for engulfment.
In certain embodiments, the Tim. binding domain is derived from human Tim 1 and/or Tim4. An exemplary human Timl molecule is provided in Uniprot.
Ref.
Q96D42 (S:EQ ID NO:36). An exemplary human Timl binding domain comprises or consists of an amino acid sequence of SEQ ID NO:37 or SEQ ID NO:43. In certain embodiments, the Timl binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:37 or SEQ ID NO:43.
In certain embodiments, the Tim .1 binding domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:37 or SEQ ID NO:43.
An exemplary human Tim4 molecule is provided in Uniprot. Ref Q96H15 (S:EQ ID NO:1). An exemplary human Tim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:42. An exemplary mouse Tim4 binding domain comprises or consists of an amino acid sequence of SEQ
ID NO:24 or amino acids 23-279 of SEQ ID NO:24. In certain embodiments, the Tim4 binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:2 or SEQ ID NO:42, or SEQ ID NO:24 or amino acids 23-279 of SEQ ID NO:24. In certain embodiments, the Tim4 binding domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:42, or SEQ
ID NO:24 or amino acids 23-279 of SEQ ID NO:24.
In embodiments, the Tim binding domain comprises an IgV domain from Tim 1. An exemplary human Tim 1 18V domain is provided in SEQ ID NO:38.
In some embodiments, the Timl IgV domain is a modified Tim! IgV domain comprising a R66G substitution in SEQ ID NO:38. The 12.66G substitution (e.g., amino acids of SEQ ID NO:34) confers a RGD domain in Tim 1. IgV domain, which may regulate integrin binding as a co-receptor for engulfment. In particular embodiments, the modified Tim] IgV domain comprises or consists of the amino acid sequence of SEQ
ID NO:41. In some embodiments, this modified Timl domain may increase phagocytic activity while preserving Tirni sensitivity. In certain embodiments, the Tiinl IgV
domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94 4, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%
identity to SEQ ID NO:38, SEQ ED .NO:38 with a R66G substitution, or SEQ ID
NO:4.I. In certain embodiments, the Timl 1.8V comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an.
amino acid sequence of SEQ ID NO:38, SEQ ID NO:38 with a R66G substitution, or SEQ ID
NO:41.
In other embodiments, the Tim binding domain comprises an IgV
domain from Tim4. An exemplary human Tim4 :I8V domain is provided in SEQ ID
NO:34. In certain embodiments, the Tim4 18V domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to S:EQ ED NO:34. in certain embodiments, the Tim4 IgV domain comprises an amino acid sequence having at least about 1,2, 3,4, 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:34.
In embodiments, the Tim binding domain comprises a mucin domain from Timl. An exemplary human Timl mucin domain is provided in SEQ ID NO:39.
In certain embodiments, the Timl mucin domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A, 99%, 99.5%, or 100% identity to SEQ ID NO:39. In certain embodiments, the Timl mucin domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:39.
In other embodiments, the Tim binding domain comprises a mucin domain from Tim4. An exemplary human Tim4 mucin domain is provided in SEQ ID
NO:35. In certain embodiments, the Tim4 mucin domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:35. In certain embodiments, the Tim4 mucin domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, .19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:35.
In some embodiments, the Tim binding domain comprises a Tim! IgV
domain and a Timl. mucin domain. In a particular embodiment, the Tim! IgV
domain comprises the amino acid sequence set forth in SEQ ID NO:38 and the Timl mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In another particular embodiment, the Timl IgV domain comprises the amino acid sequence set forth in SEQ ID NO:38 with a R66G substitution and the Timl mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In a further particular embodiment, the Tim! IgV domain comprises the amino acid sequence set forth in SEQ
ID NO:41 and the Timl mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In som.e embodiments, the Tim! IgV domain and Tim I mucin domain together comprise or consist of the amino acid sequence set forth in SEQ ID
NO:37 or SEQ ID NO:43.
In some embodiments, the Tim binding domain comprises a Tim4 IgV
domain and a Tim4 mucin domain. In a particular embodiment, the Tim4 IgV
domain comprises the amino acid sequence set forth in SEQ ID NO:34 and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the Tim4 IgV domain and Tim4 mucin domain together comprise or consist of the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:42.
In some embodiments, the Tim binding domain comprises a Timl IgV
domain and a Tim4 mucin domain. In a particular embodiment, the Timl IgV
domain comprises the amino acid sequence set forth in SEQ ID NO:38 and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In another particular embodiment, the Timl IgV domain comprises the amino acid sequence set forth in SEQ ID .NO:38 with a R66G substitution and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In a further particular embodiment, the Timl IgV domain comprises the amino acid sequence set forth in SEQ
113 .NO:41 and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ NO:35. In some embodiments, the Timl IgV domain further comprises the Timl signal sequence of SEQ ID NO:40.
In still further embodiments, the Tim binding domain comprises a Tim4 IgV domain and a Timl mucin domain. In a particular embodiment, the Tim4 IgV
domain comprises the amino acid sequence set forth in SEQ ID .N0:34 and the Timl mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In some embodiments, the Tim4 IgV domain further comprises the Tim4 signal sequence of SEQ ID NO:11.
In certain embodiments, the extracellular domain optionally comprises an extracellular, non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable proper cell/cell contact, binding, and activation. When included in a chimeric receptor as described herein, an extracellular spacer domain is generally located between the extracellular binding domain and the transmembranc domain of the chimeric Tim receptor. The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest et al Irnmunother.
28:203-11, 2005; PCT Publication No. WO 2014/031687). In certain embodiments, an extracellular spacer domain is an immunoglobulin hinge region (e.g., IgGl, IgG2, IgG3, IgG4, igA, 1gD). An immunoglobulin hinge region may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region.
An altered IgG4 hinge region is described in PCT Publication No. WO
2014/031687, which hinge region is incorporated herein by reference in its entirety. In a particular embodiment, an extracellular spacer domain comprises a modified IgG4 hinge region having an amino acid sequence of ESKYGPPCPPCP (SEQ ID NO:3). Other examples of hinge regions that may be used in the chimeric Tim receptors described herein include the hinge region from the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof.
In a particular embodiment, an extracellular spacer domain comprises a CD28 hinge region having an amino acid sequence of SEQ ID NO:32. In further embodiments, an extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from. a CH1 domain, a CH2 domain, a CH3 domain, or combinations thereof (see, e.g., PCT Publication W02014/031687, which spacers are incorporated herein by reference in their entirety). In yet further embodiments, an extracellular spacer domain may comprise a stalk region of a type H C-lectin (the extracellular domain located between the C-type lectin domain and the transmembrane domain). Type H C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.
In certain embodiments, an extracellular domain comprises an amino acid sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof. In certain embodiments, an extracellular domain is murine, human, or chimeric.
The intracellular signaling domain of a chimeric Tim receptor as described herein is an intracellular effector domain and is capable of transmitting functional signals to a cell in response to binding of the extracellular domain of the chimeric Tim receptor and phosphatidylserine. The signals transduced by the intracellular signaling domain promote effector function of the chimeric Tim receptor containing cell. Examples of effector function include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, expansion, engulfment of a target cell or particle expressing phosphatidylserine on its surface, antigen capture, antigen processing, antigen presentation, or any combination thereof.
An intracellular signaling domain comprises a primary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a primary intracellular signaling domain and a secondary intracellular signaling domain.
In some embodiments, an intracellular signaling domain comprises a primary intracellular signaling domain, a secondary intracellular signaling domain, and a tertiary intracellular signaling domain. The primary, secondary, and/or tertiary intracellular signaling domains may independently be any portion of a signaling molecule that retains sufficient signaling activity. In some embodiments, a full length signaling molecule or full length intracellular component of a signaling molecule is used. In some embodiments, a truncated portion of a signaling molecule or intracellular component of a signaling molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In some embodiments, a signaling domain is a variant of a whole or truncated portion of a signaling molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).
In some embodiments, the primary intracellular signaling domain comprises a Timl signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3 signaling domain, 4-1BB signaling domain, TLR2 signaling domain, or a TLR8 signaling domain.
In embodiments, the secondary intracellular signaling domain comprises a Timl signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAPS signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CDR', signaling domain, 4-1BB signaling domain, TLR2 signaling domain, or a lilts signaling domain.
In some embodiments, the tertiary intracellular signaling domain comprises a Timl signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP1.2 signaling domain, a CD3C signaling domain, 4-1BB signaling domain, TLR2 signaling domain or a ILR.8 signaling domain.
In some embodiments, the primary intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) containing signaling domain; the secondary intracellular signaling domain comprises a costimulatory signaling domain, Ti ml signaling domain or Tim4 signaling domain; and the tertiary intracellular signaling domain comprises a TLR signaling domain.
An ITAM containing signaling domain generally contains at least one (one, two, three, four, or more) 1TAM.s, which refer to a conserved motif of YXXL/1.-X6.8-YXXL/I. An ITAM containing signaling domain may initiate T cell activation signaling following antigen binding or ligand engagement. ITAM-signaling domains include, for example, intracellular signaling domains of CD3y, CUM, CD3c, CD34.;, CD5, CD22, CD79a, CD278 (ICOS), DAP12, FcRy, and CD66d. A costimulatory signaling domain, which, when activated in conjunction with a primary or classic (e.g., ITAM-driven) activation signal, promotes or enhances T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or combinations thereof. Costimulatory signaling domains for use in chimeric Tim receptors include, for example, CD27, CD28, CD4OL, GITR, NKG2C, CARD!, CD2, CD7, CD27, CD30, CD40, CD54 (1CAM), CD83, Cal 34 (0X-40), CD1.37 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HI/EM), CD273 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, SLP76, TRIM, ZAP70, or any combination thereof. In some embodiments, the costimulatory signaling domain comprises a 0X40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a/CD18), ICOS
(CD278), or 4-1BB (CD137) signaling domain. A TLR signaling domain may be a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TI.R8, or TLR9 signaling domain. In some embodiments, the TLR signaling domain is a TLR2 signaling domain or TLR8 signaling domain.
As used herein, the designation of primary, secondary, and tertiary intracellular signaling domains includes but is not limited to arrangements of the primary intracellular signaling domain at the N-terminus, secondary intracellular signaling domain in the middle, and tertiary intracellular signaling domain at the C-terminus of the intracellular portion of the chimeric Tim receptor. Thus, designation of the primary intracellular signaling domain does not limit the use of the selected intracellular signaling domain at the N-terminus of the intracellular portion of the chimeric Tim receptor. Designation of the secondary intracellular signaling domain does not limit the use of the selected intracellular signaling domain in the middle (or at the C-terminus for those chimeric Tim receptors only having two intracellular signaling domains) of the intracellular portion of the chimeric Tim receptor.
Designation of the tertiary intracellular signaling domain does not limit the use of the selected intracellular signaling domain at the C-terminus of the intracellular portion of the chimeric Tim receptor. Thus, different arrangements of the primary, secondary, and/or tertiary intracellular signaling domains within the intracellular portion of the chimeric Tim receptor are contemplated.
An exemplary Timl signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:44. An exemplary Tim4 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:45, SEQ ID NO:224, or SEQ ID
NO:225. An exemplary TRAF2 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:48. An exemplary TRAF6 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:46. An exemplary CD28 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:4 or 26.
An exemplary DAP12 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:9. An exemplary CD3C, signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:27 or 5. An exemplary 4-1BB signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:122. An exemplary TLR2 signaling domain comprises or consists of the amino acid sequence of SEQ
ID
NO:222. An exemplary TLR8 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:47.
In a particular embodiment, the Timl signaling domain comprises the amino acid sequence set forth in SEQ ID NO:44. In another particular embodiment, the Tim4 signaling domain comprises the amino acid sequence set forth in SEQ ID
NO:45 SEQ ID NO:224, or SEQ ID NO:225. In another particular embodiment, the TRAF2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:48 In another particular embodiment, the TRAF6 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:46. In another particular embodiment, the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:4.
In another particular embodiment, the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:26. In another particular embodiment, the signaling domain comprises the amino acid sequence set forth in SEQ ID NO:9.
In another particular embodiment, the CD3c, signaling domain comprises the amino acid sequence set forth in SEQ ID NO:27. In another particular embodiment, the CD3C
signaling domain comprises the amino acid sequence set forth in SEQ ID NO:5.
In another particular embodiment, the 4-1 BB signaling domain comprises the amino acid sequence of SEQ ID NO:122. In another particular embodiment, the TI,R2 signaling domain comprises of the amino acid sequence of SEQ ID NO:222. In another particular embodiment, or the TIM signaling domain comprises the amino acid sequence set forth in SEQ ID NO:47.
In embodiments, the primary, secondary, and/or tertiary signaling domain (if present) comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 995%, or 100% identity to any one of SEQ TD NOS:4, 5, 9, 26, 27, or 44-48. In certain embodiments, the primary, secondary, and/or tertiary signaling domains comprises an amino acid sequence haying at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of any one of SEQ ID NOS:4, 5, 9, 26, 27, 44-48, 122, and 222. In embodiments, the primary signaling domain, secondary, and/or tertiary intracellular signaling domain arc the same. In some embodiments, two or three of the primary, secondary, and tertiary intracellular signaling domains are different.
In certain embodiments, an intracellular signaling domain comprises a Timi intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a Tim4 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD3C intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TRAF6 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TRAF2 intracellular signaling domain. In some embodiments, an.
intracellular signaling domain comprises a TLR2 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TLR8 intracellular signaling domain.
In certain embodiments, an intracellular signaling domain comprises a Timl primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In other embodiments, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In other embodiments, an intracellular signaling domain comprises a TLR8 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In other embodiments, an intracellular signaling domain comprises a Tut2 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In other embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain and a DAP12 secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a CD3C tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3C secondary intracellular signaling domain, and a TLR2 tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a ILR8 secondary intracellular signaling domain, and a CD3C, tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3C secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TLR2 primary intracellular signaling domain and a secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD3C primary intracellular signaling domain and a secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TLR8 primary intracellular signaling domain and a secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD3C, primary intracellular signaling domain and a TLR8 secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TRAF6 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD3C primary intracellular signaling domain and a TR AF6 secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CO28 primary intracellular signaling domain, a -171.112 secondary intracellular signaling domain, and a cD3r, tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3C, secondary intracellular signaling domain, and a TLR2 tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a mRs secondary intracellular signaling domain, and a CD3C tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain. In some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a TLR2 secondary intracellular signaling domain. In some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a TLR8 secondary intracellular signaling domain. In some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a CD3C secondary intracellular signaling domain, in some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a TRAF6 secondary intracellular signaling domain. in some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a CD3C tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a CD3C, secondary intracellular signaling domain, and a TLR2 tertiary intracellular signaling domain. in some embodiments, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a TLR8 secondary intracellular signaling domain, and a CD3C tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a 1'im4 primary intracellular signaling domain, a CD3C secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain.
In a particular embodiment, an intracellular signaling domain comprises a Timl primary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:44 and a CDR', secondary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:27 or 5. In another particular embodiment, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:45, SEQ ID NO:224, or SEQ ID
NO:225, and a CD3( secondary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:27 or 5. In a further particular embodiment, an intracellular signaling domain comprises a TLR8 primary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:47 and a CDR; secondary intracellular signaling domain comprising an amino acid sequence of SEQ ID
NO:27 or 5. In another particular embodiment, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:4 or 26 and a DAP12 secondary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:9. In some embodiments, an intracellular signaling domain comprises a combination of primary, secondary, and optionally tertiary intracellular signaling domain sequences as shown in Table 10.
Intracellular signaling domains may be derived from a mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
The transmembrane domain of a chimeric Tim receptor connects and is positioned between the extracellular domain and the intracellular signaling domain.
The transmembrane domain is a hydrophobic alpha helix that transverses the host cell membrane. The transmembrane domain may be directly fused to the binding domain or to the extracellular spacer domain if present. In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g., receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, or the like). In one embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, a chimetic Tim receptor may comprise a Tit/14 binding domain and a Tim4 transmembrane domain. In another example, a chimeric Tim receptor may comprise a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules; the transmembrane domain and the intracellular signaling domain are derived from different molecules; or the transmembrane domain, extracellular domain, and intracellular signaling domain are all derived from different molecules Examples of transmembrane domains that may be used in chimeric Tim receptors of the present disclosure include transmembrane domains from Timl.
Tim4, and CD28. An exemplary Timl transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:8. An exemplary Tim4 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:6 or 23. An exemplary CD28 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:7. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID
NOS:6-8 or 23. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletion, additions, substitutions) to an amino acid sequence of any one of SEQ ID NOS:6-8 or 23.
Transmembrane domains may derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
in certain embodiments, a chimeric Tim receptor comprises polynucleotide sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof In certain embodiments, a chimeric Tim receptor is murine, chimeric, human, or humanized.
It is understood that direct fusion of one domain to another domain of a chimeric Tim receptor described herein does not preclude the presence of intervening junction amino acids. Junction amino acids may be natural or non-natural (e.g., resulting from the construct design of a chimeric protein). For example, junction amino acids may result from restriction enzyme sites used for joining one domain to another domain or cloning polynucleotides encoding chimeric Tim receptors into vectors.
Exemplary Chimeric Ti in Receptors The component parts of a chimeric Tim receptor as disclosed herein can be selected and arranged in various combinations to provide a desired specificity and effector phenotype to a host cell.
Exemplary chimeric Tim receptors of the present disclosure are described in Table 4.
Table 4.
Construct IgV
M:ucin Transineinbrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain I Tim I Tim I Tim 1 TimI CD3c, --7 urn! Tim I Tirn1 'fim4 -- CD31-, _________________ 3 Tim! Tim I --- CD28 CD28 _________ 4 Tim I Tim I Tim I TRAF6 5 Tim I Tim I CD28 TRAF6 6 Tim 1 Timl Timl TRAF2 7 Tim! Tim! ____ CO28 TRAF2 8 Timl Timl Tim l TI..R8 CD3 c _________________ 9 Tim! ________ Tim! CD28 CD28 DAPI2 i 10 Tim! Tim I Tim I CD28 DAPI2 i Further exemplary chimeric Tim receptors are described in Table 5.
Table 5.
Construct IgN7 Mucin Transmembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 1' SEQ ID SEQ ID SEQ ID NO:8 SEQ ID SEQ ID
.NO:38 NO:39 NO:44 NO:5 7= SEQ ID SEQ ID SEQ ID 'NO:8 SEQ ID SEQ ID
----------------------- NO:38 NO:39 NO:45 NO:5 -3" SEQ ID SEQ ID SEQ ID 'NO:7 SEQ ID
NO:38 NO:39 .N0:4 4' SEQ ID SEQ :ID SEQ
ID NO:8 SEQ ID
_______________________ .N0:38 NO:39 __________________ NO:46 5' SEQ ID SEQ
ID SEQ ID NO:7 SEQ ID
----------------------- NO:38 NO:39 NO:46 ...
6" SEQ ID SEQ ID SEQ ID 'NO:8 SEQ ID
1 NO:38 NO:39 NO:48 Construct IgV
Mucin Transrnernbrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 7' SEQ ID SEQ
ID SEQ ID NO:7 SEQ ID
NO:38 NO:39 NO:48 8' SEQ ID SEQ
ID SEQ II) NO:8 SEQ ID SEQ ID
NO:38 NO:39 NO:47 NO:5 9' SEQ ID SEQ
ID SEQ ID NO:7 SEQ ID SEQ ID
NO:38 NO:39 NO:4 NO:9 10' SEQ ID SEQ
ID SEQ ID NO:8 SEQ ID SEQ 1D
NO:38 NO:39 NO:4 NO:9 In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 4. In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 5.
In some embodiments, a chimeric Tim receptor of Construct 1 or Construct 1" comprises amino acids 21-456 of SEQ ID NO:49. In some embodiments, a chimeric Tim receptor of Construct 1 or Construct l' comprises an amino acid sequence of SEQ ID .NO:49.
In some embodiments, a chimeric Tim receptor of Construct 2 or Construct 2' comprises amino acids 21-471 of SEQ ID NO:50. In some embodiments, a chimeric Tim receptor of Construct 2 or Construct 2 comprises an amino acid sequence of SEQ ID NO:50.
In some embodiments, a chimeric Tim receptor of Construct 3 or Construct 3' comprises amino acids 21-363 of SEQ ID NO:51. In some embodiments, a chimeric Tim receptor of Construct 3 or Construct 3' comprises an amino acid sequence of SEQ .N0:51.
In some embodiments, a chimeric Tim receptor of Construct 4 or Construct 4' comprises amino acids 21-590 of SEQ ID NO:52. In some embodiments, a chimeric Tim receptor of Construct 4 or Construct 4' comprises an amino acid sequence of SEQ ID:NO:52.
In some embodiments, a chimeric Tim receptor of Construct 5 or Construct 5' comprises amino acids 21-596 of SEQ ID NO:53. In some embodiments, a chimeric Tim receptor of Construct 5 or Construct 5' comprises an amino acid sequence of SEQ ID NO:53.
In some embodiments, a chimeric Tim receptor of Construct 6 or Construct 6' comprises amino acids 21-619 of SEQ ID NO:54. In some embodiments, a chimeric Tim receptor of Construct 6 or Construct 6' comprises an amino acid sequence of SEQ ID NO:54.
In some embodiments, a chimeric Tim receptor of Construct 7 or Construct 7' comprises amino acids 21-625 of SEQ ID NO:55. In some embodiments, a chimeric Tim receptor of Construct 7 or Construct 7' comprises an amino acid sequence of SEQ ID NO:55.
In some embodiments, a chimeric Tim receptor of Construct 8 or Construct 8 comprises amino acids 21-621 of SEQ ID NO:56. In some embodiments, a chimeric Tim receptor of Construct 8 or Construct 8' comprises an amino acid sequence of SEQ B3 NO:56.
In some embodiments, a chimeric Tim receptor of Construct 9 or Construct 9' comprises amino acids 21-415 of SEQ NO:57. In some embodiments, a chimeric Tim receptor of Construct 9 or Construct 9' comprises an amino acid sequence of SEQ ID NO:57.
In some embodiments, a chimeric Tim receptor of Construct 10 or Construct 10' comprises amino acids 21-409 of SEQ ID NO:58. In some embodiments, a chimeric Tim receptor of Construct 10 or Construct 10' comprises an amino acid sequence of SEQ 113 NO:58.
Further exemplary chimeric Tim receptors of the present disclosure are described in Table 6.
Table 6.
Construct IgV Mucin Transmembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 11 Tim4 Tim4 Tim4 Tim4 CD3cõ
12 Tim4 1'irn4 Tim4 Tim 1 CD3c
In another embodiment, an intracellular signaling domain comprises a CD28 costimulatory signaling domain and a DAP12 signaling domain. In another embodiment, an intracellular signaling domain comprises a 4-1 BB costimulatory signaling domain and a DAP12 signaling domain. In yet another embodiment, an intracellular signaling domain comprises a CD27 costimulatory signaling domain and a DAP12 signaling domain. In another embodiment, an intracellular signaling domain comprises a ICOS costimulatory signaling domain and a DAP12 signaling domain.
In another embodiment, an intracellular signaling domain comprises a I,FA-1 costimulatory signaling domain and a DAP12 signaling domain. In another embodiment, an intracellular signaling domain comprises an 0X40 costimulatory signaling domain and a DAP12 signaling domain. In yet another embodiment, an intracellular signaling domain comprises a CD2 costimulatory signaling domain and a DAP12 signaling domain. In still another embodiment, an intracellular signaling domain comprises an ICAM-1 costimulatory signaling domain and a DAP12 signaling domain.
Intracellular signaling domains may be derived from a mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g., receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, or the like). In one embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, a chimeric 711m4 receptor may comprise a Tim4 binding domain and a Tim4 transmembrane domain. In another example, a chimeric Tim4 receptor may comprise a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules; the transmembrane domain and the intracellular signaling domain are derived from different molecules; or the transmembrane domain, extracellular domain, and intracellular signaling domain are all derived from different molecules. Examples of transmembrane domains that may be used in chimeric Tim4 receptors of the present disclosure include transmembrane domains from Tim4, CD3, CD3y, CD38, CD3e, CD28, CD45, CD4, CD5, CD8, CD9, CD1.6, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3. An exemplary Tim4 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:144 or 23. An exemplary CD28 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:181. An exemplary 4-1 BB transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:182. An exemplary 0X40 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO: 183. An exemplary CD27 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID
NO:184. An exemplary ICOS transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:185. An exemplary CD2 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:186. An exemplary LFA-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:187. An exemplary CD30 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:188. An exemplary CD40 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:189. An exemplary PD-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:190. An exemplary CD7 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:191. An exemplary LIGHT transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:192. An exemplary NKG2C transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:193. An exemplary 1B7-H3 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:194. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOS:23, 144, and 181-194. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletion, additions, substitutions) to an amino acid sequence of any one of SEQ113 NOS:23, 144, and 181-194.
Transmembrane domains may derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
In certain embodiments, a chimeric Tirn4 receptor comprises polynucleoti de sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof. In certain embodiments, a chimeric Tim4 receptor is murine, chimeric, human, or humanized.
It is understood that direct fusion of one domain to another domain of a chimeric Tim4 receptor described herein does not preclude the presence of intervening junction amino acids. Junction amino acids may be natural or non-natural (e.g., resulting from the construct design of a chimeric protein). For example, junction amino acids may result from restriction enzyme sites used for joining one domain to another domain or cloning polynucleotides encoding chimeric Tim4 receptors into vectors.
Exemplary components, configurations and chimeric Tim4 receptor sequences of the present disclosure are provided in Table 2.
Table 2.
SEQ
Name Amino Acid Sequence ID
NO:
human Tim4 MSKEPL1LWLMIEFWWLYLTPVTSETVVTEVLGHRVTLP
binding CLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGMRV 2 domain (amino TSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEV
acids 1-24 PGWFNDVKINVRLNLQRASTT'FHERTATTTTRRTTTTSPT
signal peptide) TTRQMTITPAALPTTvvrTpDLTTGTPLQMTTIAVF17TA
NTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESETVLPSDS
WSSVESTSADTVLLTSKESKVWDLPSTSHVSMWKTSDS
VSSPQPGASDTA.VPEQNKTTKTGQMDGIPMSMKNEMPI
SQ
mouse Tim4 MSKGLLLLWLVTELWWLYLTPAASEDTIIGFLGQPVTLP A
binding CH Y LS W SQ SRN SMC W GKGSCPN SKC N
AELLRTDGTRIIS
domain (amino RKSTKYTLI.,GKVQFGEVSLTISNTNRGDSGVYCCRIEV.P
acids 1-22 GWFNDVKKNVRLELRRATTTKICPTITTRPTTTPYVITTT
signal peptide) PELLPTitv-mTTSVLPTTTPPQTLATTAFS'FAvrrcPS`11-PG
SFSQETTKGSAFTTESETI,PASNITSQRSMMTISTDIAVIR
PTGSNPGILPSTSQLTTQKTTLTTSESLQKTTKSHQINSRQ
.1' Modified IgG4 ESKYGPPCPPCP
hinge Wildtype RSKRSR1_,LIISDYMNMTPRRPGPTRKETYQPY.APPRDFA A 164 costimulatory YRS
signaling domain CD28 RSKRS RGGH S DY?vINM TPRRPGPTRKI 1 YQPY APPRD FA
A
costimulatory YRS
signaling domain with substitutions (positions reference to full length protein) mulatory ALYLLRRDQRLPPDAHK..PPGG(3 SF RTRIQEEQADAHSTL 166 costi signaling MCI
domain CD2 KRK KQRSRRNDEELETRAHR.VA.TEERGRKPHQIPASTPQ
costimulatory NPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPP 167 signaling APSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN
domain 4-1BB ....................... KRGRKKLLYEFKQPFMRPVQTTQEEDGCSCRFPEEEEG-G
________________ costimulatory CEL
signaling domain CD27 QRR.K YR SNK
_____________________________________ GESPVEP AEPCHYSCPREEEG STIPIQEDYR
16 ) costimulatory KPEPAC SP
signaling domain costimulatory signaling domain ________________ costimulatory MNPKFAES
signaling domain ICOS
TCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVT
costimulatory L
signaling domain costimulatory LRSGASVTEPVAEERGLMSQPLIVIETCHSVGAAYLESLPL "
signaling QDA SPA GGPS S PRDLPEPRV STEHTNNKIEK WIMKADTV
domain IV GTVKAELPEGRGLAGPAEPELEEELEADHTPH YPEQE-1.
EPPLGSC SDVMLS VEEEGKEDPLpT A ASGK
costimutatory LHGCQPVTQEDGKESRISVQERQ
signaling domain PD-1 C SRAAR.GT IGARRTGQ PLK EDP S A.VPVF S VDYGE LDFQ
costi mul atory WREKTPEPPVPCYPEQTEVATIVFPSGMGTSSP ARR GS A
signaling DGPRSAQPLRPEDGH C SW PL
domain costimulatory Q
signaling domain LIGHT MEE SVVRP SWVVDGQTDIPFTRLGRSHRRQ SC SVAR
costimul atory signaling domain costimulatory QVELNLQNP SLNHQGIDKIYDC QGLLPPPEK
signaling domain costi m ulatory KEDDGQEI A
signaling domain ___________________________ VYSDLNTQRPYYK
Tim4 LLIVIIIAPSLGFVLF ALFVAFL
transmembrane domain transmembrane domain transmembrane domain __________________________ transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane domain transmembrane 1)0 domain transmembrane domain LIGHT VGLGLLLLLMGAGLAVQGWFL
transmembrane domain transmembrane domain transmembrane domain Tim4 binding MSKEPLILWLMIEFWWLYLTPVTSETVVTEVLGIIRATTLP
domain-CD28 CLYS SW SI-LNSNSMCWGKDQCPYSGCKEALIRTDGMRV
hinge-CD28 TSRKSAK YRLQGT IPRGDVSLTILNPSESDSGVYCCRIE V
transmembrane PGVVFNDVKINVRINLQRASTTTEIRTATT.TTRRTTTTSPT
costimulatory NTCLSLTPSTLPEEA'FGLLTPEPSKEGP1LTAESETVLPSDS
signaling WSSVESTSADTVLLTSKESKVWDLPSTSHVSMWKTSDS
domain- VS SPQPGASDTAVPEQNKTTIC17GQMDGIPMSMKNEMPI
DAP12 SQIEVMYPPPYLDNEKSNGTHHVKGKHLCPSPLITGP,S'KPF
signaling W VL V V VGGVLACY SLINTVAFIIFWVIZSKRSRLDISDIM
domain, amino AMTPRIZPGPTRKHYQPYAPPRDFAA YRS'YFLGRINPRGRG
acids 1-24 are AAEAATICKORITETESPYQELQGQRSDVYSDLNTQRPYY
signal peptide K
(CTX140) Ti m4 binding MSKEPLILWLMIEFWWLYL'IPVTSETVVTEVLGIIRVTLP nr_ domain- CLYS SW SI-INSN SMCW GKDQCPYSGCKEALIRTDGMRV 17`"
modified Igth TSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEV
hinge-CD28 PGWFNDVKINVRLNLQR A STTMRTATTITRICITTTSPT
transmembrane TTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAVFTTA
costimulatory WSSVESTSADTVLLTSKESKVWDLPSTSHVSMWKTSDS
signaling VS SPQPGASDTAVPEQNKTTKTGQMDGIPMSMKNEMPI
domain- SQESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF
DAP12 WV R,S'KRS'RLLHSDYMNMTPRI?PGP7RKHYOPYAPPRDPAA
signaling YRSYFLG.RLVPRGRGAAEAATRKQ.RITETESPYQELQGQ
domain, amino RSDVYSDLNTQRPYYK
acids 1-24 are signal peptide (CTX143) Tim4 binding MSKEPLILWLM1EFWWLYLTPVTSETVVTEVLGHRVTLP
domain-CD28 CLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGMRV
transmembrane TSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEV
-CD28 PGWFINIDVKINVRLNLQRASTT'FHRTATTTTRRTTTTSPT
costimulatory TTRQMTTTPAALPTTVVTTPDLTTGTVLQMTTIAVFTTA
signaling NTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESETVLPSDS
domain- WSSVESTSADTVLLTSKE:SKVWDLPSTSHVSMWKTSDS
signaling SQFWVLVVVGGVLACYSLLVTVAFIEFWVRS'KR,SRUHSD
domai n, amino YAMMTPRRPGPTRKHYQPYAPPRIVA A YRS'YFLGRINPRG
acids 1:24 are RGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRP
signal peptide , YYK
(CTX137) 1 Tim4 binding MSKEPLILWLMIEFWWLYLTPVTSETVVTEVLGHRVTLP 198 domain ¨ Tim4 CLYSSWSHNSNSMCWGKDQCPYSGCKEAL1RTDGMRV
DAP12; ammo PGWFNDVKINVRLNLQRA STT1'1-1WrATTITRRTT: [1 SP
acids 1-24 are TTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAVFTTA
signal peptide NTCLSLTPSTLPEEATGLLTPEPSKEGPlurAESET'VLPSDS
(CTX256) WSSVESTSADTVULTSKESKVWDLPSTSHVSMWKTSDS
VSSPQPGAS:DTAVPEQNKTTKTGQIVEDGIPMSMKNEMP1 SQII,MIIAPSLGFVLFALEVAFLRSKRSRLLI-ISDYMNMIP
RRPGPTRKHYQPYAPPRDFAAYRSYFLGRLVPRGRGAA
EAAIRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK
In embodiments, a chimeric Tim receptor comprises a single chain chimeric protein, the single chain chimeric protein comprising: (a) an extracellular domain comprising a binding domain comprising: (i) a Timl IgV domain or Tim4 IgV
domain and (ii) a Timl mucin domain or Tim4 mucin domain; (b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) a transmembrane domain positioned between and connecting the extracellular domain and the intracellular signaling domain.
In particular embodiments, the binding domain comprises: (i) a Tim!
IgV domain and a Timl mucin domain; (ii) a Tim4 IgV domain and a Tim4 mucin domain; (iii) a Timl IgV domain and a Tim4 mucin domain; or (iv) a Tim4 IgV
domain and a Timl mucin domain. In certain embodiments, the extracellular domain of the chimeric Tim receptors described herein optionally includes an extracellular spacer domain positioned between and connecting the binding domain and transmembrane domain When expressed in a host cell, chimeric Tim receptors of the present disclosure can confer a phosphatidylserine-specific, cytotoxic phenotype to the modified host cell (e.g., the host cell becomes cytotoxic to a stressed, damaged, injured, apoptotic, or necrotic cell expressing phosphatidylserine on its surface). In certain embodiments, the chimeric Tim receptors induce apoptosis in targeted cells via release of granzymes, perforin, granulysin, or any combination thereof. In further embodiments, cells expressing a chimeric Tim receptor according to the present description exhibit an engulfment phenotype specific to phosphatidylserine presenting cells.
The intracellular signaling domain can include one or more effector (also referred to as "costimulatory signaling") domains that costimulate the modified host cell. Signaling by the intracellular signaling domain(s) is triggered by binding of the extracellular domain to phosphatidylserine. In certain embodiments, the intracellular signaling domain comprises a first intracellular signaling domain. In further embodiments, the intracellular signaling domain comprises a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a first intracellular signaling domain, a second intracellular signaling domain, and a third intracellular signaling domain.
Chimeric Tim receptors according to the present disclosure can be used in a variety of therapeutic methods where clearance of apoptotic, necrotic, damaged, or stressed cells is beneficial, while providing costimulation that enhances cellular immune response, reduces immune cell exhaustion, or both.
As described herein, a chimeric Tim receptor corn prises an extracellular domain comprising a Tim binding domain. The Tim binding domain confers specificity to phosphatidylserine (PtdSer), which is a phospholipid with a negatively charged head-group and a component of the cell membrane. In healthy cells, phosphatidylserine is preferentially found in the inner leaflet of the cell membrane. However, when cells are stressed, damaged or undergo apoptosis or necrosis, phosphatidylserine is exposed on the outer leaflet of the cell membrane. Thus, phosphatidylserine may be used as a marker to distinguish stressed, damaged, apoptotic, necrotic, pyroptotic, or oncotic cells. Binding of phosphatidylserine by the Tim binding domain may block the interaction between the phosphatidylserine and another molecule and, for example, interfere with, reduce or eliminate certain functions of the phosphatidylserine (e.g., signal transduction). In some embodiments, the binding of a phosphatidylserine may induce certain biological pathways or identify the phosphatidylserine molecule or a cell expressing phosphatidylserine for elimination.
A Tim binding domain suitable for use in a chimeric Tim receptor of the present disclosure may be any polypeptide or peptide derived from a Tim I
and/or Tim4 molecule that specifically binds phosphatidylserine. In embodiments, a Tim binding domain comprises an IgV domain from Timl or Tim4, and a mucin domain from Tim I
or Tim4. For example, a Tim binding domain may comprise a Timl IgV domain and a Tim I mucin domain. In another example, a Tim binding domain may comprise a Tim I
IgV domain and a Tim4 mucin domain. In another example, a Tim binding domain may comprise a Tim4 IgV domain and a Tim 1 mucin domain. In another example, a Tim binding domain may comprise a Tim4 IgV domain and a Tim4 mucin domain.
Phosphatidylserine binding is generally regulated by the I8\' domain.
The core phosphatidylserine binding domain is a four amino acid sequence in the 18V
domain (e.g., amino acids 95-98 of SEQ ID NO:34 or amino acids 92-95 of SEQ
NO:38). A Tim4 binding domain binds minimally to cells with low phosphatidylserine density. A 717im I. binding domain binds more strongly to a lower phosphatidylserine density, resulting in a lower threshold for response. A summary of Timl and Tim4 binding to phosphatidylserine is provided in Table 3. By combining Timi IgV
domain and Tim4 mucin domain, or Tim4 IgV domain and Timl rnucin domain, the binding affinity of the binding domain to phosphatidylserine can be modulated.
Further, such a combination in the Tim binding domain also provides a combination of the sensitivity to phosphatidylserine of Tim4 and the stability in protein expression of Tim 1.
Table 3. .
Low PtdSer 1 Moderate PtdSer High PtdSer i Cooperativity Tim-1 Moderate binding Strong binding Strong binding I
Tim-4 Minimal binding Moderate binding Strong binding I.
1 Yes Additionally, an RGD domain (e.g., amino acids 68-70 of SEQ ID
NO:34) in an IgV domain may regulate integrin binding as a co-receptor for engulfment.
In certain embodiments, the Tim. binding domain is derived from human Tim 1 and/or Tim4. An exemplary human Timl molecule is provided in Uniprot.
Ref.
Q96D42 (S:EQ ID NO:36). An exemplary human Timl binding domain comprises or consists of an amino acid sequence of SEQ ID NO:37 or SEQ ID NO:43. In certain embodiments, the Timl binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:37 or SEQ ID NO:43.
In certain embodiments, the Tim .1 binding domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:37 or SEQ ID NO:43.
An exemplary human Tim4 molecule is provided in Uniprot. Ref Q96H15 (S:EQ ID NO:1). An exemplary human Tim4 binding domain comprises or consists of an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:42. An exemplary mouse Tim4 binding domain comprises or consists of an amino acid sequence of SEQ
ID NO:24 or amino acids 23-279 of SEQ ID NO:24. In certain embodiments, the Tim4 binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:2 or SEQ ID NO:42, or SEQ ID NO:24 or amino acids 23-279 of SEQ ID NO:24. In certain embodiments, the Tim4 binding domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:42, or SEQ
ID NO:24 or amino acids 23-279 of SEQ ID NO:24.
In embodiments, the Tim binding domain comprises an IgV domain from Tim 1. An exemplary human Tim 1 18V domain is provided in SEQ ID NO:38.
In some embodiments, the Timl IgV domain is a modified Tim! IgV domain comprising a R66G substitution in SEQ ID NO:38. The 12.66G substitution (e.g., amino acids of SEQ ID NO:34) confers a RGD domain in Tim 1. IgV domain, which may regulate integrin binding as a co-receptor for engulfment. In particular embodiments, the modified Tim] IgV domain comprises or consists of the amino acid sequence of SEQ
ID NO:41. In some embodiments, this modified Timl domain may increase phagocytic activity while preserving Tirni sensitivity. In certain embodiments, the Tiinl IgV
domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94 4, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%
identity to SEQ ID NO:38, SEQ ED .NO:38 with a R66G substitution, or SEQ ID
NO:4.I. In certain embodiments, the Timl 1.8V comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an.
amino acid sequence of SEQ ID NO:38, SEQ ID NO:38 with a R66G substitution, or SEQ ID
NO:41.
In other embodiments, the Tim binding domain comprises an IgV
domain from Tim4. An exemplary human Tim4 :I8V domain is provided in SEQ ID
NO:34. In certain embodiments, the Tim4 18V domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to S:EQ ED NO:34. in certain embodiments, the Tim4 IgV domain comprises an amino acid sequence having at least about 1,2, 3,4, 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:34.
In embodiments, the Tim binding domain comprises a mucin domain from Timl. An exemplary human Timl mucin domain is provided in SEQ ID NO:39.
In certain embodiments, the Timl mucin domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A, 99%, 99.5%, or 100% identity to SEQ ID NO:39. In certain embodiments, the Timl mucin domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:39.
In other embodiments, the Tim binding domain comprises a mucin domain from Tim4. An exemplary human Tim4 mucin domain is provided in SEQ ID
NO:35. In certain embodiments, the Tim4 mucin domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:35. In certain embodiments, the Tim4 mucin domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, .19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of SEQ ID NO:35.
In some embodiments, the Tim binding domain comprises a Tim! IgV
domain and a Timl. mucin domain. In a particular embodiment, the Tim! IgV
domain comprises the amino acid sequence set forth in SEQ ID NO:38 and the Timl mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In another particular embodiment, the Timl IgV domain comprises the amino acid sequence set forth in SEQ ID NO:38 with a R66G substitution and the Timl mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In a further particular embodiment, the Tim! IgV domain comprises the amino acid sequence set forth in SEQ
ID NO:41 and the Timl mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In som.e embodiments, the Tim! IgV domain and Tim I mucin domain together comprise or consist of the amino acid sequence set forth in SEQ ID
NO:37 or SEQ ID NO:43.
In some embodiments, the Tim binding domain comprises a Tim4 IgV
domain and a Tim4 mucin domain. In a particular embodiment, the Tim4 IgV
domain comprises the amino acid sequence set forth in SEQ ID NO:34 and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the Tim4 IgV domain and Tim4 mucin domain together comprise or consist of the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:42.
In some embodiments, the Tim binding domain comprises a Timl IgV
domain and a Tim4 mucin domain. In a particular embodiment, the Timl IgV
domain comprises the amino acid sequence set forth in SEQ ID NO:38 and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In another particular embodiment, the Timl IgV domain comprises the amino acid sequence set forth in SEQ ID .NO:38 with a R66G substitution and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In a further particular embodiment, the Timl IgV domain comprises the amino acid sequence set forth in SEQ
113 .NO:41 and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ NO:35. In some embodiments, the Timl IgV domain further comprises the Timl signal sequence of SEQ ID NO:40.
In still further embodiments, the Tim binding domain comprises a Tim4 IgV domain and a Timl mucin domain. In a particular embodiment, the Tim4 IgV
domain comprises the amino acid sequence set forth in SEQ ID .N0:34 and the Timl mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In some embodiments, the Tim4 IgV domain further comprises the Tim4 signal sequence of SEQ ID NO:11.
In certain embodiments, the extracellular domain optionally comprises an extracellular, non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable proper cell/cell contact, binding, and activation. When included in a chimeric receptor as described herein, an extracellular spacer domain is generally located between the extracellular binding domain and the transmembranc domain of the chimeric Tim receptor. The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest et al Irnmunother.
28:203-11, 2005; PCT Publication No. WO 2014/031687). In certain embodiments, an extracellular spacer domain is an immunoglobulin hinge region (e.g., IgGl, IgG2, IgG3, IgG4, igA, 1gD). An immunoglobulin hinge region may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region.
An altered IgG4 hinge region is described in PCT Publication No. WO
2014/031687, which hinge region is incorporated herein by reference in its entirety. In a particular embodiment, an extracellular spacer domain comprises a modified IgG4 hinge region having an amino acid sequence of ESKYGPPCPPCP (SEQ ID NO:3). Other examples of hinge regions that may be used in the chimeric Tim receptors described herein include the hinge region from the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof.
In a particular embodiment, an extracellular spacer domain comprises a CD28 hinge region having an amino acid sequence of SEQ ID NO:32. In further embodiments, an extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from. a CH1 domain, a CH2 domain, a CH3 domain, or combinations thereof (see, e.g., PCT Publication W02014/031687, which spacers are incorporated herein by reference in their entirety). In yet further embodiments, an extracellular spacer domain may comprise a stalk region of a type H C-lectin (the extracellular domain located between the C-type lectin domain and the transmembrane domain). Type H C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.
In certain embodiments, an extracellular domain comprises an amino acid sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof. In certain embodiments, an extracellular domain is murine, human, or chimeric.
The intracellular signaling domain of a chimeric Tim receptor as described herein is an intracellular effector domain and is capable of transmitting functional signals to a cell in response to binding of the extracellular domain of the chimeric Tim receptor and phosphatidylserine. The signals transduced by the intracellular signaling domain promote effector function of the chimeric Tim receptor containing cell. Examples of effector function include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, expansion, engulfment of a target cell or particle expressing phosphatidylserine on its surface, antigen capture, antigen processing, antigen presentation, or any combination thereof.
An intracellular signaling domain comprises a primary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a primary intracellular signaling domain and a secondary intracellular signaling domain.
In some embodiments, an intracellular signaling domain comprises a primary intracellular signaling domain, a secondary intracellular signaling domain, and a tertiary intracellular signaling domain. The primary, secondary, and/or tertiary intracellular signaling domains may independently be any portion of a signaling molecule that retains sufficient signaling activity. In some embodiments, a full length signaling molecule or full length intracellular component of a signaling molecule is used. In some embodiments, a truncated portion of a signaling molecule or intracellular component of a signaling molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In some embodiments, a signaling domain is a variant of a whole or truncated portion of a signaling molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).
In some embodiments, the primary intracellular signaling domain comprises a Timl signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3 signaling domain, 4-1BB signaling domain, TLR2 signaling domain, or a TLR8 signaling domain.
In embodiments, the secondary intracellular signaling domain comprises a Timl signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAPS signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CDR', signaling domain, 4-1BB signaling domain, TLR2 signaling domain, or a lilts signaling domain.
In some embodiments, the tertiary intracellular signaling domain comprises a Timl signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP1.2 signaling domain, a CD3C signaling domain, 4-1BB signaling domain, TLR2 signaling domain or a ILR.8 signaling domain.
In some embodiments, the primary intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) containing signaling domain; the secondary intracellular signaling domain comprises a costimulatory signaling domain, Ti ml signaling domain or Tim4 signaling domain; and the tertiary intracellular signaling domain comprises a TLR signaling domain.
An ITAM containing signaling domain generally contains at least one (one, two, three, four, or more) 1TAM.s, which refer to a conserved motif of YXXL/1.-X6.8-YXXL/I. An ITAM containing signaling domain may initiate T cell activation signaling following antigen binding or ligand engagement. ITAM-signaling domains include, for example, intracellular signaling domains of CD3y, CUM, CD3c, CD34.;, CD5, CD22, CD79a, CD278 (ICOS), DAP12, FcRy, and CD66d. A costimulatory signaling domain, which, when activated in conjunction with a primary or classic (e.g., ITAM-driven) activation signal, promotes or enhances T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or combinations thereof. Costimulatory signaling domains for use in chimeric Tim receptors include, for example, CD27, CD28, CD4OL, GITR, NKG2C, CARD!, CD2, CD7, CD27, CD30, CD40, CD54 (1CAM), CD83, Cal 34 (0X-40), CD1.37 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HI/EM), CD273 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, SLP76, TRIM, ZAP70, or any combination thereof. In some embodiments, the costimulatory signaling domain comprises a 0X40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a/CD18), ICOS
(CD278), or 4-1BB (CD137) signaling domain. A TLR signaling domain may be a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TI.R8, or TLR9 signaling domain. In some embodiments, the TLR signaling domain is a TLR2 signaling domain or TLR8 signaling domain.
As used herein, the designation of primary, secondary, and tertiary intracellular signaling domains includes but is not limited to arrangements of the primary intracellular signaling domain at the N-terminus, secondary intracellular signaling domain in the middle, and tertiary intracellular signaling domain at the C-terminus of the intracellular portion of the chimeric Tim receptor. Thus, designation of the primary intracellular signaling domain does not limit the use of the selected intracellular signaling domain at the N-terminus of the intracellular portion of the chimeric Tim receptor. Designation of the secondary intracellular signaling domain does not limit the use of the selected intracellular signaling domain in the middle (or at the C-terminus for those chimeric Tim receptors only having two intracellular signaling domains) of the intracellular portion of the chimeric Tim receptor.
Designation of the tertiary intracellular signaling domain does not limit the use of the selected intracellular signaling domain at the C-terminus of the intracellular portion of the chimeric Tim receptor. Thus, different arrangements of the primary, secondary, and/or tertiary intracellular signaling domains within the intracellular portion of the chimeric Tim receptor are contemplated.
An exemplary Timl signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:44. An exemplary Tim4 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:45, SEQ ID NO:224, or SEQ ID
NO:225. An exemplary TRAF2 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:48. An exemplary TRAF6 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:46. An exemplary CD28 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:4 or 26.
An exemplary DAP12 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:9. An exemplary CD3C, signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:27 or 5. An exemplary 4-1BB signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:122. An exemplary TLR2 signaling domain comprises or consists of the amino acid sequence of SEQ
ID
NO:222. An exemplary TLR8 signaling domain comprises or consists of an amino acid sequence of SEQ ID NO:47.
In a particular embodiment, the Timl signaling domain comprises the amino acid sequence set forth in SEQ ID NO:44. In another particular embodiment, the Tim4 signaling domain comprises the amino acid sequence set forth in SEQ ID
NO:45 SEQ ID NO:224, or SEQ ID NO:225. In another particular embodiment, the TRAF2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:48 In another particular embodiment, the TRAF6 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:46. In another particular embodiment, the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:4.
In another particular embodiment, the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:26. In another particular embodiment, the signaling domain comprises the amino acid sequence set forth in SEQ ID NO:9.
In another particular embodiment, the CD3c, signaling domain comprises the amino acid sequence set forth in SEQ ID NO:27. In another particular embodiment, the CD3C
signaling domain comprises the amino acid sequence set forth in SEQ ID NO:5.
In another particular embodiment, the 4-1 BB signaling domain comprises the amino acid sequence of SEQ ID NO:122. In another particular embodiment, the TI,R2 signaling domain comprises of the amino acid sequence of SEQ ID NO:222. In another particular embodiment, or the TIM signaling domain comprises the amino acid sequence set forth in SEQ ID NO:47.
In embodiments, the primary, secondary, and/or tertiary signaling domain (if present) comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 995%, or 100% identity to any one of SEQ TD NOS:4, 5, 9, 26, 27, or 44-48. In certain embodiments, the primary, secondary, and/or tertiary signaling domains comprises an amino acid sequence haying at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to an amino acid sequence of any one of SEQ ID NOS:4, 5, 9, 26, 27, 44-48, 122, and 222. In embodiments, the primary signaling domain, secondary, and/or tertiary intracellular signaling domain arc the same. In some embodiments, two or three of the primary, secondary, and tertiary intracellular signaling domains are different.
In certain embodiments, an intracellular signaling domain comprises a Timi intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a Tim4 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD3C intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TRAF6 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TRAF2 intracellular signaling domain. In some embodiments, an.
intracellular signaling domain comprises a TLR2 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TLR8 intracellular signaling domain.
In certain embodiments, an intracellular signaling domain comprises a Timl primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In other embodiments, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In other embodiments, an intracellular signaling domain comprises a TLR8 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In other embodiments, an intracellular signaling domain comprises a Tut2 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In other embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain and a DAP12 secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a CD3C tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3C secondary intracellular signaling domain, and a TLR2 tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a ILR8 secondary intracellular signaling domain, and a CD3C, tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3C secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TLR2 primary intracellular signaling domain and a secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD3C primary intracellular signaling domain and a secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TLR8 primary intracellular signaling domain and a secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD3C, primary intracellular signaling domain and a TLR8 secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a TRAF6 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD3C primary intracellular signaling domain and a TR AF6 secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CO28 primary intracellular signaling domain, a -171.112 secondary intracellular signaling domain, and a cD3r, tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3C, secondary intracellular signaling domain, and a TLR2 tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a mRs secondary intracellular signaling domain, and a CD3C tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain. In some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a TLR2 secondary intracellular signaling domain. In some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a TLR8 secondary intracellular signaling domain. In some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a CD3C secondary intracellular signaling domain, in some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a TRAF6 secondary intracellular signaling domain. in some embodiments, an intercellular signaling domain comprises a primary intracellular signaling domain and a CD3C secondary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a CD3C tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a CD3C, secondary intracellular signaling domain, and a TLR2 tertiary intracellular signaling domain. in some embodiments, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a TLR8 secondary intracellular signaling domain, and a CD3C tertiary intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises a 1'im4 primary intracellular signaling domain, a CD3C secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain.
In a particular embodiment, an intracellular signaling domain comprises a Timl primary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:44 and a CDR', secondary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:27 or 5. In another particular embodiment, an intracellular signaling domain comprises a Tim4 primary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:45, SEQ ID NO:224, or SEQ ID
NO:225, and a CD3( secondary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:27 or 5. In a further particular embodiment, an intracellular signaling domain comprises a TLR8 primary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:47 and a CDR; secondary intracellular signaling domain comprising an amino acid sequence of SEQ ID
NO:27 or 5. In another particular embodiment, an intracellular signaling domain comprises a CD28 primary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:4 or 26 and a DAP12 secondary intracellular signaling domain comprising an amino acid sequence of SEQ ID NO:9. In some embodiments, an intracellular signaling domain comprises a combination of primary, secondary, and optionally tertiary intracellular signaling domain sequences as shown in Table 10.
Intracellular signaling domains may be derived from a mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
The transmembrane domain of a chimeric Tim receptor connects and is positioned between the extracellular domain and the intracellular signaling domain.
The transmembrane domain is a hydrophobic alpha helix that transverses the host cell membrane. The transmembrane domain may be directly fused to the binding domain or to the extracellular spacer domain if present. In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g., receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, or the like). In one embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, a chimetic Tim receptor may comprise a Tit/14 binding domain and a Tim4 transmembrane domain. In another example, a chimeric Tim receptor may comprise a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules; the transmembrane domain and the intracellular signaling domain are derived from different molecules; or the transmembrane domain, extracellular domain, and intracellular signaling domain are all derived from different molecules Examples of transmembrane domains that may be used in chimeric Tim receptors of the present disclosure include transmembrane domains from Timl.
Tim4, and CD28. An exemplary Timl transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:8. An exemplary Tim4 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:6 or 23. An exemplary CD28 transmembrane domain comprises or consists of an amino acid sequence of SEQ
ID NO:7. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID
NOS:6-8 or 23. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletion, additions, substitutions) to an amino acid sequence of any one of SEQ ID NOS:6-8 or 23.
Transmembrane domains may derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.
in certain embodiments, a chimeric Tim receptor comprises polynucleotide sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof In certain embodiments, a chimeric Tim receptor is murine, chimeric, human, or humanized.
It is understood that direct fusion of one domain to another domain of a chimeric Tim receptor described herein does not preclude the presence of intervening junction amino acids. Junction amino acids may be natural or non-natural (e.g., resulting from the construct design of a chimeric protein). For example, junction amino acids may result from restriction enzyme sites used for joining one domain to another domain or cloning polynucleotides encoding chimeric Tim receptors into vectors.
Exemplary Chimeric Ti in Receptors The component parts of a chimeric Tim receptor as disclosed herein can be selected and arranged in various combinations to provide a desired specificity and effector phenotype to a host cell.
Exemplary chimeric Tim receptors of the present disclosure are described in Table 4.
Table 4.
Construct IgV
M:ucin Transineinbrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain I Tim I Tim I Tim 1 TimI CD3c, --7 urn! Tim I Tirn1 'fim4 -- CD31-, _________________ 3 Tim! Tim I --- CD28 CD28 _________ 4 Tim I Tim I Tim I TRAF6 5 Tim I Tim I CD28 TRAF6 6 Tim 1 Timl Timl TRAF2 7 Tim! Tim! ____ CO28 TRAF2 8 Timl Timl Tim l TI..R8 CD3 c _________________ 9 Tim! ________ Tim! CD28 CD28 DAPI2 i 10 Tim! Tim I Tim I CD28 DAPI2 i Further exemplary chimeric Tim receptors are described in Table 5.
Table 5.
Construct IgN7 Mucin Transmembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 1' SEQ ID SEQ ID SEQ ID NO:8 SEQ ID SEQ ID
.NO:38 NO:39 NO:44 NO:5 7= SEQ ID SEQ ID SEQ ID 'NO:8 SEQ ID SEQ ID
----------------------- NO:38 NO:39 NO:45 NO:5 -3" SEQ ID SEQ ID SEQ ID 'NO:7 SEQ ID
NO:38 NO:39 .N0:4 4' SEQ ID SEQ :ID SEQ
ID NO:8 SEQ ID
_______________________ .N0:38 NO:39 __________________ NO:46 5' SEQ ID SEQ
ID SEQ ID NO:7 SEQ ID
----------------------- NO:38 NO:39 NO:46 ...
6" SEQ ID SEQ ID SEQ ID 'NO:8 SEQ ID
1 NO:38 NO:39 NO:48 Construct IgV
Mucin Transrnernbrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 7' SEQ ID SEQ
ID SEQ ID NO:7 SEQ ID
NO:38 NO:39 NO:48 8' SEQ ID SEQ
ID SEQ II) NO:8 SEQ ID SEQ ID
NO:38 NO:39 NO:47 NO:5 9' SEQ ID SEQ
ID SEQ ID NO:7 SEQ ID SEQ ID
NO:38 NO:39 NO:4 NO:9 10' SEQ ID SEQ
ID SEQ ID NO:8 SEQ ID SEQ 1D
NO:38 NO:39 NO:4 NO:9 In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 4. In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 5.
In some embodiments, a chimeric Tim receptor of Construct 1 or Construct 1" comprises amino acids 21-456 of SEQ ID NO:49. In some embodiments, a chimeric Tim receptor of Construct 1 or Construct l' comprises an amino acid sequence of SEQ ID .NO:49.
In some embodiments, a chimeric Tim receptor of Construct 2 or Construct 2' comprises amino acids 21-471 of SEQ ID NO:50. In some embodiments, a chimeric Tim receptor of Construct 2 or Construct 2 comprises an amino acid sequence of SEQ ID NO:50.
In some embodiments, a chimeric Tim receptor of Construct 3 or Construct 3' comprises amino acids 21-363 of SEQ ID NO:51. In some embodiments, a chimeric Tim receptor of Construct 3 or Construct 3' comprises an amino acid sequence of SEQ .N0:51.
In some embodiments, a chimeric Tim receptor of Construct 4 or Construct 4' comprises amino acids 21-590 of SEQ ID NO:52. In some embodiments, a chimeric Tim receptor of Construct 4 or Construct 4' comprises an amino acid sequence of SEQ ID:NO:52.
In some embodiments, a chimeric Tim receptor of Construct 5 or Construct 5' comprises amino acids 21-596 of SEQ ID NO:53. In some embodiments, a chimeric Tim receptor of Construct 5 or Construct 5' comprises an amino acid sequence of SEQ ID NO:53.
In some embodiments, a chimeric Tim receptor of Construct 6 or Construct 6' comprises amino acids 21-619 of SEQ ID NO:54. In some embodiments, a chimeric Tim receptor of Construct 6 or Construct 6' comprises an amino acid sequence of SEQ ID NO:54.
In some embodiments, a chimeric Tim receptor of Construct 7 or Construct 7' comprises amino acids 21-625 of SEQ ID NO:55. In some embodiments, a chimeric Tim receptor of Construct 7 or Construct 7' comprises an amino acid sequence of SEQ ID NO:55.
In some embodiments, a chimeric Tim receptor of Construct 8 or Construct 8 comprises amino acids 21-621 of SEQ ID NO:56. In some embodiments, a chimeric Tim receptor of Construct 8 or Construct 8' comprises an amino acid sequence of SEQ B3 NO:56.
In some embodiments, a chimeric Tim receptor of Construct 9 or Construct 9' comprises amino acids 21-415 of SEQ NO:57. In some embodiments, a chimeric Tim receptor of Construct 9 or Construct 9' comprises an amino acid sequence of SEQ ID NO:57.
In some embodiments, a chimeric Tim receptor of Construct 10 or Construct 10' comprises amino acids 21-409 of SEQ ID NO:58. In some embodiments, a chimeric Tim receptor of Construct 10 or Construct 10' comprises an amino acid sequence of SEQ 113 NO:58.
Further exemplary chimeric Tim receptors of the present disclosure are described in Table 6.
Table 6.
Construct IgV Mucin Transmembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 11 Tim4 Tim4 Tim4 Tim4 CD3cõ
12 Tim4 1'irn4 Tim4 Tim 1 CD3c
13 Tim4 Tim4 CD28 CD28 Construct IgV Mucin Transmembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 13A Tim4 Tim4 CD28 CD28 CD3?.;
14 Tim4 Tim4 Tim4 TRAF6
15 Tim4 Tim4 CD28 TRAF6
16 Tim4 Tim4 Tim4 TRAF2
17 Tim4 Tim4 CD28 TRAF2
18 Tim4 Tim4 Tim 1 TLR8 CD3C, Further exemplary chimeric Tim receptors are described in Table 7.
Table 7.
Construct IgV Mucin Transmembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 11' SEQ SEQ
ID SEQ ID NO:6 SEQ ID SEQ ID
NO:34 NO:35 NO:45 NO:5 12' SEQ
ID SEQ ID SEQ ID NO:6 SEQ ID SEQ ID
_____________________________ NO:34 __ NO:35 NO:44 NO:5 13' SEQ ID SEQ ID
SEQ ID NO:7 SEQ ID
NO:34 NO:35 NO:4 14' SEQ SEQ ID
SEQ ID NO:6 SEQ ID
NO:34 NO:35 NO:46 15' SEQ ID SEQ ID SEQ ID NO:7 SEQ ID
NO:34 NO:35 N0:46 16' SEQ SEQ ID
SEQ ID NO:6 SEQ ID
NO:34 NO:35 NO:48 17' SEQ ID SEQ ID
SEQ ID NO:7 SEQ ID
NO:34 N0:35 NO:48 18' SEQ ID SEQ
ID SEQ ID NO:8 SEQ 11) SEQ ID
NO:34 NO:35 NO:47 NO:5 In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 6. In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 7.
In some embodiments, a chimeric Tim receptor of Construct 11 or Construct 11' comprises amino acids 25-490 of SEQ ID NO:59. In some embodiments, a chimeric Tim receptor of Construct 11 or Construct 11' comprises an amino acid sequence of SEQ ID NO:59.
In some embodiments, a chimeric Tim receptor of Construct 12 or Construct 12' comprises amino acids 25-495 of SEQ ID NO:60. In some embodiments, a chimeric Tim receptor of Construct 12 or Construct 12' comprises an amino acid sequence of SEQ ID NO:60.
In some embodiments, a chimeric Tim receptor of Construct 13 or Construct 13' comprises amino acids 25-382 of SEQ ID NO:61. In some embodiments, a chimeric Tim receptor of Construct 13 or Construct 13' comprises an amino acid sequence of SEQ ID NO:61.
In some embodiments, a chimeric Tim receptor of Construct 13A (TiM4 binding domain-CD28 TM ¨ CD28 costim- CD3t,' (amino acids 1-24 signal peptide)) comprises amino acids 25-383 of SEQ ID NO:71. In some embodiments, a chimeric Tim receptor of Construct 13A comprises an amino acid sequence of SEQ ID NO:
71.
In some embodiments, a chimeric Tim receptor of Construct 14 or Construct 14' comprises amino acids 25-609 of SEQ ID NO:62. In some embodiments, a chimeric Tim receptor of Construct 14 or Construct 14' comprises an amino acid sequence of SEQ ID NO:62.
In some embodiments, a chimeric Tim receptor of Construct 15 or Construct 15' comprises amino acids 25-615 of SEQ ID NO:63. In some embodiments, a chimeric Tim receptor of Construct 15 or Construct 15' comprises an amino acid sequence of SEQ ID NO:63.
In some embodiments, a chimeric Tim receptor of Construct 16 or Construct .16' comprises amino acids 25-638 of SEQ ID NO:64. In some embodiments, a chimeric Tim receptor of Construct 16 or Construct 16' comprises an amino acid sequence of SEQ ID NO:64.
In some embodiments, a chimeric Tim receptor of Construct 17 or Construct 17' comprises amino acids 25-644 of SEQ ID NO:65. In some embodiments, a chimeric Tim receptor of Construct 17 or Construct 17' comprises an amino acid sequence of SEQ ID NO:65.
In some embodiments, a chimeric Tim receptor of Construct 18 or Construct 18' comprises amino acids 25-640 of SEQ ID NO:66 in some embodiments, a chimeric Tim receptor of Construct 18 or Construct 18' comprises an amino acid sequence of SEQ ID NO:66.
Further exemplary chimeric Tim receptors of the present disclosure are described in Table 8.
Table 8.
Construct IgV
Mucin Transrnembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain
Table 7.
Construct IgV Mucin Transmembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 11' SEQ SEQ
ID SEQ ID NO:6 SEQ ID SEQ ID
NO:34 NO:35 NO:45 NO:5 12' SEQ
ID SEQ ID SEQ ID NO:6 SEQ ID SEQ ID
_____________________________ NO:34 __ NO:35 NO:44 NO:5 13' SEQ ID SEQ ID
SEQ ID NO:7 SEQ ID
NO:34 NO:35 NO:4 14' SEQ SEQ ID
SEQ ID NO:6 SEQ ID
NO:34 NO:35 NO:46 15' SEQ ID SEQ ID SEQ ID NO:7 SEQ ID
NO:34 NO:35 N0:46 16' SEQ SEQ ID
SEQ ID NO:6 SEQ ID
NO:34 NO:35 NO:48 17' SEQ ID SEQ ID
SEQ ID NO:7 SEQ ID
NO:34 N0:35 NO:48 18' SEQ ID SEQ
ID SEQ ID NO:8 SEQ 11) SEQ ID
NO:34 NO:35 NO:47 NO:5 In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 6. In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 7.
In some embodiments, a chimeric Tim receptor of Construct 11 or Construct 11' comprises amino acids 25-490 of SEQ ID NO:59. In some embodiments, a chimeric Tim receptor of Construct 11 or Construct 11' comprises an amino acid sequence of SEQ ID NO:59.
In some embodiments, a chimeric Tim receptor of Construct 12 or Construct 12' comprises amino acids 25-495 of SEQ ID NO:60. In some embodiments, a chimeric Tim receptor of Construct 12 or Construct 12' comprises an amino acid sequence of SEQ ID NO:60.
In some embodiments, a chimeric Tim receptor of Construct 13 or Construct 13' comprises amino acids 25-382 of SEQ ID NO:61. In some embodiments, a chimeric Tim receptor of Construct 13 or Construct 13' comprises an amino acid sequence of SEQ ID NO:61.
In some embodiments, a chimeric Tim receptor of Construct 13A (TiM4 binding domain-CD28 TM ¨ CD28 costim- CD3t,' (amino acids 1-24 signal peptide)) comprises amino acids 25-383 of SEQ ID NO:71. In some embodiments, a chimeric Tim receptor of Construct 13A comprises an amino acid sequence of SEQ ID NO:
71.
In some embodiments, a chimeric Tim receptor of Construct 14 or Construct 14' comprises amino acids 25-609 of SEQ ID NO:62. In some embodiments, a chimeric Tim receptor of Construct 14 or Construct 14' comprises an amino acid sequence of SEQ ID NO:62.
In some embodiments, a chimeric Tim receptor of Construct 15 or Construct 15' comprises amino acids 25-615 of SEQ ID NO:63. In some embodiments, a chimeric Tim receptor of Construct 15 or Construct 15' comprises an amino acid sequence of SEQ ID NO:63.
In some embodiments, a chimeric Tim receptor of Construct 16 or Construct .16' comprises amino acids 25-638 of SEQ ID NO:64. In some embodiments, a chimeric Tim receptor of Construct 16 or Construct 16' comprises an amino acid sequence of SEQ ID NO:64.
In some embodiments, a chimeric Tim receptor of Construct 17 or Construct 17' comprises amino acids 25-644 of SEQ ID NO:65. In some embodiments, a chimeric Tim receptor of Construct 17 or Construct 17' comprises an amino acid sequence of SEQ ID NO:65.
In some embodiments, a chimeric Tim receptor of Construct 18 or Construct 18' comprises amino acids 25-640 of SEQ ID NO:66 in some embodiments, a chimeric Tim receptor of Construct 18 or Construct 18' comprises an amino acid sequence of SEQ ID NO:66.
Further exemplary chimeric Tim receptors of the present disclosure are described in Table 8.
Table 8.
Construct IgV
Mucin Transrnembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain
19 Tim4 Tim 'Hint rLRs CD3C
20 'Fim4 Timi Timl CD28 DAP12
21 Tim4 Timl CD28 CD28 DAP1.2 Further exemplary chimeric Tim receptors are described in Table 9.
Table 9.
Construct loV
Mucin Transmembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 19 SEQ ID SEQ ID i SEQ ID NO:8 SEQ SEQ
ID
NO:34 NO:39 g NO:47 NO:5 20 SEQ ID SEQ ID SEQ ID NO:8 SEQ ID SEQ
ID
NO:34 NO:39 NO:4 .N0:9 21 S:EQ 1D SEQ ID SEQ ID NO:7 SEQ ID SEQ
ID
NO:34 NO:39 NO:4 NO:9 in some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 8. In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 9.
In some embodiments, a chimeric Tim receptor of Construct 19 or Construct 19' comprises amino acids 25-628 of SEQ ID NO:67. In some embodiments, a chimeric Tim receptor of Construct 19 or Construct 19' comprises an amino acid sequence of SEQ ID NO:67.
In some embodiments, a chimeric Tim receptor of Construct 20 or Construct 20' comprises amino acids 25-416 of SEQ ID NO:68. In some embodiments, a chimeric Tim receptor of Construct 20 or Construct 20' comprises an amino acid sequence of SEQ ID .N0:68.
In some embodiments, a chimeric Tim receptor of Construct 21 or Construct 21 comprises amino acids 25-422 of SEQ ID NO:69. In some embodiments, a chimeric Tim receptor of Construct 21 or Construct 21' comprises an amino acid sequence of SEQ ID NO:69.
Further exemplary chimeric 'Tim receptors are described in Table 10. In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 10. In some embodiments, a chimeric Tim receptor of the present disclosure does not include a chimeric Tim receptor having the amino acid sequence of SEQ ID NO:227, 238, 249, 72, or 73. In some embodiments, a chimeric Tim receptor of the present disclosure does not include a chimeric Tim receptor having the combination of components as described for constructs SEQ ID NO:227, 238, 249, 72, or 73.
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:127 or the amino acid sequence of S:EQ ID NO:127 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO: 128 or the amino acid sequence of SEQ ED NO.128 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID
NO:129 or the amino acid sequence of SEQ ID NO:129 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:130 or the amino acid sequence of SEQ ID
NO: .130 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:131 or the amino acid sequence of SEQ ID NO:131 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:132 or the amino acid sequence of SEQ ID NO:132 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:133 or the amino acid sequence of SEQ ID NO:133 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:134 or the amino acid sequence of SEQ ID NO:134 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:135 or the amino acid sequence of SEQ ID NO:135 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:136 or the amino acid sequence of SEQ ID NO:136 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:137 or the amino acid sequence of SEQ ID NO:137 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:138 or the amino acid sequence of SEQ ID NO:138 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:139 or the amino acid sequence of SEQ ID NO:139 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:140 or the amino acid sequence of SEQ ID NO:140 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:141 or the amino acid sequence of SEQ ID NO:141 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:142 or the amino acid sequence of SEQ ID NO:142 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:143 or the amino acid sequence of SEQ ID NO: 143 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:144 or the amino acid sequence of SEQ ID NO:144 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:145 or the amino acid sequence of SEQ ID NO:145 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:146 or the amino acid sequence of SEQ ID NO: 146 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:147 or the amino acid sequence of SEQ ID NO:147 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ NO:148 or the amino acid sequence of SEQ ID NO:148 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:149 or the amino acid sequence of SEQ ID NO:149 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:150 or the amino acid sequence of SEQ ID NO:150 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:151 or the amino acid sequence of SEQ ID NO:151 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:152 or the amino acid sequence of SEQ ID NO:152 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:153 or the amino acid sequence of SEQ 1D NO:153 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:154 or the amino acid sequence of SEQ ID NO:154 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:155 or the amino acid sequence of SEQ ID NO:155 absent the signal sequence (amino acids 1-24).
Le ..
'0 ie ..
A
be Table 10.
c be Construct Construct Description Signal Extracellular Transmembrane Intracellular -- Intracellular --Intracellular -- re .....
o Number Peptide Domain Domain Domain #1 Domain #2 Domain N3 ce) ON
be pCTX1161 hTIM4-TIM4-CT)28tm- 117144 TIM4 CD28 C7D28 CD3z Ct CA
[SEQ ID CD281cd-CD3z1CD [SEQ ID [SEQ ID [SEQ ID [SEQ
ID NO:41 [SEQ ID NO:27]
N.0:227] NO:218] NO:219] NO:220]
.
.
pCD.C1.162 hTIM4-TIM4-CD28tm- IITIM4 TIM4 CD28 [SEQ ID CD28icd-TLR2ICD- [SEQ ID [SEQ ID [SEQ ID CD28 [SEQ ID CD3z NO:228] CD3zICD NO:218]
NO:219] NO:2201 [SEQ ID NO:4] NO:222] [SEQ ID NO:27]
pCIX1163 hTIM4-TIM4-CD28tm- hunt TIM4 . CD28 [SEQ ID CD28icd-CD3zICD- [SEQ ID [SEQ ID [SEQ ID CD28 CD3z [SEQ ID
NO:229] TLR2ICD NO:218]
NO:219] NO:2201 [SEQ ID NO:41 [SEQ ID NO:27] NO:2221 pCIX1164 hTIN14-TIM4-CD28tm- hum4 TIM4 . CD28 [SEQ ID CD28icd-TLR8ICD- [SEQ ID [SEQ ID [SEQ ID CD28 TLR8 CDly NO:230] CD3zICD NO:2181 NO:219] NO:2201 [SEQ ID NO:41 [SEQ ID NO:47] [SEQ ID NO:271 --) 00 pCTX1165 hTLM4-TLM4-CD28tm- IIIIM4 TLM4 CD28 [SEQ ID CD28ial-CD3zTCD- [SEQ ID [SEQ 1D [SEQ ED CD28 CD3z TLR8 NO:231] TI.R8ICD NO:218]
NO:219] NO:2201 [SEQ ID NO:4] [SEQ ID NO:27] [SEQ ID NO:471 pCTX1166 urnvu TIM4 CD28 TLR2 [SEQ ID IITIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID CD3z NO:232] -11.1121CD-CD3z1CD
NO:218] NO:219] NO:220] NO:222] [SEQ ID NO:27]
pCTX1167 bTIM4 TIM4 CD28 [SEQ ID liTIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID CD3z [SEQ ID
NO:233] CD3zICD-TLR21CD
NO:218] NO:219] NO:220] [SEQ ID
NO:27] NO:222] oci pCTX1168 IMM4 TIM4 CD28 n ; 3 [SEQ ID liTIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID TLR8 CD3z NO:234] TLR81CD-CD3zICD
NO:218] NO:219] NO:220] [SEQ ID
NO:47] [SEQ ID NO:27] b4 o b.) pCTX1169 Mil& MN CD28 O' [SEQ ID 1iTIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID CD3z TLR8 A
o NO:235] CD3zICD-TLR81CD
NO:218] NO:219] NO:220] [SEQ ID
NO:27] [SEQ ID NO:47] o I-.
Le ..-'0 ..-A
pCTX1170 hum4 TIM4 CD28 be [SEQ ID IfTIN,14-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID TRAF6 CD3z o be re NO:236] TRAF6ICD-CD3zICD
NO:218] NO:219] NO:220] [SEQ ID
NO:46] [SEQ ID NO:27] -...
a .
ce) pCTX1171 IfI1M4 TThil4 - CD28 ON
b.) CIO
[SEQ ID hTIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ ID
CD3z TRAF6 CA
NO:237] CD3z1CD-TRAF6ICD
NO:218] NO:219] NO:2201 [SEQ ID NO:27] [SEQ ID NO:46]
pCTX1172 hTIM4 TIM4 TIM4 [SEQ ID hTIM4-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ ID
CD28 CD3z NO:238] CD28ial-CD3zICD
NO:218] NO:219] NO:221] [SEQ ID NO:41 [SEQ ID NO:27]
pCTX1173 IITIM4-TIM4-TIM4tm- hTIM4 TIM4 TIM4 [SEQ ID CD28ial-TLR2ICD- [SEQ ID [SEQ ID [SEQ ID CD28 [SEQ ID CD3z NO:239] CD3zICD NO:218]
NO:219] NO:2211 [SEQ ID NO:41 NO:222] [SEQ ID NO:27]
pCTX1174 hTIM4-TIM4-TIM4tin- hTIM4 TIM4 TIM4 [SEQ ID CD28ied-CD3zICD- [SEQ ID [SEQ ID [SEQ ID CD28 CD3z [SEQ ID
NO:2401 Tut21c0 NO:2181 1 NO:2191 NO:2211 1SEQ ID NO:41 1SEQ ID NO:271 NO:2221 -..) pCTX1175 hTIM4-TIM4-TIM4un- hTIM4 TIM4 TIM4 kr) [SEQ ID CD28ic4-ILR8ICD- [SEQ ID [SEQ ID [SEQ ID CO28 TLR8 CD3z NO:241] CD3zICD NO:218]
NO:219] NO:221] [SEQ ID NO:41 [SEQ ID NO:47] [SEQ ID NO:27]
pCTX1176 hTIM4-TIM4-TIM4lin- hTIM4 TIM4 TIM4 [SEQ ID CD28icd-CD3zICD- [SEQ ID [SEQ ID [SEQ ID CD28 CD3z TLR8 NO:242] TLR8ICD NO:218]
NO:219] NO:221] [SEQ ID NO:4] [SEQ ID NO:27] [SEQ ID NO:471 ......
pCTX1177 hTEM4 TIM4 TIM4 TLR2 [SEQ ID hTD/14-TIM4-TIM4lm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID CD3z NO:243] TLR2ICD-CD3zICD
NO:218] NO:219] NO:221] NO:222] [SEQ ID NO:27]
pCTX1178 hTIM4 TIM4 TIM4 TLR2 oci n [SEQ ID hTIM4-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ ID
CD3z [SEQ ID
NO:244] CD3zICD-TLR2ICD
NO:218] NO:219] NO:221] [SEQ ID
NO:27] NO:222] ri2 I
be pCTXI179 hTIM4 TIM4 TIM4 =
b.) [SEQ ID hTIN14-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ
ID TLR8 CD3z =..
O' N.0:245] 1 ILR8TCD-C)3zICD NO:218] NO:219] NO:2211 [SEQ
ID NO:47] [SEQ ID NO:27] A
A
I.+
Le .-'0 .-A
pCTX1180 num4 TIM4 TIM4 be [SEQ ID hTLM4-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ ID
CD3z TLR8 o be re NO:246] CD3zICD-TLR8ICD
NO:2181 NO:2191 NO:221] [SEQ ID
NO:27] [SEQ ID NO:47] .....
a pCTX1181 IiIIM4 TLM4 - TIM4 ON
b.) CIO
[SEQ ID hTIM4-TIM4-TIM40n- [SEQ ID [SEQ ID [SEQ ID
TRAF6 CD3z CA
NO:247] TRARICD-CD3z1CD
NO:218] NO:219] NO:221] [SEQ ID NO:461 [SEQ ID NO:27]
pCTX1182 hTIM4 TIM4 TIM4 [SEQ ID hTIM4-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ ID
CD3z TRAF6 NO:248] CD3zICD-TRAF6ICD
NO:218] NO:219] NO:221) [SEQ ID NO:271 [SEQ ID NO:46]
pCTX1183 hTIM4 TIM4 TIM4 1IM4 [SEQ ID liTIM4-TIM4-TINI4hn- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID
NO:249] TiM4ICD NO:218]
NO:219] NO:221] NO:2251 pCTX1184 hTIM4 TIM4 TIN% 1IM4 [SEQ ID linv14-TIM4-TINI4lin- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID TLR2 CD3/
NO:2501 TIM4ICD-TLR2-CD3z NO:2181 1 NO:2191 NO:2211 NO:2241 1SEQ ID NO:871 ISE) ID NO:271 oo pCTX1185 hTIM4 TIM4 TIM4 1IM4 o [SEQ ID INIM4-TIM4-TINI4tm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID CD3z [SEQ ID
NO:251] TIM4ICD-CD3z-TLR2 NO:218] NO:219] NO:221] NO:224] [SEQ ID NO:27] NO:222]
pCTX1186 hTIM4 TIM4 TIM4 TIM4 [SEQ ID hTIN14-TIM4-TIM4lm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID TLR8 CD3z NO:252] TIM4ICD-TLR8-CD3z NO:218] NO:219] NO:221] NO:224] [SEQ ID NO:47] [SEQ ID NO:27]
pCTX1187 hTEM4 TIM4 TIM4 TIM4 [SEQ ID hTIN14-TIN14-TIM4lm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID CD3z TLR8 NO:253] TIM4ICD-CD3z.-TLR8 NO:218] NO:219] NO:221] NO:224] [SEQ ID NO:27] [SEQ ID NO:4'7]
pCTX1189 hTIM4 TIM4 TIM4 TLR2 oci n [SEQ ID IITIM4-TIM4-TIM40n- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID
NO:72] TLR2ICD NO:218]
NO:219] NO:221] NO:222] ri2 I
be pCTX1190 hTIM4 TIM4 T1M4 b.) [SEQ ID hT1M4-TIM4-TIM4tin- [SEQ ID [SEQ ID [SEQ
ID TLR8 =..
O' NO:73] 1 ILR8TCD NO:218]
NO:219] NO:2211 [SEQ ID NO:47] A
A
I.+
Polynucleotides, Vectors, and Host Cells In certain aspects, the present disclosure provides nucleic acid molecules that encode any one or more of the chimeric Tim receptors described herein. A
nucleic acid may refer to a single- or double-stranded DNA, cDNA, or RNA, and may include a positive and a negative strand of the nucleic acid which complement one another, including antisense DNA, cDNA, and RNA. A nucleic acid may be naturally occurring or synthetic forms of DNA or RNA.. The nucleic acid sequences encodi rig a desired chimeric Tim receptor can be obtained or produced using recombinant methods known in the art using standard techniques, such as by screening libraries from cells expressing the desired sequence or a portion thereof, by deriving the sequence from a vector known to include the same, or by isolating the sequence or a portion thereof directly from cells or tissues containing the same as described in, for example, Sambrook etal.
(1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology, 2003). Alternatively, the sequence of interest can be produced synthetically, rather than being cloned.
Polynucleotides encoding the chimeric Tim receptor compositions provided herein may be derived from any animal, such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, or a combination thereof.
In certain embodiments, a polynucleotide encoding the chimeric Tim receptor is from the same animal species as the host cell into which the polynucleotide is inserted.
The polynucleotides encoding chimeric Tim receptors of the present disclosure may be operatively linked to expression control sequences.
Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak consensus sequences);
sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
In certain embodiments, a polynucleotide encoding a chimeric Tim receptor comprises a sequence encoding a signal peptide (also referred to as leader peptide or signal sequence) at the 5'-end for targeting of the precursor protein to the secretory pathway. The signal peptide is optionally cleaved from the N-terminus of the extracellular domain during cellular processing and localization of the chimeric Tim receptor to the host cell membrane. A polypeptide from which a signal peptide sequence has been cleaved or removed may also be called a mature polypeptide.
Examples of signal peptides that may be used in the chimeric Tim receptors of the present disclosure include signal peptides derived from endogenous secreted proteins, including, e.g., GM-CSF (amino acid sequence of SEQ ID NO:10), Timl (amino acid sequence of SEQ ID NO:40), or Tim4 (amino acid sequence of SEQ ID NO:11 or 25).
In certain embodiments, a polynucleotide sequence encodes a mature chimeric Tim receptor polypeptide, or a polypeptide sequence comprises a mature chimeric Tim receptor polypeptide. It is understood by persons of skill in the art that for sequences disclosed herein that include a signal peptide sequence, the signal peptide sequence may be replaced with another signal peptide that is capable of trafficking the encoded protein to the extracellular membrane.
In certain embodiments, a chimeric Tim receptor encoding polynucleotide of the present disclosure is codon optimized for efficient expression in a target host cell comprising the polynucleotide (see, e.g, Scholten et al., (Fin. Innnunol.
119:135-145 (2006)). As used herein, a "codon optimized" polynucleotide comprises a heterologous polynucleotide having codons modified with silent mutations corresponding to the abundances of tRNA in a host cell of interest.
A single polynucleotide molecule may encode one, two, or more chimeric Tim receptors according to any of the embodiments disclosed herein. A
polynucleotide encoding more than one transgene may comprise a sequence (e.g., IRES, viral 2A peptide) disposed between each gene for multicistronic expression.
Polynucleotides encoding at least two transgenes (e.g., a chimeric Tim receptor and CAR) provided in the present disclosure may be used to compose tandem expression cassettes. A tandem expression cassette refers to a component of a vector nucleic acid comprising at least two transgenes under the control of, or operatively linked to, the same set of regulatory sequences for tandem or co-expression of the at least two transgenes. Regulatory sequences that may be used in tandem expression cassettes of the present disclosure include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak consensus sequences);
sequences that enhance protein stability; sequences that enhance protein secretion, or any combination thereof.
In one aspect, the present disclosure provides a tandem expression cassette comprising a polynucleotide encoding a chimeric Tim receptor of the present disclosure and a polynucleotide encoding a cellular immunotherapy agent (e.g., a CAR, TCR, etc.).
In certain embodiments, a tandem expression cassette can be constructed to optimize spatial and temporal control. For example, a tandem expression cassette can include promoter elements to optimize spatial and temporal control. In some embodiments, a tandem expression cassette includes tissue specific promoters or enhancers that enable specific induction of a tandem expression cassette to an organ, a cell type (e.g., immune cell), or a pathologic rnicroenvironment, such as a tumor or infected tissue. An "enhancer" is an additional promoter element that can function either cooperatively or independently to activate transcription. In certain embodiments, a tandem expression cassette includes a constitutive promoter. An exemplary constitutive promoter for use in tandem expression cassettes of the present disclosure is an EF-la promoter. In certain embodiments, a tandem expression cassette includes an inducible promoter. In certain embodiments, a tandem expression cassette includes a tissue specific promoter.
The at least two transgenes contained within the tandem expression cassettes may be in any order. For example, a tandem expression cassette comprising a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a CAR
may be arranged from 5' to 3': chimeric Tim receptor-CAR, or CAR- chimeric Tim receptor.
In certain embodiments, receptors that comprise two or more polypeptide chains that associate to form a multimer or complex may be encoded by two or more polynucleotide molecules within a tandem expression construct.
Exemplary multimeric receptors contemplated for expression in tandem expression constructs of the present disclosure include multichain CARs, TCRs, TCR-CARs, and TRuCTm constructs. Accordingly, exemplary tandem expression cassette embodiments encoding a chimeric 'rim receptor and a TCR may comprise a polynucleotide encoding a chimeric Tim receptor, a polynucleotide encoding a TCRa chain polypeptide, and a polynucleotide encoding a TCRI3 chain polypeptide.
In certain embodiments, tandem expression cassettes of the present disclosure may comprise an internal ribosome entry site (IRES) or peptide cleavage site such as a furin cleavage site or viral 2A peptide, disposed between each polynucleotide contained within the tandem expression cassette to allow for co-expression of multiple proteins from a single mRNA. For example, an :ERES, furin cleavage site, or viral 2A
peptide may be disposed between a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a CAR within a tandem expression cassette. In another example, an IRES, furin cleavage site, or viral 2A peptide may be disposed between each of a polynucleotide encoding a chimeric Tim receptor, a polynucleotide encoding a TCRa chain polypeptide, and a polynucleotide encoding a TCRI3 chain polypeptide.
In certain embodiments, a viral 2A peptide is a porcine teschovirus-1 (P2A), ihosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variant thereof. An exemplary T2A peptide comprises an amino acid sequence of any one of SEQ ID NOs:12, 28, 29, or 30. An exemplary P2A peptide comprises an amino acid sequence of SEQ ID NO: 13 or 31. An exemplary E2A peptide sequence comprises an amino acid sequence of SEQ ID NO: 14. An exemplary F2A peptide sequence comprises an amino acid sequence of SEQ ID NO:15.
Certain embodiments of tandem expression cassettes of the present disclosure comprise a polynucleotide encoding a CAR/or TCR specific for a target antigen (e.g., tumor antigen) and a polynucleotide encoding a chimeric Tim receptor of the present disclosure. Upon binding a target cell expressing the target antigen by the CAR/or TCR, a cell modified to express such a tandem expression cassette induces apoptosis of the target cell. Apoptosis induces exposure of pro-engulfment markers on the target cell, such as phosphatidylserine, which may then target the damaged or apoptotic cells for engulfment by the chimeric Tim receptor.
A polynucleotide encoding a desired chimeric Tim receptor can be inserted into an appropriate vector, e.g., a viral vector, non-viral plasmid vector, and non-viral vectors, such as lipid-based DNA vectors, modified mRNA (morIRNA), self-amplifying mRNA, CELiD, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty), for introduction into a host cell of interest (e.g., an immune cell).
Polynucleotides encoding a chimeric Tim receptor of the present disclosure can be cloned into any suitable vector, such as an expression vector, a replication vector, a probe generation vector, or a sequencing vector. In certain embodiments, a polynucleotide encoding the extracellular domain, a polynucleotide encoding the transmembmne domain, and a polynucleotide encoding the intracellular signaling domain are joined together into a single polynucleotide and then inserted into a vector.
In other embodiments, a polynucleotide encoding the extracellular domain, a polynucleotide encoding the transmembrane domain, and a polynucleotide encoding the intracellular signaling domain may be inserted separately into a vector such that the expressed amino acid sequence produces a functional chimeric Tim receptor. A
vector that encodes a chimeric Tim receptor is referred to herein as a "chimeric Tim receptor vector."
In certain embodiments, a vector comprises a polynucleotide encoding one chimeric Tim receptor. In certain embodiments, a vector comprises one polynucleotide encoding two or more chimeric Tim receptors. In certain embodiments, a single polynucleotide encoding two or more chimeric Tim receptors is cloned into a cloning site and expressed from a single promoter, with each chimeric Tim receptor sequence separated from each other by an internal ribosomal entry site (IRES), furin cleavage site, or viral 2A peptide to allow for co-expression of multiple genes from a single open reading frame (e.g., a multi ci stronic vector). In certain embodiments, a viral 2A peptide is a porcine teschovirus-1 (P2A), Thosea asigncr virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variant thereof. An exemplary T2A peptide comprises an amino acid sequence of SEQ DD NO:12, 28, 29, or 30. An exemplary P2A peptide comprises an amino acid sequence of SEQ ID
NO:13 or 31. An exemplary E2A peptide sequence comprises an amino acid sequence of SEQ
ID NO:14. An exemplary F2A peptide sequence comprises an amino acid sequence of SEQ ID NO:15.
In certain embodiments, a vector comprises two Or more polynucleotides, each polynucleotide encoding a chimeric Tim receptor. The two or more polynucleotides encoding chimeric Tim receptors may be cloned sequentially into a vector at different cloning sites, with each chimeric Tim receptor expressed under the regulation of different promoters. In certain embodiments, vectors that allow long-term integration of a transgene and propagation to daughter cells are utilized.
Examples include viral vectors such as, adenovirus, adeno-associated virus, vaccinia virus, herpes viruses, cytomegalovirus, pox virus, or retroviral vectors, such as lentiviral vectors.
Vectors derived from lentivirus can be used to achieve long-term gene transfer and have added advantages over vectors including the ability to transduce non-proliferating cells, such as hepatocytes, and low immunogenicity.
A vector that encodes a core virus is referred to herein as a "viral vector." There are a large number of available viral vectors suitable for use with the compositions of the instant disclosure, including those identified for human gene therapy applications (see Pfeifer and \Terme, Ann. Rev. Genomics Hum. Genet.
2:177, 2001). Suitable viral vectors include vectors based on RNA viruses, such as retrovirus-derived vectors, e.g., Maloney murine leukemia virus (MI,V)-derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, Fly, equine infectious anemia virus, S1V, and Maedi-Visna virus (ovine lentivirus). M:ethods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing chimeric receptor transgencs are known in the art and have been previous described, for example, in U.S. Patent 8,119,772; Walehli et al., PLoS One 6:327930, 2011;
Zhao et at, J. lmmunol. /74:4415, 2005; Engels et al., Hum. Gene filer. 14:1155, 2003;
Frecha etal., Mot iher. 18:1748, 2010; Verhoeyen et al., Methods Mol. Biol. 506:97, 2009.
Retroviral and lenti viral vector constructs and expression systems are also commercially available.
In certain embodiments, a viral vector is used to introduce a non-endogenous polynucleotide encoding a chimeric Tim receptor to a host cell. A
viral vector may be a retroviral vector or a lentiviral vector. A viral vector may also include a nucleic acid sequence encoding a marker for transduction. Transduction markers for viral vectors are known in the art and include selection markers, which may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. In particular embodiments, a viral vector further comprises a gene marker for transduction comprising a fluorescent protein (e.g., green, yellow), an extracellular domain of human CD2, or a truncated human EGFR (EGFRt or tEGFR; see Wang etal., Blood 118:1255, 2011). An exemplary tEGFR comprises an amino acid sequence of SEQ ID NO:16. When a viral vector genome comprises a plurality of genes to be expressed in a host cell as separate proteins from a single transcript, the viral vector may also comprise additional sequences between the two (or more) genes allowing for multicistronic expression.
Examples of such sequences used in viral vectors include internal ribosome entry sites (RES), furin cleavage sites, viral 2A peptides (e.g., T2A, P2A, E2A, F2A), or any combination thereof.
Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Iher. 5: 1517, 1998).
Other viral vectors recently developed for gene therapy uses can also be used with the compositions and methods of this disclosure. Such vectors include those derived from baculoviruses and a-viruses. (Jolly, D J. 1999. Emerging Viral Vectors.
pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).
In certain embodiments, a chimeric Tim receptor vector can be constructed to optimize spatial and temporal control. For example, a chimeric Tim receptor vector can include promoter elements to optimize spatial and temporal control.
In some embodiments, a chimeric Tim receptor vector includes tissue specific promoters or enhancers that enable specific induction of a chimeric Tim receptor to an organ, a cell type (e.g., immune cell), or a pathologic microenvironment, such as a tumor or infected tissue. An "enhancer" is an additional promoter element that can function either cooperatively or independently to activate transcription. In certain embodiments, a chimeric Tim receptor vector includes a constitutive promoter.
In certain embodiments, a chimeric Tim receptor vector includes an inducible promoter.
In certain embodiments, a chimeric Tim receptor vector includes a tissue specific promoter.
In certain embodiments, a chimeric Tim receptor vector can include a gene encoding a homing receptor, such as CCR4 or CXCR4, to improve homing and antitumor activity in viiv.
Where temporal control is desired, a chimeric Tim receptor vector may include an element that allows for inducible depletion of transduced cells.
For example, such a vector may include an inducible suicide gene. A suicide gene may be an apoptotic gene or a gene that confers sensitivity to an agent (e.g., a drug).
Exemplary suicide genes include chemically inducible caspase 9 (iCASP9) (U.S. Patent Publication No. 2013/0071414), chemically inducible Fas, or Herpes simplex virus thymidine kinase (HSV-TK), which confers sensitivity to ganciclovir. In further embodiments, a chimeric Tim receptor vector can be designed to express a known cell surface antigen that, upon infusion of an associated antibody, enables depletion of transduced cells.
Examples of cell surface antigens and their associated antibodies that may be used for depletion of transduced cells include CD20 and Rituximab, RQR.8 (combined CD34 and CD20 cpitopes, allowing CD34 selection and anti-CD20 deletion) and Rituximab, and EGFR and Cetuximab.
Inducible vector systems, such as the tetracycline (Tet)-On vector system which activates transgene expression with doxycycline (Heinz et al., Hum. Gene Ther.
2011, 22:166-76) may also be used for inducible chimeric Tim receptor expression.
Inducible chimeric Tim receptor expression may be also accomplished via retention using a selective hook (RUSH) system based on streptavidin anchored to the membrane of the endoplasmic reticulum through a hook and a streptavidin binding protein introduced into the chimeric Tim receptor structure, where addition of biotin to the system leads to the release of the chimeric Tim receptor from the endoplasmic reticulum (Agaugue et al., 2015, Mot. Ther. 23(Suppl. 1):S88).
In certain embodiments, a cell, such as an immune cell, obtained from a subject may be engineered into a non-natural or recombinant cell (e.g., a non-natural or recombinant immune cell) by introducing a polynucleotide that encodes a chimeric Tim receptor as described herein, whereby the cell expresses a cell surface localized chimeric Tim receptor. In certain embodiments, a host cell is an immune cell, such as a myeloid progenitor cell or a lymphoid progenitor cell. Exemplary immune cells that may be modified to comprise a polynucleotide encoding a chimeric Tim receptor or a vector comptising a polynucleotide encoding a chimeric Tim receptor include a T cell, a natural killer cell, a B cell, a lymphoid precursor cell, an antigen presenting cell, a dendritic cell, a Langerhans cell, a myeloid precursor cell, a mature myeloid cell, a monocyte, or a macrophage.
In certain embodiments, a B cell is genetically modified to express one or more chimeric Tim receptors. B cells possess certain properties that may be advantageous as host cells, including: trafficking to sites of inflammation, capable of internalizing and presenting antigen, capable of costimulating T cells, highly proliferative, and self-renewing (persist for life). In certain embodiments, a chimeric Tim receptor modified B cell is capable of digesting an engulfed target cell or engulfed target particle into smaller peptides and presenting them to T cells via an MHC
molecule. Antigen presentation by a chimeric Tim receptor modified B cell may contribute to antigen spreading of the immune response to non-targeted antigens. B
cells include progenitor or precursor cells committed to the B cell lineage (e.g., pre-pro-B cells, pro-B cells, and pre--B cells); immature and inactivated B cells; or mature and functional or activated B cells. In certain embodiments, B cells may be naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cell, plasmablast cell, memory B cells, or any combination thereof. Memory B cells may be distinguished from naive B cells by expression of CD27, which is absent on naive B cells. In certain embodiments, the B cells can be primary cells or cell lines derived from human, mouse, rat, or other mammals.
B cell lines are well known in the art. If obtained from a mammal, a B cell can be obtained from numerous sources, including blood, bone marrow, spleen, lymph node, or other tissues or fluids. A B cell composition may be enriched or purified.
In certain embodiments, a T cell is genetically modified to express one or more chimeric Tim receptors. Exemplary T cells include CD4' helper, CDS+
effector (cytotoxic), naive (CD45 RA+, CCR7+, CD62L+, CD27+, CD45R0-), central memory (CD45R0+, CD62L+, CD8+), effector memory (CD45RA+, CD45R0-, CCR7-, CD62L-, CD27-), T memory stem, regulatory, mucosal-associated invariant (MNIT), y5 (gd), tissue resident T cells, natural killer T cells, or any combination thereof. In certain embodiments, the 'I' cells can be primary cells or cell lines derived from human, mouse, rat, or other mammals. If obtained from a mammal, a T cell can be obtained from numerous sources, including blood, bone marrow, lymph node, thymus, or other tissues or fluids. A T cell composition may be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In certain embodiments, the T cells lack endogenous expression of a TCRa gene, TC1113 gene, or both. Such T cells may naturally lack endogenous expression of 'TCRa and 13 chains, or may have been modified to block expression (e.g., T cells from a transgenic mouse that does not express TCR a and 13 chains or cells that have been manipulated to inhibit expression of TCR a and 13 chains) or to knockout a TCRa chain, a TCI213 chain, or both genes.
In certain embodiments, host cells expressing a chimeric Tim protein of this disclosure on the cell surface arc not T cells or cells of a T cell lineage, but cells that are progenitor cells, stem cells or cells that have been modified to express cell surface anti-CD3.
In certain embodiments, gene editing methods are used to modify the host cell genome to comprise a polynucleotide encoding a chimeric Tim receptor of the present disclosure. Gene editing, or genome editing, is a method of genetic engineering wherein DNA is inserted, replaced, or removed from a host cell's genome using genetically engineered endonucleases. The nucleases create specific double-stranded breaks at targeted loci in the genome. The host cell's endogenous DNA repair pathways then repair the induced break(s), e.g., by non-homologous ending joining (NEIEJ) and homologous recombination. Exemplary endonucleases useful for gene editing include a zinc finger nuclease (ZEN), a transcription activator-like effector (TALE) nuclease, a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system (e.g., CRISPR-Cas9), a meganuclease, or combinations thereof. Methods of disrupting or knocking out genes or gene expression in immune cells including :B cells and T cells, using gene editing endonucleases are known in the art and described, for example, in PCT Publication Nos. WO 2015/066262;
WO 2013/074916; WO 2014/059173; Cheong et al., Nat. COMM. 2016 7:10934; Chu et al., Proc. Natl. Acad. Sci. USA. 2016 113:12514-12519; methods from. each of which are incorporated herein by reference in their entirety.
In certain embodiments, expression of an endogenous gene of the host cell is inhibited, knocked down, or knocked out. Examples of endogenous genes that may be inhibited, knocked down, or knocked out in a B cell include IGH, lGiç
ICA, or any combination thereof. Examples of endogenous genes that may be inhibited, knocked down, or knocked out in a T cell include a TCR gene (TRA or TRB), an HLA
gene (HLA class I gene or HLA class II gene), an immune checkpoint molecule (PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, .A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, or PVRIG/CD1.12R), or any combination thereof. Expression of an endogenous gene may be inhibited, knocked down, or knocked out at the gene level, transcriptional level, translational level, or a combination thereof. Methods of inhibiting, knocking down, or knocking out an endogenous gene may be accomplished, for example, by an RNA interference agent (e.g., siRNA, shRNA, miRNA, etc.) or an engineered endonuclease (e.g., CRISPR/Cas nuclease system, a zinc finger nuclease (ZFN), a Transcription Activator Like Effector nuclease (TALEN), a meganuclease), or any combination thereof. in certain embodiments, an endogenous B cell gene (e.g., IGH, IGK, or 10.) is knocked out by insertion of a polynucleotide encoding a chimeric Tim receptor of the present disclosure into the locus of the endogenous B cell gene, such as via an engineered endonuclease.
In certain embodiments, an endogenous T cell gene (e.g., a TCR gene, an HLA
gene, or an immune checkpoint molecule gene) is knocked out by insertion of a polynucleotide encoding a chimeric Tim receptor of the present disclosure into the locus of the endogenous 1' cell gene, such as via an engineered endonuclease.
In certain embodiments, a host cell may be genetically modified to express one type of chimeric Tim receptor. In other embodiments, a host cell may express at least two or more different chimeric Tim receptors.
The present disclosure also provides a composition comprising a population of chimeric Tim receptor modified host cells. In certain embodiments, the population of chimeric Tim receptor modified host cells may be a population of B cells, a population of T cells, a population of natural killer cells, a population of lymphoid precursor cells, a population of antigen presenting cells, a population of dendritic cells, a population of Langerhans cells, a population of myeloid precursor cells, a population of mature myeloid cells, or any combination thereof. Furthermore, a population of chimeric Tim receptor modified host cells of a particular cell type may be composed of one or more subtypes. For example, a population of B cells may be composed of chimeric Tim receptor modified naive B cells, plasma cells, regulatory B
cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablast cells, memory B
cells, or any combination thereof. In another example, a population of T cells may be composed of chimeric Tim receptor modified CD4+ helper T cells, CD8 effector (cytotoxic) T cells, naive (CD45 RA+, CCR7+, CD62L+, CD27+, CD45R0-) T cells, central memory (CD45R0+, CD62L+, CDS+) T cells, effector memory (CD45RA -I-, CD45R0-, CCR7-, CD62L-, CD27-) T cells, T memory stem cells, regulatory T
cells, mucosal-associated invariant T cells (MAIT), T6 (gd) cells, tissue resident T
cells, natural killer T cells, or any combination thereof.
In certain embodiments, a population of host cells is composed of cells that each expresses the same chimeric Tim receptor(s). In other embodiments, a population of host cells is composed of a mixture of two or more subpopulation of host cells, wherein each subpopulation expresses a different chimeric Tim receptor or set of chimeric Tim receptors.
In certain embodiments, when preparing chimeric Tim receptor modified host cells, e.g., B cells or T cells, one or more growth factor cytokines that promotes proliferation of the host cells, e.g., B cells or T cells, may be added to the cell culture.
The cytokines may be human or non-human. Exemplary growth factor cytokines that may be used to promote T cell proliferation include 1L-2, 1L-15, or the like.
Exemplary growth factor cytokines that may be used to promote B cell proliferation include CD4OL, 1L-2, IL-4, IL-15, 1L-21, BAIT, or the like.
Prior to genetic modification of the host cells with a chimeric Tim receptor vector, a source of host cells (e.g., T cells, B cells, natural killer cells, etc.) is obtained from a subject (e.g., whole blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue), from which host cells are isolated using methods known in the art. Specific host cell subsets can be collected in accordance with known techniques and enriched or depleted by known techniques, such as affinity binding to antibodies, flow cytometry and/or immunomagnetic selection. After enrichment and/or depletion steps and introduction of a chimeric Tim receptor, in vitro expansion of the desired modified host cells can be carried out in accordance with known techniques, or variations thereof that will be apparent those skilled in the art.
Chimeric Tim receptors of the present disclosure confer cytotoxic activity to host cells expressing the chimeric Tim receptors that is specific for phosphatidylserine. Thus, upon binding phosphatidylscrine exposed on the surface of a target cell, a host cell expressing a chimeric Tim receptor is capable of inducing apoptosis of the target cell. In certain embodiments, the host cell expressing the chimeric Tim receptor induces apoptosis of the target cell via: release of granzymes, perforins, granulysin, or any combination thereof; Fas ligand-Fas interaction;
or both.
In further embodiments, the chimeric Tim receptor further confers phosphatidylserine specific engulfment activity to host cells expressing the chimeric Tim receptor. in yet further embodiments, the host cell does not naturally exhibit an engulfment phenotype prior to modification with the chimeric Tim receptor.
Chimeric Tim receptors of the present disclosure may also be capable of costimulating T cells via at least one signaling pathway. In certain embodiments, chimeric Tim receptors provide costimulatory signals to T cells via at least two distinct signaling pathways (e.g., via the selected costimulatory signaling domain(s) in the chimeric Tim receptor). For example, a chimeiic Tim receptor comprising a CD28 costimulatory signaling domain may be capable of providing a costimulatory signal via CD28 and Tim 1. In certain embodiments, host immune cells expressing the chimeric Tim receptors exhibit reduction or inhibition of immune cell exhaustion. In certain embodiments, the host immune cell is a T cell or NK cells. In certain embodiments, exhausted T cells exhibit; (a) increased expression of PD-1, LAG3, TIM:3, or any combination thereof; (b) decreased production of IFN-y, TNE-a, or any combination thereof; or both (a) and (b). In certain embodiments, exhausted NK
cells exhibit; (a) increased expression of PD-1, NKG2A, TIM3, or any combination thereof;
(b) decreased production of IFN-,y, TN. F-a, or both; or both (a) and (h).
In certain embodiments, host cells expressing the chimeric Tim receptors exhibit an enhanced effector response (e.g., tumor specific). In certain embodiments, the effector response is enhanced T cell proliferation, cytokine production (e.g., IFN-y, :IL-2, TNF-a), cytotoxic activity, persistence, or any combination thereof.
In certain embodiments host cells expressing the chimeric Tim receptors exhibit a reduced immunosuppressive response to phosphatidylserine.
Phosphatidylserine is one of the primary apoptotic cell ligands that signal "eat me" to phagocytes. The removal of apoptotic cells by phagocytes generally reduces or prevents an inflammatory response via secretion of anti-inflammatory cytokines and TGF-f3 and the decrease of secretion of inflammatory cytokines TNF-a, IL-Ifl, and IL-12. Thus, phosphatidylserine may act as an immunosuppressive signal during the clearance of apoptotic cells. In certain embodiments, upon binding phosphatidylserine, a chimeric Tim receptor modified host cell exhibits increased antigen-specific cytokine production (e.g., IFN-y,1L-2, TNF-a), thereby reducing the immunosuppressive response to phosphatidylserine.
The expression of chimeric Tim receptors on host cells may be functionally characterized according to any of a large number of art-accepted methodologies for assaying host cell (e.g.. T cell) activity, including determination of T cell binding, activation or induction and also including determination of T
cell responses that are antigen-specific. Examples include determination of T cell proliferation, T cell cytokine release, antigen-specific 1' cell stimulation, (717.L activity (e.g., by detecting 51Cr or Europium release from pre-loaded target cells), changes in T cell phenotypic marker expression, and other measures of T cell functions.
Procedures for performing these and similar assays are may be found, for example, in Lefkovits (Immunology Methods. Manual: The Comprehensive Sourcebook of Techniques, 1998). See, also, Current! Protocols in Immunology; Weir, Handbook of Experimental Immunology, Black-well. Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA. (1979); Green and Reed, Science 281:1309 (1998) and references cited therein. Cytokine levels may be determined according to methods known in the art, including for example, ELISA, ELISPOT, intracellular cytokine staining, flow cytometry, and any combination thereof (e.g., intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion resulting from an antigen-specific elicitation or stimulation of an immune response may be determined by isolating lymphocytes, such as circulating lymphocytes in samples of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and/or cell viability, such as by incorporation of vitiated thymidinc or non-radioactive assays, such as MTT assays and the like.
In certain embodiments, a chimeric Tim receptor modified host cell has a phagocytic index of about 20 to about 1,500 for a target cell. A "phagocytic index" is a measure of phagocytic activity of the transduced host cell as determined by counting the number of target cells or particles ingested per chimeric Tim receptor modified host cell during a set period of incubation of a suspension of target cells or particles and chimeric Tim receptor modified host cells in media. Phagocyfic index may be calculated by multiplying [total number of engulfed target cells/total number of counted chimeric Tim receptor modified cells (e.g., phagocytic frequency)] x [average area of target cell or particle staining per chimeric Tim receptor + host cell x 100 (e.g., hybrid capture)] or [total number of engulfed particles/total number of counted chimeric Tim receptor modified host cells] x [number of chimeric Tim receptor modified host cells containing engulfed particles/ total number of counted chimeric Tim receptor cells] x 100. In certain embodiments, a chimeric Tim receptor modified cell has a phagocytic index of about 30 to about 1,500; about 40 to about 1,500; about 50 to about 1,500;
about 75 to about 1,500; about 100 to about 1,500; about 200 to about 1,500;
about 300 to about 1,500; about 400 to about 1,500; about 500 to about 1,500; about 20 to about 1,400; about 30 to about 1,400; about 40 to about 1,400; about 50 to about 1,400; about 100 to about 1,400; about 200 to about 1,400; about 300 to about 1,400; about 400 to about 1,400; about 500 to about 1,400; about 20 to about 1,300; about 30 to about 1,300; about 40 to about 1,300; about 50 to about 1,300; about 100 to about 1,300;
about 200 to about 1,300; about 300 to about 1,300; about 400 to about 1,300;
about 500 to about 1,300; about 20 to about 1,200; about 30 to about 1,200; about 40 to about 1,200; about 50 to about 1,200; about 100 to about 1,200; about 200 to about 1,200;
about 300 to about 1,200; about 400 to about 1,200; about 500 to about 1,200;
about 20 to about 1,100; about 30 to about 1,100; about 40 to about 1,100; about 50 to about 1,100; about 100 to about 1,100; about 200 to about 1,100; about 300 to about 1,100;
about 400 to about 1,100; or about 500 to about 1,100; about 20 to about 1,000; about to about 1,000; about 40 to about 1,000; about 50 to about 1,000; about 100 to about 1,000; about 200 to about 1,000; about 300 to about 1,000; about 400 to about 1,000; or 30 about 500 to about 1,000; about 20 to about 750; about 30 to about 750;
about 40 to about 750; about 50 to about 750; about 100 to about 750; about 200 to about 750;
about 300 to about 750; about 400 to about 750; or about 500 to about 750;
about 20 to about 500; about 30 to about 500; about 40 to about 500; about 50 to about 500; about 100 to about 500; about 200 to about 500; or about 300 to about 500. In further embodiments, the incubation time is from about 2 hours to about 4 hours, about 2 hours, about 3 hours, or about 4 hours. In yet further embodiments, a chimeric Tim receptor modified cell exhibits phagocytic index that is statistically significantly higher than a cell transduced with truncated EGET. control. Phagocytic index may be calculated using methods known in the art and as further described in the Examples and PCT
Application No. PCT/US2017/053553 (incorporated herein by reference in its entirety), including quantification by flow cytometry or fluorescence microscopy.
Host cells may be from an animal, such as a human, primate, cow, horse, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, or a combination thereof In a preferred embodiment, the animal is a human. Host cells may be obtained from a healthy subject or a subject having a disease associated with expression or overexpressi on of an antigen.
PARP Inhibitors Any suitable PARP inhibitor may be used in the compositions and methods of the disclosure. Exemplary PARP inhibitors include talazoparib, niraparib, rucaparib, olaparib (AZ 2281, KU59436), veliparib (ABT 888), CEP 9722, E7016, AG014699, MK4827, BMN-673, and Painiparib (BGB-290).
In a particular embodiment, the PARP inhibitor comprises niraparib. In a particular embodiment, the PARP inhibitor comprises talazoparib. In a particular embodiment, the PARP inhibitor comprises rucaparib. In a particular embodiment, the PARP inhibitor comprises olaparib. In a particular embodiment, the PARP
inhibitor comprises veliparib. In a particular embodiment, the PARP inhibitor comprises CEP
9722. In a particular embodiment, the PARP inhibitor comprises E7016. In a particular embodiment, the PARP inhibitor comprises AG014699. In a particular embodiment, the PARP inhibitor comprises MK4827. In a particular embodiment, the PARP
inhibitor comprises BMN-673. In a particular embodiment, the PARP inhibitor comprises Pamiparib.
Methods of Treatment A chimeric Tim receptor, a polynucleotides encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor, in combination with a PARP inhibitor according to any of the embodiments provided herein may be used in a method of treating a subject suffering from a disease, disorder or undesired condition. Embodiments of these methods include administering to a subject (i) a therapeutically effective amount of a pharmaceutical composition including one or more chimeric Tim receptors, polynucleotides encoding one or more chimefic Tim receptors, vectors comprising polynucleotides encoding one or more chimeric Tim receptors, or a population of host cells genetically modified to express one or more chimeric Tim receptors according to the present description; and (ii) a therapeutically effective amount of a pharmaceutical composition comprising a PARP
inhibitor.
The chimeric Tim receptor compositions as described herein may be administered before PARP inhibitor therapy (e.g., 1 day to 30 days or more before the PARP inhibitor therapy), concurrently with PARP inhibitor therapy (on the same day), or after PARP inhibitor therapy (e.g., 1 day ¨ 30 days or more after the PARP
inhibitor therapy). In certain embodiments, the chimeric Tim receptor modified cells are administered after administration of the one or more additional therapies. In further embodiments, the chimeric Tim receptor modified cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration of the PARP inhibitor. In still further embodiments, the chimeric Tim receptor modified cells are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week after administration of the PARP inhibitor therapy.
Where the PARP inhibitor therapy involves multiple doses, the chimeric Tim receptor modified cells may be administered after the initial dose of the one or more additional therapies, after the final dose of the one or more additional therapies, or in between multiple doses of the one or more additional therapies.
Diseases that may be treated with cells expressing a chimeric Tim receptor as described in the present disclosure include cancer. Adoptive immune and gene therapies are promising treatments for various types of cancer (Morgan etal., Science 314:126, 2006; Schmitt et al., Hum. Gene They. 20:1240, 2009; June, J.
Clin.
Invest. 117:1466, 2007) and infectious disease (Kitchen etal., PLoS One 4:38208, 2009; Rossi et al., Nat. Biatechnol. 25:1444, 2007; Zhang et al., PLoS Palhog.
6:el 001018, 2010; Luo et al., J. A/161. Med 89:903, 2011).
A wide variety of cancers, including solid tumors and leukemias are amenable to the compositions and methods disclosed herein. Exemplary cancers that may be treated using the receptors, modified host cells, and composition described herein include adenocarcinoma of the breast, prostate, and colon; all forms of bronchogenic carcinoma of the lung; myeloid leukemia; melanoma; hepatoma;
neuroblastoma; papilloma; apudoma; choristoma; branchiorna; malignant carcinoid syndrome; carcinoid heart disease; and carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, :Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell). Additional types of cancers that may be treated using the receptors, modified host cells, and composition described herein include histiocytic disorders; malignant histiocytosis; leukemia; Hodgkin's disease;
immunoproliferative small; non-Hodgkin's lymphoma; plasmacytoma; multiple myelorna; chronic myeloid leukemia (CML); acute myeloid leukemia (AML); plasrnacytoma;
reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosareoma;
fibroma; fibrosarcoma; giant cell tumors; histiocytoma; lipoma; liposarcoma;
mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma;
craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma;
myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymorna;
trophoblastic tumor. Further, the following types of cancers arc also contemplated as amenable to treatment using the receptors, modified host cells, and composition described herein: adenoma; cholangioma; cholesteatoma; cyclindroma;
cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma;
hepatoma;
hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma; myoblastoma; myomma; myosarcoma;
rhabdomyoma.; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma;
medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma;
neurofibroma; neuroma; paraganglioma; paraganglioma nonehromaffin. The types of cancers that may be treated also include angiokeratoma; angiolymphoid hyperplasia with eosi nophili a; angioma sclerosing; angiornatosis; glomangioma;
hemangioendothelioma; hemangiorna; hernangiopericytoma; hernangiosarcorna;
lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; careinosarcoma;
chondrosarcorna; cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma;
leiomyosarcoma; leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma;
myxosarcoma; ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasms;
nerofibromatosis; cervical dysplasia; and peritoneal cancer.
In some embodiments, compositions and methods of the present disclosure are useful to treat solid tumors. In some embodiments, the solid tumor cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian cancer, peritoneal cancer, or prostate cancer. In some embodiments, the breast cancer is triple negative breast cancer. In some embodiments, the ovarian cancer is advanced ovarian cancer. In some embodiments, the prostate cancer is advanced prostate cancer. In some embodiments, the solid tumor cancer is melanoma. In some embodiments, the solid tumor cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer.
In additional embodiments, the cancer is a Breast cancer (BRCA) mutated cancer. In particular embodiments, the cancer is a BRCA1 mutated cancer, a BRCA2 mutated cancer, or both.
A chimeric Tim receptor of the present disclosure may be administered to a subject in cell-bound form (e.g., gene therapy of target cell population). Thus, for example, a chimeric Tim receptor of the present disclosure may be administered to a subject expressed on the surface of T cells, Natural Killer Cells, Natural Killer T cells, B cells, lymphoid precursor cells, antigen presenting cells, dendritic cells, Langerhans cells, myeloid precursor cells, mature myeloid cells, including subsets thereof, or any combination thereof. In certain embodiments, methods of treating a subject comprise administering an effective amount of chimeric Tim receptor modified cells (i.e., recombinant cells that express one or more chimeric Tim receptors). The chimeric Tim receptor modified cells may be xenogeneic, syngeneic, allogeneic, or autologous to the subject.
Chimeric Tim receptor modified cells and PARP inhibitors may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as the condition of the patient, size, weight, body surface area, age, sex, type and severity of the disease, particular therapy to be administered, particular form of the active ingredient, time and the method of administration, and other drugs being administered concurrently. The present disclosure provides pharmaceutical compositions comprising chimeric Tim receptor modified cells and a pharmaceutically acceptable carrier, diluent, or excipient. Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. Other suitable infusion medium can be any isotonic medium formulation, including saline, Normosol R (Abbott), Plasina-Lyte A (Baxter), 5% dextrose in water, or Ringer's lactate.
A treatment effective amount of cells in a pharmaceutical composition is at least one cell (for example, one chimeiic Tim receptor modified T cell) or is more typically greater than 102 cells, for example, up to 106, up to 10, up to 108 cells, up to 109 cells, up to 10' cells, or up to 101' cells or more. In certain embodiments, the cells are administered in a range from about 106 to about 10" cells/m2, preferably in a range of about 10 to about 109 cells/m2. The number of cells will depend upon the ultimate use for which the composition is intended as well the type of cells included therein. For example, a composition comprising cells modified to contain a chimeric Tim receptor will comprise a cell population containing from about 5% to about 95% or more of such cells. in certain embodiments, a composition comprising chimeric Tim receptor modified cells comprises a cell population comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%
or more of such cells. For uses provided herein, the cells are generally in a volume of a liter or less, 500 mls or less, 250 mls or less, or 100 mls or less. Hence the density of the desired cells is typically greater than 104 cells/ml and generally is greater than 10' cells/ml, generally 108 cells/m1 or greater. The cells may be administered as a single infusion or in multiple infusions over a range of time. Repeated infusions of chimeric Tim receptor modified cells may be separated by days, weeks, months, or even years if relapses of disease or disease activity are present. A clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 106, 107, 108, 109, 1010, or 1011 cells. A preferred dose for administration of a host cell comprising a recombinant expression vector as described herein is about 107 cells/m2, about 5 x 107 cells/m2, about 108 cells/m2, about 5 x 108 cells/m2, about 109 cells/m2, about 5 x 109 cells/m2, about 1010 cells/m2, about 5 x 101 cells/m2, or about 10" cells/m2.
Chimeric Tim receptors and PARP inhibitors as described herein may be administered intravenously, intraperitoneally, intranasally, intratumorly, into the bone marrow, into the lymph node, and /or into cerebrospinal fluid.
In one aspect, methods of the present disclosure comprise conferring or enhancing phosphatidylserine-specific cytotoxic activity of a cell comprising introducing into a host cell a nucleic acid molecule encoding at least one chimeric Tim receptor or a chimeric Tim receptor vector according to any of the embodiments described herein; and expressing the at least one chimeric Tim receptor in the host cell, wherein the at least one chimeric Tim receptor enhances the phosphatidylserine-specific cytotoxic activity of the host cell as compared to a the host cell prior to modification to express a chimeric Tim receptor. In certain embodiments, the cytotoxic activity of the host cell is increased at least about 10%, 15%, 201?6, 25%, 30%, 35 /0, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more as compared to thc host cell prior to modification with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector. In some embodiments, the host cell is an immune cell.
In some embodiments, the host cell is a T cell or an NK cell. Methods of measuring cytotoxic activity of host cells, particularly immune cells such as T cells and NK cells, include a chromium (51Cr)-release assay, a or firefly luciferase release assay, flow cytometric methods of mediating target cell death and effector cell activity (see, e.g., Expert Rev. Vaccines, 2010, 9:601-616).
In certain embodiments, methods of the present disclosure comprise conferring or enhancing phosphatidylserine-specific cytotoxic activity of a cell further comprise conferring or enhancing phosphatidylserine-specific engulfment activity of the host cell expressing the at least one chimeric Tim receptor. In certain such embodiments, the host cell does not naturally exhibit an engulfment phenotype prior to modification with the chimeric Tim receptor. For example in certain such embodiments, the engulfment activity of the host cell is increased at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50 4, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more as compared to the host cell prior to modification to express the chimeric Tim receptor vector. In certain embodiments, the host cell does not naturally possess engulfment activity. In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell or an NK cell. Methods of measuring engulfment activity of host cells include methods as described in PCT/US2017/053553 (incorporated herein by reference in its entirety).
In another aspect, a chitneric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor according to any of the embodiments provided herein may be used in a method of enhancing effector function of the host cell. In certain embodiments, enhanced effector function comprises increased cytotoxic activity, increased antigen specific cytokine production (e.g., IFN-1, IL-2, TNF-a, or any combination thereof), increased anti-apoptotic signaling, increased persistence, increased expansion, increased proliferation, or any combination thereof. In certain embodiments, the effector function of the host cell is enhanced at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 1600/0, 170%, 180%, 190%, 200% or more as compared to a host cell that is not modified with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector. In some embodiments, the host cell is an immune cell. In certain embodiments, the host cell is a T cell or an NK cell.
In another aspect, host cells modified with chimeric Tim receptors of the present disclosure can be used in methods for inhibiting or reducing immune cell exhaustion. In some embodiments, the immune cell is a T cell or NK cell. In certain embodiments, reduced exhaustion in T cells comprises; (a) decreased expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof in T cells; (b) increased production of1FN-y, 1L-2, TNF-a, or any combination thereof in T cells; or both (a) and (b). In certain embodiments, reduced exhaustion in NK cells comprises; (a) decreased expression of PD-1, NKG2A, TI1043, or any combination thereof in NK cells; (b) increased production of IFN-y, TNF-a, or both in NK cells; or both (a) and (b). In certain embodiments, the expression of an immune checkpoint molecule is decreased at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a host immune cell expressing the chimeric 'rim receptor as compared to a host immune cell that is not modified with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector.
In certain embodiments, the expression of the cytokine is increased at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%
or more in a host immune cell expressing the chimeric Tim receptor as compared to a host immune cell that is not modified with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector.
In another aspect, a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor according to any of the embodiments provided herein may be used in a method of reducing an immunosuppressive response to phosphatidylserine in a host cell. In certain embodiments, the immunosuppressive response comprises secretion of anti-inflammatory cytokines (e.g., IL-10, TGF-0, or both), the decrease in secretion of inflammatory cytokines (e.g., TNI7-a, IL-113, and IL-12), or both. In certain embodiments, the immunosuppressive response of the host cell to phosphatidylserine is decreased at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% as compared to a host cell that is not modified with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector. In some embodiments, the host cell is an immune cell. In certain embodiments, the host cell is a T cell or an NK cell.
In yet another aspect, a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor according to any of the embodiments provided herein may be used in methods for eliminating target cells bearing surface exposed phosphatidylserine, e.g., for the elimination of cancer cells bearing surface presented phosphatidylserine. In certain embodiments, the target cells are damaged, stressed, apoptotic, necrotic cells (e.g., tumor cells) bearing surface exposed phosphatidylserine In certain embodiments, host cells expressing chimeric Tim receptors clear damaged, stressed, apoptotic, or necrotic target cells bearing surface exposed phosphatidylserine via inducing apoptosis, or both inducing apoptosis and engulfment. Host cells expressing chimeric Tim receptors may be administered to a subject alone, or in combination with other therapeutic agents, including for example CAR-T cells, TCRs, antibodies, radiation therapy, chemotherapy, small molecules, oncolytic viruses, electropulse therapy, etc.
In another aspect, a chimeric Tim receptor, a polynucleotides encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor according to any of the embodiments provided herein may be used in methods to enhance the effect of a therapeutic agent that induces cellular stress, damage, necrosis, or apoptosis. Certain therapies, such as chemotherapy, radiation therapy, UV light therapy, clectropulse therapy, adoptive cellular immunotherapy (e.g., CAR-T cells, TCRs) and oncolytic viral therapy, can induce cell damage or death to tumor cells, diseased cells, and cells in their surrounding environment. Cells expressing chimeric Tim receptors can be administered in combination with the cell damaging/cytotoxic therapy to bind to the phosphatidylserine moieties exposed on the outer leaflet of targeted cells and clear stressed, damaged, diseased, apoptotic, necrotic cells.
Chimeric Tim receptors and PARP inhibitors may be administered to a subject in combination with one or more additional therapeutic agents.
Examples of therapeutic agents that may be administered in combination with a chimeric Tim compositions according to the present description include radiation therapy, adoptive cellular immunotherapy agent (e.g., recombinant TCR, enhanced affinity TCR, CAR, TCR-CAR, scTCR fusion protein, dendritic cell vaccine), antibody therapy, immune checkpoint molecule inhibitor therapy, UV light therapy, electric pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy, or a pharmaceutical therapy, such as a chemotherapeutic agent, a therapeutic peptide, a hormone, an aptamer, antibiotic, anti-viral agent, anti-fungal agent, anti-inflammatory agent, a small molecule therapy, or any combination thereof In certain embodiments, the chimeric Tim receptor modified host cells may clear stressed, damaged, apoptotic, necrotic, infected, dead cells displaying surface phosphatidylserine induced by the one or more additional therapeutic agents.
In certain embodiments, a vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapy agent (e.g., chimeric antigen receptor, recombinant TCR, etc.). In certain embodiments, a single polynucleotide encoding the chimeric Tim receptor and cellular immunotherapy agent (e.g., CAR) is cloned into a cloning site and expressed from a single promoter, with the chimeric Tim receptor sequence and cellular immunotherapy agent (e.g., CAR) sequence separated from each other by an internal ribosomal entry site (IRES), furin cleavage site, or viral 2A peptide to allow for co-expression of multiple genes from a single open reading frame (e.g., a multicistronic vector). In certain embodiments, a viral 2A peptide is a porcine teschovirus-1 (P2A), 7hosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (172A), or variant thereof. An exemplary T2A peptide comprises an amino acid sequence of SEQ ID NO:12, 28, 29, or 30. An exemplary P2A peptide comprises an amino acid sequence of SEQ ID
NO:13 or 31. An exemplary E2A peptide sequence comprises an amino acid sequence of SEQ
ID NO:14. An exemplary F2A peptide sequence comprises an amino acid sequence of SEQ ID NO:15.
In certain embodiments, a polynucleotide encoding the chimeric Tim receptor and a polynucleotide encoding the cellular immunotherapy agent (e.g., CAR) binding protein are joined together into a single polynucleotide and then inserted into a vector. In other embodiments, a polynucleotide encoding the CER, and a polynucleotide encoding the CAR or TCR binding protein may be inserted separately into a vector in the same or different cloning sites, such that the expressed amino acid sequence produces a functional CER and CAR/or TCR. A vector that encodes a tandem expression cassette is referred to herein as a "tandem expression vector."
In certain embodiments, a vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapy agent (e.g., CAR). The pol ynucleotides encoding the chimeric Tim receptor and cellular immunotherapy agent (e.g., CAR) may be cloned sequentially into a vector at different cloning sites, with the chimeric Tim receptor and cellular immunotherapy agent (e.g., CAR) expressed under the regulation of different promoters.
In certain embodiments, a chimeric Tim receptor modified host cell may also be modified to co-express one or more small GTPases. Rho GTPases, a family of small (-21 k Da) signaling G proteins and also a subfamily of the Ras superfamily, regulate actin cytoskeleton organization in various cell types and promote pseudopod extension and phagosome closure during phagocytosis (see, e.g., Castellano et al., 2000, J. Cell Sci. 113:2955-2961). Engulfment requires F-actin recruitment beneath tethered cells or particles, and F.-actin rearrangement to allow membrane extension resulting in cell or particle internalization. RhoGTPases include RhoA, Racl, Rac2, RhoG, and CDC42. Other small GTPases, such as Rap I , is involved in regulation of complement mediated phagocytosis. Co-expression of a small GTPasc with the chimeric Tim receptor may promote target cell or particle internalization and/or phagosome formation by the host cell. In some embodiments, a recombinant nucleic acid molecule encoding a GTPase is encoded on a separate vector than the chimeric Tim receptor-containing vector. In other embodiments, a recombinant nucleic acid molecule encoding a GTPase is encoded on the same vector as the chimeric Tim receptor. The GTPase and chimeric Tim receptor may be expressed under the regulation of different promoters on the same vector (e.g., at different multiple cloning sites). Alternatively, the chimeric Tim receptor and GTPase may be expressed under the regulation of one promoter in a tnulticistronic vector. The polynucleotide sequence encoding the chimeric Tim receptor and the polynucleotide sequence encoding the small GTPase(s) may be separated from each other by an IRES or viral 2A. peptide in a multicistronic vector.
Exemplary 2A
peptides include T2A (SEQ ID NO:12), P2A (SEQ ID NO:13), E2A (SEQ ID NO:14), F2A (SEQ ID NO:15). Examples of GTPases that may be co-expressed with a chimeric Tim receptor include Racl, Rac2, Rab5 (also referred to as Rab5a), Rab7, R.apl, RhoA., RhoG, CDC42, or any combination thereof. In specific embodiments, the GTPase comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to a Racl amino acid sequence of SEQ ID NO:17, a Rab5 amino acid sequence of SEQ ID NO:18, a Rab7 amino acid sequence of SEQ ID
NO:19, a Rapl amino acid sequence of SEQ ID NO:20, a R.hoA amino acid sequence of SEQ ID NO:21, a CDC42 amino acid sequence of SEQ ED NO:22, or any combination thereof.
In certain embodiments, a chimeric Tim receptor modified host cell may also be modified to co-express a cellular immunotherapy agent (e.g., CAR, TCR, etc.).
In some embodiments, the cellular immtmotherapy agent comprises a chimeric antigen receptor (CAR). CARs are recombinant receptors that generally comprise: an extracellular domain comprising a binding domain that binds to a target antigen; an intracellular signaling domain (e.g., comprising an ITAM containing intracellular signaling domain and optionally an intracellular costimulatory domain), and a transmembrane domain positioned between and connecting the extraccllular domain and the intracellular signaling domain.
Binding domains suitable for use in CARs of the present disclosure include any antigen-binding polypeptide. A binding domain may comprise an antibody or antigen binding fragment thereof, including for example, a full length heavy chain, Fab fragment, Fab', F(ab')2, sFv, VH domain, VL domain, dAb, VI-111, CDR, and scFv.
In certain embodiments, a CAR binding domain is murine, chimeric, human, or humanized.
In certain embodiments, the binding domain of the CAR targets a cancer or tumor antigen. Exemplary antigens that a CAR may target include CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, LICAM, CD22, CDI9, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR, GD-3, }WV E6, HPV E7, MUC-1, HER2, folate receptor a, CD97, CD171, CD179a, CD44v6, WTI, VEGF-a, VEGFR1, IL-13Ral, 1L-13Ra2, It-11Ra, PSA, FcRII5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A
antigen, Lewis Y antigen, MAGE, MAGE-Al, RAGE-1, folate receptor 0, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM I, GM:3, o-acetyl-GD2, and In certain embodiments, the extracellular domain of CARs provided in the present disclosure optionally comprises an extracellular, non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain away from the host cell suiface to further enable proper cell to cell contact, binding, and activation. An extracellular spacer domain is generally located between the extracellular binding domain and the transmembrane domain of the CAR.
The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest et al., J. Immwtother. 28:203-11, 2005;
PCT
Publication No. WO 2014/031687). In certain embodiments, an extracellular spacer domain is an immunoglobulin hinge region (e.g., IgGl, IgG2, IgG3, I8G4, IgA, IgD).
An immunoglobulin hinge region may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region. An altered IgG4 hinge region is described in PCT Publication No. WO 2014/031687, which hinge region is incorporated herein by reference in its entirety. In a particular embodiment, an extracellular spacer domain comprises a modified IgG4 hinge region having an amino acid sequence of SEQ ID NO:3.
Other examples of hinge regions that may be used in the CARs described herein include the hinge region from the extracellular regions of type I
membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof. In a particular embodiment, an extracellular spacer domain comprises a CD8a hinge region having an amino acid sequence of SEQ ED NO:70. In another particular embodiment, an extracellular spacer domain comprises a CD28 hinge region having an amino acid sequence of SEQ ID NO:32. In further embodiments, an extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from: a CH1 domain, a CH2 domain, a CI-13 domain, or combinations thereof (see, e.g., PCT Publication W02014/031687, which spacers are incorporated herein by reference in their entirety). In yet further embodiments, an extracellular spacer domain may comprise a stalk region of a type El C-lectin (the extracellular domain located between the C-typelectin domain and the transmembrane domain). Type 11 C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.
CARs of the present disclosure comprise a transmembrane domain that connects and is positioned between the extracellular domain and the intracellular signaling domain. The transmembrane domain ranges in length from about 15 amino acids to about 30 amino acids. The transmembrane domain is a hydrophobic alpha helix that transverses the host cell membrane and anchors the CAR in the host cell membrane. The transmembrane domain may be directly fused to the binding domain or to the extracellular spacer domain if present. In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g, receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, or the like). The transmembrane domain can be selected from the same molecule as the extracellular domain or the intracellular signaling domain (e.g., a CAR
comprises a CD28 costimulatory signaling domain and a CD28 transmembrane domain). In certain embodiments, the transmembrane domain and the extracellular domain are each selected from different molecules. In other embodiments, the transmembrane domain and the intracellular signaling domain are each selected from different molecules. In yet other embodiments, the transmembrane domain, the extracellular domain, and the intracellular signaling domain are each selected from different molecules.
Exemplary transmembrane domains for use in CARs of the present disclosure include a CD28, CD2, CD4, CD8a, CD5, CD38, CDR.), CD3, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD7911, CD80, CD86, CD95 (Fas), CD134 (0X40), CD137 (4-IBB), CD150 (SLAMF1), CD152 (CTLA4), CD154 (CD4OL), CD200R, CD223 (LAG3), CD270 (BTLA), CD273 (PD-L2), CD274 (PD-Li), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRct, FcR13, FcRy, Fyn, GAL9, KIR, Lek, LAT, LRP, N.KG2D, NOTCH1, NOTCH2, NoTcH3, NOTCH4, P`FCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCR13, TIM3, TRIM, LPA5, and Zap70 transmembrane domain.
An exemplary CD28 transmembrane domain comprises an amino acid sequence of SEQ
113 .N0:7. In a particular embodiment, a transmembrane domain comprises a CD8a transmembrane domain having an amino acid sequence of SEQ ID NO:33.
The intracellular signaling domain of a CAR is an intracellular effector domain and is capable of transmitting functional signals to a cell in response to binding of the extracellular domain of the CAR to a target molecule (e.g., cancer antigen) and activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. In some embodiments, the CAR
induces a function of a T cell such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. The intracellular signaling domain may be any portion of an intracellular signaling molecule that retains sufficient signaling activity.
In some embodiments, the intracellular signaling domain is obtained from an antigen receptor component (e.g., TCR) or costimulatory molecule. In some embodiments, a full length intracellular signaling domain of an antigen receptor or costimulatory molecule is used.
In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor or costimulatory molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, an intracellular signaling domain is a variant of a full length or truncated portion of an intracellular signaling domain of an antigen receptor co stimulatory molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).
In certain embodiments, the intracellular signaling domain of a CAR
comprises an immunoreceptor tyrosine-based activation motif (ITAM) containing signaling domain. An ITAM containing signaling domain generally contains at least one (one, two, three, four, or more) ITAM:s, which refer to a conserved motif of YXXL/I-X6-8-YXXL/I. An ITAM containing signaling domain may initiate T cell activation signaling following antigen binding or ligand engagement. ITAM-signaling domains include, for example, intracellular signaling domains of CD37, CD38, CD3e, CD3C,, CD5, CD22, CD79a, CD278 (ICOS), DAP12, FcRy, and CD66d. Exemplary CD3C, signaling domains that may be used in CARs of the present disclosure comprise an amino acid sequence of SEQ ID NO:27 or 5.
CAR intracellular signaling domains optionally comprise a costimulatory signaling domain, which, when activated in conjunction with a primary or classic (e.g., 'TAM-driven) activation signal, promotes or enhances T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or combinations thereof. Costimulatory signaling domains for use in CARs include, for example, CD27, CD28, CD4OL, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAPIO, LAT, LFA-I , LIGHT, NKG2C, S1 ,P76, TRIM, ZAP70, or any combination thereof In a particular embodiment, the costimulatory signaling domain comprises a 0X40, CD2, CD27, CD28, LFA-1 (CD1 la/CD18), ICOS (CD278), or 4-1BB (CD137) signaling domain. Exemplary CD28 costimulatory signaling domains that may be used in CARs of the present disclosure comprise an amino acid sequence of SEQ ID NO:26 or 4. An exemplary 4-1BB costimulatory signaling domain comprises an amino acid sequence of SEQ ID NO: i22. In certain embodiments, a CAR comprises one, two, or more costimulatory signaling domains.
In some embodiments, CARs are recombinant receptors composed of an scFv binding domain derived from an antibody, a transmembrane domain, and an intracellular signaling domain(s). In some embodiments, the intracellular signaling domain(s) are derived from a TCR.
In certain embodiments, a chimeric antigen receptor comprises an amino acid sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof In certain embodiments, chimeric antigen receptor is murine, chimeric, human, or humanized.
In certain embodiments, a CAR is a first generation CAR, a second generation CAR, or a third generation CAR. A first generation CAR generally has an intracellular signaling domain comprising an intracellular signaling domain of CD3C, FcleRI, or other ITAM-containing activating domain to provide a T cell activation signal. Second generation CARs further comprise a costimulatory signaling domain (e.g., a costimulatory signaling domain from an endogenous T cell costimulatory receptor, such as CD28, 4-1I3B, or TCOS). Third generation CARs comprise an TTAM-containing activating domain, a first costimulatory signaling domain and a second costimulatory signaling domain.
In some embodiments, one or more of the extracellular domain, the binding domain, the linker, the transmembrane domain, the intracellular signaling domain, or the costimulatory domain comprises junction amino acids. "Junction amino acids" or "junction amino acid residues" refer to one or more (e.g., about 2-20) amino acid residues between two adjacent domains, motifs, regions, modules, or fragments of a protein, such as between a binding domain and an adjacent linker, between a transmembrane domain and an adjacent extracellular or intracellular domain, or on one or both ends of a linker that links two domains, motifs, regions, modules, or fragments (e.g., between a linker and an adjacent binding domain or between a linker and an adjacent hinge). Junction amino acids may result from the construct design of a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site or self-cleaving peptide sequences during the construction of a polynucleotide encoding a fusion protein). For example, a transmembrane domain of a fusion protein may have one or more junction amino acids at the amino-terminal end, carboxy -terminal end, or both.
Exemplary binding domain, extracellular spacer, transmembrane, and intracellular signaling domain sequences for use in CAR.s of the present disclosure are set forth in Table 11.
Table H.
Name Amino Acid Sequence SEQ ID
NO:
Flexible linker GGGGSGGGGSGGGGS
Flexible linker GTGG'SGGTGSGTGIS
1gG4 hinge .ESK.YGPPC.PPC.P
region (short) CD8a hinge TT.TPAPRPP-rpAPTIASQPLSLRITACRPAAGGAVErrit region GLDFACD
CD28 hinge IEVMYPPPYLDNEKSNGTI1HVKGKHLCPSPLFPGPSKP
region CD8a IYIWAPLAartGVLLLSLAIrrLYC
transmembrane region transmembrane FW VLVVVGGVLACYSLLVTVAHIFWV
region wildtype CD3 R'VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD
signaling ICRRGRDPEMGGKPQRRICNPQEGLYNELQKDKMAEA
domain YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM
QALPPR
variant CD3C, R'VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD
signaling 1CRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY
domain SE1GMKGERRRGKGHDGLYQGLsTATKDTYDALH1Q
ALPPR
wild.type CD28 RSICRSRLLHSDYNLNMTPRRPGPTRICHYOPYAPPRDFA
costimulatory AYRS
signaling domain variant CD28 RSICRSRGGHSDYNLNMTPRRPGPTRICHYQPYAPPRDF
CoStimulatory AAYRS
signaling domain with substitutions with positions in reference to full length protein costimulatory GGCEI.:
signaling domain C1MCSFR_Sign MLLVTSLLLCELPHPAFLLIP
al Peptide In certain embodiments, a chimeric Tim receptor modified host cell co-expresses a recombinant TCR. Recombinant TCR proteins include "traditional"
TCRs composed of a heterodimer of a chain polypeptide and chain polypeptide or a heterodimer of a y chain polypeptide and a 5 chain polypeptide, binding fragments and fusion proteins thereof, including for example, single chain TCRs, single domain TCRs, soluble TCR fusion TCR proteins, and TCR fusion constructs (TRuCTm). In certain embodiments, a tandem expression cassette comprises a polynucleotide encoding a recombinant TCR beta chain comprising a TCR beta variable region and a TCR
beta constant region, and a polynucleotide encoding a recombinant TCR alpha chain comprising a TCR. alpha variable region and a TCR. alpha constant region. In certain embodiments, a recombinant TCR is an enhanced affinity TCR. In one embodiment, a recombinant TCR is an enhanced affinity TCR.
In certain embodiments, a recombinant TCR binding protein is a single chain TCR (scTCR) comprising a Va joined to a vi3 by a flexible linker. In some embodiments, a scTCR comprises a Va-linker-V13 polypeptide. In other embodiments, a scTCR comprises a V134inker-Va polypeptide.
in certain embodiments, a chimeric Tim receptor modified host cell may also be modified to co-express a single chain TCR (scTCR) fusion protein. A
scTCR
fusion protein comprises a binding domain comprising a scTCR (a TcR Va domain linked to a TCR V13 domain), an optional extracellular spacer, a transmembrane domain, and an intracellular component comprising a single intracellular signaling domain providing an T cell activation signal (e.g., a CD31; ITAM-containing activating domain) and optionally a costimulatory signaling domain (see, Aggen etal., 2012, Gene Ther. 19:365-374; Stone etal., Cancer Immunol. Immunother. 2014, 63:1163-76).
In certain embodiments, a chimeric Tim receptor modified host cell may also be modified to co-express a T cell receptor-based chimeric antigen receptor (TCR-CAR). A TCR-CAR is a heterodimeric fusion protein generally comprising a soluble TCR (a polypeptide chain comprising a Vu domain and Ca domain and a polypeptide chain comprising a V13 domain and a C13 domain) wherein the v3c:13 polypeptide chain is linked to a transmembrane domain and an intracellular signaling component (e.g., an ITAM-containing activating domain and optionally a costimulatory signaling domain) (see, e.g., Wa1seng et al., 2017 Scientific Reports 7:10713).
In certain embodiments, an engineered host cell that co-expresses a chimeric Tim receptor and a cellular immunotherapy agent (e.g., CAR., 'FCR, etc.) comprises a recombinant nucleic acid encoding the chimeric Tim receptor and a recombinant nucleic acid molecule encoding the cellular immunotherapy agent on separate vectors within the engineered host cell.
In some embodiments, an engineered host cell that co-expresses a chimeric Tim receptor and a cellular immunotherapy agent (e.g., CAR., TeR, etc.) comprises a recombinant nucleic acid encoding the chimeric Tim receptor and a recombinant nucleic acid molecule encoding the cellular immunotherapy agent on the same vector as the chimeric Tim receptor within an engineered host cell. The chimeric Tim receptor and cellular immunotherapy agent may be expressed under the regulation of different promoters on the same vector (e.g., at different multiple cloning sites).
Alternatively, the chimeric Tim receptor and cellular immunotherapy agent may be expressed under the regulation of one promoter in a multicistronic vector (e.g., tandem expression vector). The polynucleotide sequence encoding the chimeric Tim receptor and the polynucleotide sequence encoding the cellular immunotherapy agent may be separated by an IRES or viral 2A peptide in a multicistronic vector.
Tandem expression cassettes, tandem expression vectors, and engineered host cells comprising the same are described in International Application Publication No. W02019/191339, which is incorporated herein by reference in its entirety.
Host cells expressing chimeric Tim receptors may be administered to a subject alone, or in combination with other therapeutic agents, including for example CAR-T cells, TCRs, antibodies, radiation therapy, chemotherapies, small molecules, oncolytic viruses, electropulse therapy, etc.
In certain embodiments, the chimeric Tim receptor and adoptive cellular immunotherapy agent (e.g., a CAR, TCR-CAR, TCR, etc. described above) are administered to the subject in the same host cell or different host cells. In certain embodiments, the chimeric Tim receptor and adoptive cellular immunotherapy agent are expressed in the same host cell from the same vector or from separate vectors. In certain embodiments, the chimeric Tim receptor and adoptive cellular immunotherapy agent are expressed in the same host cell from a multicistronic vector. In certain embodiments, the chimeric Tim receptor is expressed in the same host cell type as the adoptive cellular immunotherapy agent (e.g., the chimeric Tim receptor is expressed CD4 T cells and the CAR/or TCR is expressed in CD4 T cells, or the chimeric Tim receptor is expressed CD8 T cells and the CAR/or TCR is expressed in CD8 T
cells).
In other embodiments, the chimeric Tim receptor is expressed in a different host cell type as the adoptive immunotherapy agent (e.g., the chimeric Tim receptor is expressed CD4 T cells and the CAR/or TCR is expressed in CD8 T cells). Cellular immunotherapy compositions comprising a combination of immune cells or cellular subsets engineered with chimeric Tim receptors and a cellular immunotherapy agent (e.g., CAR, TCR, etc.), methods of making, and methods of use are described in PCT
International Publication No. W02019/191340, which is incorporated herein by reference in its entirety.
Exemplary antigens that a recombinant TCR, enhanced affinity TCR, CAR, TCR-CAR, or sc*TCR. fusion protein may target include WT-1, mesothelin, MART-1, NY-ESO-1, MAGE-A3, HPV E7, survivin, a Fetoprotein, and a tumor-specific neoantigen.
CARs of the present disclosure may target a variety of antigens, including a viral antigen, bacterial antigen, fungal antigen, parasitic antimn, tumor antigen, autoimnnune disease antigen. Exemplary antigens that a CAR may target include CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, M1JC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA.125, CD56, c-Met, EGFR, GD-3, HPV E6, IIPV E7, MUC-1, HER2, folate receptor a, CD97, CD171, CD179a, CD44v6, WTI, VEGF-a, VEGFR1, IL-13Ral, IL-13R2, IL-11R.a, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-Al, RAGE-1, folate receptor p, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GI\41, GM3, o-acetyl-GD2, and GD2.
Radiation therapy includes external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiation, 3-dimensional conformal radiation therapy, intensity-modulated radiation therapy, volumetric modulated arc therapy, particle therapy, proton therapy, and auger therapy), brachytherapy, systemic radioisotope therapy, intraoperative radiotherapy, or any combination thereof.
Exemplary antibodies for use in conjunction with the chimeric Tim compositions described herein include rituxmab, pertuzumab, trastuzumab, alenituzumab, Ibrituniornab tiuxetan, Brentuximab vedotin, cetuximab, bevacizurnab, abciximab, adalimumab, alefacept, basilizimab, belitnumab, bezlotoxumab, canakinumab, certolizumab pegol, daclizumab, denosumab, efalizumab, golimumab, olaratumab, palivizumab, panitumumab, and tocilizumab.
Exemplary inhibitors of immune checkpoint molecules that may be for use in conjunction with the chimeric Tim compositions described herein include checkpoint inhibitors targeting PD-I,1, PD-1.2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CI:LA-4, BTLA, KIR, LAG3, T1M3, A2alt, CD244/2B4, CD160, mu, LAIR-1, PVRIG/CD112R, or any combination thereof. In certain embodiments, an immune checkpoint inhibitor may be an antibody, a peptide, an RNAi agent, or a small molecule. An antibody specific for CTLA-4 may be ipilimumab or tremelimumab.
An antibody specific for PD-1 may be pidilizumab, nivolumab, or pcmbrolizumab. An antibody specific for PD-Li may be durvalumab, atezolizumab, or avelumab.
Exemplary chemotherapeutics for use in conjunction with the chimeric Tim receptor compositions described herein may include an alkylating agent, a platinum based agent, a cytotoxic agent, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor.
A chemotherapeutic includes non-specific cytotoxic agents that inhibit mitosis or cell division, as well as molecularly targeted therapy that blocks the growth and spread of cancer cells by targeting specific molecules that are involved in tumor growth, progression, and metastasis (e.g., oncogenes). Exemplary non-specific chemotherapeutics for use in conjunction with the expression cassette compositions described herein may include an alkylating agent, a platinum based agent, a cytotoxic agent, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as fol ate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA
synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), hypomethylating agent, and a DNA repair inhibitor.
Examples of chemotherapeutic agents considered for use in combination therapies contemplated herein include vemurafenib, dabrafenib, trametinib, cobimetinib, anastrozole (Arimidexe), bicalutamide (Casodex0), bleomycin sulfate (Blenoxanee), busulfan (Myleran0), busulfan injection (Busulfex0), capecitabine (Xeloda6), N4-pentoxycarbony1-5-deox y-5-fluorocyti dine, carboplatin (Paraplatine), camiustine (BiCNUO), chlorambucil (Leukeran ), cisplatin (Platinole), cladribine (Leustatin0), cyclophosphamide (Cytoxan or NeosarS), cytarabine, cytosine arabinoside (Cytosar-U0), cytarabine liposome injection (DepoCyte), dacarbazine (DTIC-Dome ), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidinee), daunorubicin citrate liposome injection (DaunoXomee), dexamethasonc, docctaxcl (Taxoterce), doxorubicin hydrochloride (Adriamycine, Rubex ), etoposi de (Vepesida), fludarabine phosphate (FludaraV), 5-fluorouracil (Adrucile, Efudexe), flutamide (Eulexine), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea (HydreaC), Idarubicin (Idamycine), ifosfamide (IFEM.)), irinotecan (Camptosare), L-asparaginase (ELSPARO), leucovorin calcium, melphalan (Alkeran0), 6-mercaptopurine (Purinethole), methotrexate (Folexe), mitoxantrone (Novantrone ,), myiotarg, paclitaxel (Taxo10), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadele),fdabra tamoxifen citrate (Nolvadex0), teniposide (Vumone), 6-thioguanine, thiotepa, tirapazamine (Tirazone0), topotecan hydrochloride for injection (Ilycamptine), vinblastine (Velban0), vincristine (Oncovine), ibrutinib, venetociax, crizotinib, aiectinib, brigatinib, ceritinib, and vinorelbine (Navelbine0).
Exemplary alkylating agents for use in combination therapies contemplated herein include nitrogen mustards, ethyleni mine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard , Chlorethaminacile, Demethyldopan , Desmethyldopan , Haemanthamine , Nordopan , lifracil nitrogen Mustard , Uracilloste, Uracilmostaza , Uramustin , Uramustinee), chiormethine (Mustargene), cycl ophosphami de (Cytoxane, Neosar , Clafene, Endoxane, :Procytox , Revi rninuneTm), ifosfamitle (Mitoxana0), melphalan (Alkeran0), Chloramtbucil (Leukerane), pipobroman (Amedel , Vercyte ), triethylenemelamine (Hemel , Hexa.lene, Hexastate), triethylenethiophosphoramine, Temozolomide (Tem od a re), thiotepa (Thiopl ex ), busulfan (Busilvex , Mylerang), carmustine (BiCNUO), lomustine (CeeNUO), streptozocin (Zanosare), and Dacarbazine (DTIC:-Dome ). Additional exemplary alkylating agents for use in combination therapies contemplated herein include, without limitation, Oxaliplatin (Eloxatine); Temozolomide (Temodare and Temodale); Dactinomycin (also known as actinomycin-D, Cosmegen0); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran0); Altretamine (also known as hexamethylmelamine (11M114), Hexalen0); Carmustine (BiCNUID); Bendamustine (Treandae); Busulfan (Busulfex and Mylerame); Carboplatin (Paraplatine); Lomustine (also known as CCNU, CecNUO); Cisplatin (also known as CDDP, Platinol and Platinol -AQ);
Chiorambucil (LeukeranS); Cycl ophosphami de (Cytoxan and Neosar0);
Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-DomeS); Altretamine (also known as hexamethylmelamine (IIMM), Hexalen8); Ifosfamide (Ifex0);
Prednumustine; Procarbazine (Matulane6); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen0); Streptozocin (Zanosar0); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex )); Cyclophosphamide (Endoxan , CytoxanCiD, Neosar , Procytox , Revimmunee); and I3endamustine HCl (Treanda0).
Exemplary platinum based agents for use in combination therapies contemplated herein include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthriplatin, and triplatin tetranitrate.
Exemplary hypomethylating agents for use in combination therapies include azacitidine and decitabine.
Exemplary molecularly targeted inhibitors for use in conjunction with the chimeric Tim receptor compositions described herein include small molecules that target molecules involved in cancer cell growth and survival, including for example, receptor tyrosine kinase inhibitors, RAF inhibitors, BCL-2 inhibitors, ABL
inhibitors, TRK inhibitors, c-K:IT inhibitors, c-MET inhibitors, CDK4/6 inhibitors, FAK
inhibitors, FGFR inhibitors, FLT3 inhibitors, IDIII inhibitors, IDF12 inhibitors, PDGFRA inhibitors, and RE!' inhibitors Exemplary molecularly targeted therapy includes hormone antagonists, signal transduction inhibitors, gene expression inhibitors (e.g., translation inhibitors), apoptosis inducers, angiogenesis inhibitors (e.g., a VEGF pathway inhibitor), tyrosine kinase inhibitors (e.g., an EGF/EGFR pathway inhibitor), growth factor inhibitors, GTPase inhibitors, serine/threonine kinase inhibitors, transcription factor inhibitors, inhibitors of driver mutations associated with cancer, B-Raf inhibitors, RAF
inhibitors, a MEK inhibitors, mTOR inhibitors, adenosine pathway inhibitors, EGFR
inhibitors, PI3K inhibitors, BCL2 inhibitors, VEGFR inhibitors, MET inhibitors, MYC
inhibitors, BCR-ABL inhibitors, ABL inhibitors, HER2 inhibitors, IT-RAS inhibitors, K-RAS
inhibitors, PDGFR inhibitors, ALK inhibitors, ROSI inhibitors, BTK inhibitors, TRK
inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4/6 inhibitors, FAK
inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PARP
inhibitors, PARP inhibitors, PDGFRA inhibitors, and RET inhibitors. In certain embodiments, use of molecularly targeted therapy comprises administering a molecularly targeted therapy specific for the molecular target to a subject identified as having a tumor that possesses the molecular target (e.g., driver oncogene). in certain embodiments, the molecular target has an activating mutation. In certain embodiments, use of chimeric Tim receptor modified cells in combination with a molecularly targeted inhibitor increases the magnitude of anti-tumor response, the durability of anti-tumor response, or both. In certain embodiments, a lower than typical dose of molecularly targeted therapy is used in combination with chimeric Tim receptor modified cells.
Exemplary angiogenesis inhibitors include, without limitation A6 (Angstrom Pharmaceuticals), ABT-510 (Abbott Laboratories), AB`17-627 (Atrasentan) (Abbott Laboratories/Xinlay), ABT-869 (Abbott Laboratories), Actimid (CC4047, Pomalidomide) (Celgene Corporation), AdGVPEDF.11D (GeriVec), ADH-1 (Exherin) (Adherex Technologies), AEE788 (Novartis), AG-013736 (Axitinib) (Pfizer), (Prinomastat) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX5 I
(AngioGenex), ALN-VSP (ALN-VSP 02) (Alnylaxn Pharmaceuticals), AMG 386 (Amgen), AMG706 (Amgen), Apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573 (Ridaforolimus/MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ
197 (ArQule), ASA404 (Novartis/Antisoma), Ati pri mod (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals), Avastin (Bev acizumab) (Genentech), AZD2171 (Cediranib/Recentin) (AstraZeneca), BAY 57-9352 (Telati nib) (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim Pharmaceuticals), 13113W 2992 (Boehringer Ingelheim Pharmaceuticals), :BMS-275291 (Bristol-Myers Squibb), BM:S-582664 (Brivanib) (Bristol-Myers Squibb), BMS-690514 (Bristol-Myers Squibb), Calcitriol, CCI-779 (Torisel) (Wyeth), CDP-791 (ImClone Systems), Ceflatonin (Homoharringtonine/HHT) (ChemGenex Therapeutics), Celebrex (Celecoxib) (Pfizer), CEP-7055 (Ccphalon/Sanofi), CHIR-265 (Chiron Corporation), NGR-TNF, COL-3 (Mctastat) (Collagenex Pharmaceuticals), Combretastatin (Oxigene), CP-751,871(Figitumumab) (Pfizer), CP-547,632 (Pfizer), CS-7017 (Daiichi Sankyo Pharma), CT-322 (Angiocept) (Adnexus), Curcumin, Dalteparin (Fragmin) (Pfizer), Disulfiram (Antabuse), (Eisai Limited), E7080 (Eisai Limited), EMD 121974 (Cilengitide) (EMD
Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), Endostar (Simcere), Erbitux (linClone/I3ristol-Myers Squibb), EZN-2208 (Enzon Pharmaceuticals), EZN-2968 (Enzon Pharmaceuticals), GC1008 (Genzyme), Genistein, GSK1363089 (Foretinib) (GlaxoSmithKline), GW786034 (Pazopanib) (GlaxoSmithKline), GT-111 (Vascular Biogenics Ltd.), IMC-112113 (Ramucirumab) (imClone Systems), IMC-18F1 (imClone Systems), IMC-3G3 (ImClone LLC), INCB007839 (Incyte Corporation), INGN 241 (Introgen Therapeutics), Iressa (ZD1839/Gefitinib), LBH589 (Faridak/Panobinostst) (Novartis), Lucentis (Ranibizumab) (Genentech/Novartis), LY317615 (Enzastaurin) (Eli Lilly and Company), Macugen (Pegaptanib) (Pfizer), MEDI522 (Abegrin) (MedImmune), MLN518 (Tandutinib) (Millennium), Neovastat (AE941/Benefin) (Aeterna Zentaris), Nexavar (Bayer/Onyx), NM-3 (Genzyme Corporation), Noscapine (Cougar Biotechnology), NPI-2358 (Nereus Pharmaceuticals), OSI-930 (051), Pal amid 529 (Paloma Pharmaceuticals, Inc.), Parizein Capsules (2ME2) (EntreMed), Panzem NCD
(2ME2) (EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen lndustries/Medigen Biotechnology), PKC412 (Novartis), Polyphenon E (Green Tea Extract) (Polypheno E International, Inc.), PPI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (Vatalanib) (Novartis), PXD101 (Belinostat) (CuraGen Corporation), ltADOO I (Everolimus) (Novartis), RAF265 (Novartis), Regorafenib (BAY73-4506) (Bayer), Revlimid (Celgene), Retaane (Alcon Research), SN38 (Liposomal) (Neopharm), SNS-032 (BMS-387032) (Sunesis), S0M230 (Pasireotide) (Novartis), Squalamine (Genaera), Suramin, Sutent (Pfizer), Tarceva (Genentech), TB-403 (Thrombogenics), Tempostatin (Collard Biopharmaceuticals), Tetrathiomolybdate (Sigma-Aldrich), TG100801 (TargeGen), Thalidomide (Celgene Corporation), Tinzaparin Sodium, TKI258 (Novartis), IRC093 (Tracon Pharmaceuticals Inc.), VEGF Trap (Aflibercept) (Regencron Pharmaceuticals), VEGF
Trap-Eye (It egeneron Pharmaceuticals), Veglin (VasGene Therapeutics), Bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), Vorinostat (Merck), and ZSTK474.
Exemplary B-Raf inhibitors include vemurafenib, dabrafenib, and encorafenib.
Exemplary MEK inhibitors include binimetinib, cobimetinib, refameti nib, selumetinib, and trameti nib.
Exemplary BTK inhibitors include ibrutinib. Loxo-305, tirabrutinib, GDC-0853, acalabrutinib, ONO-4059, spebrutinib, BGB-3111, 1-1M71224, and M7583.
Exemplary TRK inhibitors include entrectinib, larotrectinib, CH7057288, ONO-7579, LOX0-101, lestaurtinib, and LOX0-195.
Exemplary c-KIT inhibitors include imatinb, sunitinb, and ponatinib.
Exemplary c-MET inhibitors include capmatinib, crizotinib, tivantinib, onartuzumab, INCB28060, AMG-458, savolitinib, and tepotinib.
Exemplary CDK4/6 inhibitors include palbociclib, ribociclib, abemaciclib, and trilaciclib Exemplary FAK inhibitors include defactinib, GSK2256098, B1853520, and PF-00562271.
Exemplary FGFR inhibitors include erdafitinib, pemigatinib, infigratinib, rogaratinib, AZD4547, BG.1398, FP-1039, and ARQ 087.
Exemplary FLT-3 inhibitors include quizartinib, crenolanib, gilteritinib, midostaurin, and lestaurtinib.
Exemplary I1)111 inhibitors include ivosidenib, BAY-1436032, and AGI-5198.
An exemplary IDH2 inhibitor includes enasidenib.
Exemplary PDGFRA inhibitors include imatinib, regorafenib, crenolanib, and olaratumab.
Exemplary pan-RAF inhibitors include belvarafenib, LX11254, LY3009120, INU-152, and HM95573.
Exemplary RET inhibitors include lenvatinib, alectinib, vandetanib, cabozantinib, BLU-667, and LOX0-292.
Exemplary ROS1 inhibitors include ceritinib, lorlatinib, entrectinib, crizotinib, TPX-0005, and DS-6051b.
Exemplary Vascular Endothelial Growth Factor (VEGF) receptor inhibitors include, but are not limited to, Bevacizumab (Avasting), axitinib (Inlyta0);
Brivanib alaninate (BMS-582664, (S) ......... ((R)-1-(4-(4-Fluoro-2-methy1-1H-indo1-5-yloxy)-5-methylpyrrolo[2,1-][1,2,4]triazin-6-yloxy)propan-2-y1)2-aminopropanoate);
Sorafenib (Nexavar0); Pazopanib (Votriente); Sunitinib malate (Sutent0);
Cediranib (AZD2171, CAS 288383-20-1); Vargatef (BIBF1120, CAS 928326-83-4); :Foretinib (GSK1363089); Telatinib (BAY57-9352, CAS 332012-40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib (Gleevece); Ponatinib (AP24534, CAS 943319-70-8);
Tivozanib (AV951, CAS 475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Vatalanib dihydrochloride (PTK787, CAS 212141-51-0); Brivanib (BMS-540215, CAS 649735-46-6); Vandetanib (Caprelsa or AZD6474); M:otesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethy1-1H-indo1-6-y1)-2-[(4-pyridinylmethyl)arnino]-3-pyridinecarboxamide, described in PCT Publication No. WO
02/066470); Dovitinib dilactic acid (TK1258, CAS 852433-84-2); Linfanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 849217-68-1); Lestaurtinib (CAS
111358-88-4); N15-M5-(1, 1-Di methylethyl)-2-oxazolylimethyl ]thio]-2-thiazoly1]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-Amino-1-044(3-methoxyphenyl)am in o)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)pi peridin-3-ol (BMS690514); N-(3,4-Di chloro-2-11 uoroph en yl )-6-methoxy-7-[[(3aa,513,6aa)-octahydro-2-methylcyclopenta[c]pyrrol-5-Amethoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); 4-Methy1-34[1-methyl-6-(3-pyridiny1)-1H-pyrazolo[3,4-d]pyrimidin-4-yliamino]-N-[3-(trifluoromethypphenyl]-benzamide (BHG712, CAS
940310-85-0); and Aflibercept (Eylea0).
Exemplary EGF pathway inhibitors include, without limitation tyrphostin 46, EKB-569, crlotinib (Tarccvaa), gcfitinib (Ircssa0), crbitux, ninnotuzumab, lapatinib (Tykerbe), cetuximab (anti-EGFR mAb), 188Re-1abe1ed nimotuzumab (anti-EGFR mAb), and those compounds that are generically and specifically disclosed in WO 97/02266, EP 0 564 409, WO 99/03854, EP 0 520 722, EP
0 566 226, EP 0 787 722, EP 0 837 063, U.S. Pat. No. 5,747,498, WO 98/10767, WO
97/30034, WO 97/49688, WO 97/38983 and WO 96/33980. Exemplary EGFR
antibodies include, but are not limited to, Cetuximab (Erbitux ); Panitumumab (Vectibix ); Matuzumab (EMD-72000); Trastuzumab (Herceptin0); Nimotuzumab (fiR3); Zalutumumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1). Exemplary Epidermal growth factor receptor (EGFR) inhibitors include, hut not limited to, Erlotinib hydrochloride (Tarceva0);
ceritinib;
brigatinib; osimeritinib; icotinib; Gefitnib (Iressa0); N44-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3"S")-tetrahydro-3-furanyfloxy]-6-quinazoliny1]-4(diinethylamino)-2-butenainide, 17ovoke); Vandetanib (Caprelsa0); Lapatinib (Tykerb ); (3R,4R)-4-Amino-1-04-((3-methoxyphenypamino)pyrrolo[2.,1-f][1,2,4]triazin-5-yOmethyl)piperidin-3-ol (BMS690514); Canertinib dihydrochloride (C1-1033); 6-[4-[(4-Ethyl-1-pi perazinypmethyl ]pheny1]-N-[(1R)-1-pheny I
ethyl]-7H-Pyrrolo[2,3-d]pyrimidin-4-amine (AF:E788, CAS 497839-62-0); Mubritinib (TAK165);
Pelitinib (EKB569); Afatinib (BIEW2992); Netatinib (HK1-272); N-[44[1-[(3-Fluorophenyl)methy1]-1H-indazol-5-yflaminoj-5-methylpyrrolo[2,1-1][1,2,4]triazin-6-y1]-carbamic acid, (3S)-3-morpholinylmethyl ester (B1\4S599626); N-(3,4-Dichloro-2-fluoropheny1)-6-methoxy-7-[[(3aa,50,6aa)-octahydro-2-methylcyclopenta[c]pyrrol-yllmethoxyl-4-quinazolinamine (X1,647, CAS 781613-23-8); 4-[44(1R)-1-Phenylethyliamino]-7H-pyrrolo[2,3-d]pyrimidin-6-y1Fphenol (PK1166, CAS 187724-61-4); rocelitinib Exemplary mTOR inhibitors include, without limitation, rapamycin (RaparnuneS), and analogs and derivatives thereof; SDZ-RAD; Temsirolimus (Torisele; also known as CCI-779); Ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,95,12S,15R,16E,18R,19Rõ21R,23 S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hcxamethy1-2,3,10,14,20-pcntaoxo-11,36-dioxa-azatri cycl o[30 .3.1 . 04'9]hex atri aconta-16,24,26,28-tetraen-12-yl]propy1]-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); Everolimus (Afinitor or RAD001); Rapamycin (AY22989, SirolimusS); Simapimod (CAS 164301-51-3); (5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-y1}-2-methoxyphenyl)methanol (AZD8055); 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridiny1)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N241,4-dioxot[4-(4-oxo-8-pheny1-4H-1-benzopyran-2-yOmorpholinium-4-yl]methoxyibuty1]-1..-arginylglycyl-L-a-aspartylL-serine-, inner salt (SF1126, CAS 936487-67-1).
Exemplary Phosphoinositide 3-kinase (PI3K) inhibitors include, but are not limited to, duvelisib, idelalisib, 442-(1H-Indazol-4-y1)-64[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-ylimorpholine (also known as GDC 0941 and described in PCT Publication Nos. WO 09/036082 and WO
09/055730); 2-Methy1-244-[3-methyl-2-oxo-8-(quinolin-3-y1)-2,3-dihydroimidazo[4,5-clquinolin-l-yl]phenylipropionitrile (also known as BEZ 235 or NVP-BEZ 235, and described in PCT Publication No. WO 06/122806); 4-(trifluoromethyl)-5-(2,6-ditnorpholinopyritnidin-4-yl)pridin-2-amine (also known as BKM120 or N VP-BKM120, and described in PCT Publication No. W02007/084786); Tozasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)-54[4-(4-Pyridiny1)-6-quinolinyl]methyleneF
2,4-thiazolidinedi one (GSK1059615, CAS 958852-01-2); (1E,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-14(di-2-propenylamino)methylenei-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(tnethoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtlio[1,2-c]pyran-2,7,10(11-1)-trione (PX866, CAS 502632-66-8); and 8-Pheny1-2-(morpholin-4-y1)-chromen-4-one (LY294002, CAS 154447-36-6). Exemplary Protein Kinase B (PKB) or AKT inhibitors include, but are not limited to. 8-[4-(1-Aminocyclobutyl)pheny1]-9-pheny1-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one (MK-2206, CAS 1032349-1); Perifosine (KRX0401); 4-Dodecyl-N-1,3,4-thiadiazol-2-y1 -benzenesulfonamide (PHT-427, CAS 1191951-57-1); 442-(4-Amino-1,2,5-oxadiazo1-3-y1)-1-ethy1-7-[(3S)-3-piperidinylmethoxy]-1H-imidazo[4,5-c]pyridin-4-y1]-2-mothyl-3-butyn-2-ol (GSK690693, CAS 937174-76-0); 8-(1-Hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6-one (palomid 529, P529, or SG-00529); Tricirbine (6-Amino-4-methy1-8-(13-D-ribofuranosyl)-4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine), (ctS)-a-[[[5-(3-Methyl-1H-indazol-5-yl)-3-pyridinyl]oxylmethyl:1-benzeneethanamine (A674563, CAS 552325-73-2); 4-[(4-Chlorophenyl)methy1]-147H-pyrrolo[2,3-d]pyrimidin-4-y1)-4-piperidinamine (CCT128930, CAS 885499-61-6); 4-(4-Chloropheny1)-444-(1H pyrazol-4-yl)pheny1]-piperidine (AT7867, CAS 857531-00-1); and Archexin (RX-0201, CAS 663232-27-7).
In certain embodiments, a tyrosine kinase inhibitor used in combination with chimeric Tim receptor modified cells is an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK inhibitors include crizotinib, ceritinib, alectinib, bnigatinib, dalantercept, entrectinib, and lorlatinib.
In certain embodiments where chimeric 'I'im receptor modified cells are administered in combination with one or more additional therapies, the one or more additional therapies may be administered at a dose that might otherwise be considered subtherapeutic if administered as a monotherapy. In such embodiments, the chimeric Tim receptor composition may provide an additive or synergistic effect such that the one or more additional therapies can be administered at a lower dose.
Combination therapy includes administration of a chimeric Tim receptor compositions as described herein before an additional therapy (e.g., 1 day to 30 days or more before the additional therapy), concurrently with an additional therapy (on the same day), or after an additional therapy (e.g., 1 day - 30 days or more after the additional therapy). In certain embodiments, the chimeric Tim receptor modified cells are administered after administration of the one or more additional therapies. In further embodiments, the chimeric Tim receptor modified cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration of the one or more additional therapies. In still further embodiments, the chimeric Tim receptor modified cells are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week after administration of the one or more additional therapies. Where the one or more additional therapies involves multiple doses, the chimeric Tim receptor modified cells may be administered after the initial dose of the one or more additional therapies, after the final dose of the one or more additional therapies, or in between multiple doses of the one or more additional therapies.
In certain embodiments, methods of the present disclosure include a depletion step. A depletion step to remove chimeric Tim receptors from the subject may occur after a sufficient amount of time for therapeutic benefit in order to mitigate toxicity to a subject. In such embodiments, the chimeric Tim receptor vector may include an inducible suicide gene, such as 1CASP9, inducible Fas, or HSV-TK.
Similarly, a chimeric Tim receptor vector may be designed for expression of a known cell surface antigen such as CD20 or truncated EGFR (SEQ TD NC):16) that facilitates depletion of transduced cells through infusion of an associated monoclonal antibody (mAb), for example, Rituximab for CD20 or Cetuximab for EGFR. Alemtuzumab, which targets CD52 present on the surface of mature lymphocytes, may also be used to deplete transduced B cells, T cells, or natural killer cells.
Subjects that can be treated by the compositions and methods of the present disclosure include animals, such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs The subject may be male or female, and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled.
Accordingly, the claims are not limited by the disclosure.
EXAMPLES
EVALUATING TIM-CER-T CELLS FOR SYNTHETIC LETHAL PARP INHIBITOR SYNERGISMS
IN OVARIAN CANCER
Kuramochi ovarian cancer cells expressing an mCherry-NLS plasmid were incubated with 0.78 to 100 uM of the PARP inhibitor Niraparib. The number of Kuramochi cells / well was tracked via imaging. At t-50, the number of Kuramochi cells in culture decreases monotonically with increasing Niraparib concentration. The results are shown in FIG. IA.
Similar experiments were performed at various doses of the PARP
inhibitors Niraparib, Olaparib, Talazoparib, Veliperib, and Rucaparib, and the number of Kuramochi cells was plotted at t=50 h. Talazoparib and Niraparib exhibit the most sensitive EC50 values, followed by Rucaparib and Olaparib. Veliparib has the least impact on the number of detected Kuramochi cells of the five PARP inhibitors tested.
Niraparib and Rucaparib almost eliminate the Kuramochi culture by t=50h. The results are shown in FIG. 1B.
PAM' INHIBITOR UFREGULATES SURFACE PHOSPHATIDYLSERINE AND HAS NEGLIGIBLE
Kuramochi cells expressing a mCherry-NLS plasmid were incubated with various concentrations of Niraparib, a Tim4-Fc chimera, and an anti-mouse IgG2a antibody conjugated to Alexa488. The number of Tim4+ Kuramochi cells (i.e., green objects) increases significantly above the IgG only control in response to incubation with even 1.56 pivI Niraparib (FIGS. 2A-2B). The number of Kuramochi cells increases in response to 1.56-12.5 l.LM Niraparib (FIG. 2C), indicating that PARP
inhibition induces surface phosphatidylserine without killing Kuramochi cells.
Co-cultures of primary human CD4+ and CD8+ T cells were incubated with various concentrations of Niraparib and T cell viability, diameter, and density were measured using a ViCell cell counter on days 7, 8, and 9 after activation with TransAct, corresponding to days 1, 2, and 3 after incubation with Niraparib. T cell viability, average diameter, and expansion from day 7 to day 9 exhibit negligible changes as a function of Niraparib concentration (FIG. 3).
CHIMERIC TIM RECEPTOR/ NIRAPARM SYNERGISTICALLY KILL K.URAMOCHI CELLS /N
HMO AT DIFFERENT DOSES
Kuramochi cells expressing a mCherry-NLS plasmid were incubated for hours with 25, 12.5, or 6.25 M. Niraparib. The next day the Niraparib concentration was reduced to 0.521.IM and co-cultures of primary human CD4+ and CD8+ T cells expressing chimeric Tim receptor construct 13A (TIM4 binding domain-CD28 15 transmembrane domain ¨ CD28 costimulatory domain- CD3t; signaling domain) (SEQ
ID NO:162) were added to the Kuramochi culture. T cell cultures expressing chimeric Tim receptor construct 13A significantly reduce the number of Kuramochi cells in culture below the mock transduction control (control-T), depending on the experimental conditions. The number of Kuramochi cells expressing mCherry-NLS was monitored 20 via IncuCyte. The results are shown in FIG. 4. Combination therapy with chimeric Tim receptor Construct 13A and niraparib synergistically kill Kuramochi cells at different doses.
Kuramochi cells expressing a mCherry-NLS plasmid were incubated for ¨20 hours with 1.56 p,M Niraparib. The next day, media were removed from cells and replaced with fresh media with 0 1.1.M Niraparib, and primary human CD4+ and CD8+ T
cells expressing chimeric Tim receptor CTX140 (Tim4 binding domain-CD28 hinge-CD28 transmembrane-CD28 costimulatory domain-DAP12)(SEQ ID NO:195) or CTX156 (tEGFR control) were added to the Kuramochi culture. The number of Kuramochi cells expressing mCherry-NLS was monitored via IncuCyte. The results are shown in FIG. 5. T cell cultures expressing chimeric Tim receptor construct significantly reduce the number of Kuramochi cells in culture below the mock transduction control.
2,500 Kuramochi ovarian cancer cells expressing an mCherry-NLS
plasmid were plated on day 0. 25, 12.5, or 6.25 !AM Niraparib were added to cell culture -4 hours later. -20 hours after initial Niraparib dose, primary human CD4+ and CD8-F- T cells expressing chimeric Tim receptor CTX137 (Tim4 binding domain-transmembrane-CD28 costirnulatory domain-DAP12) (SEQ ID NO:197) or CTX156 (tEGFR control) and maintenance dose of 0.52 pM Niraparib were added. The results for 25 M. Niraparib pre-treatment are shown in FIG. 6. The results for 25, 12.5, or 6.25 pM Niraparib pre-treatment with varying effector:target (E:T) ratios are shown in FIG.
7. T cell cultures expressing chimeric Tim receptor construct CTX137 significantly reduce the number of Kuramochi cells in culture below the mock. transduction control.
To induce Ptd-Ser exposure, BRCA-2 mutated Kuramochi cell line was treated with therapeutic doses of the PARP inhibitor Niraparib. Brief exposure to Niraparib elicits changes in membrane phospholipid symmetry, in a dose dependent manner, and is effective in inhibiting growth of Kuramochi cells (HG. 8A). The addition of chimeric Tim4 receptor pCTX133 (Tim4-TLR2-CD3z), at low effector:
target ratios (1:1), enhanced the potency of Niraparib in vitro compared to transduced-controls, demonstrating the ability of pCTX133 to elicit direct cytotoxic effects on target cells (HG. 8B).
T cell transduction and culturing: CD4 and CD8 T cells were isolated from frozen healthy donor PBMCs using Miltenyi CD4+ and CD8+ isolation kits.
and CD8 T cells were then mixed in a 1:1 ratio and incubated overnight in a flat bottom 24 well plate in OpTmizer medium supplemented with cell serum replacement, L-Glutamine, GlutaMAX, and IL-2, 1L-7, and IL-15 cytokincs, and TransAct diluted 1:50.
16-24 hours past activation with TransAct the T cells were transduced with the appropriate virus at an MO1¨ 10 with 81.1g/mL polybrene and 1:50 TransAct in a flat bottom 96 well plate. ¨24 hours after transduction the T cells were transferred to a 24 well GRex plate in OpTmizer medium supplemented with cell serum replacement, L-Glutamine, GlutaMAX, and 1L-2, 1L-7, and 1L-15 cytokines. T cells were counted and supplemented with fresh medium approximately every two days thereafter.
Cell line Engineering: A2780 (human ovarian cancer cell line) and Kuramochi (BRCA deificient human ovarian cancer cell line) cells were transduced with lentiviral vectors encoding a mCherry-Nuclear localization sequence (NLS) gene and then sorted according to mCherry expression. A2780-mCherry-NLS cell lines were clonally sorted, expanded, and banked, whereas Kuramochi-mCherry-NLS cells were expanded and banked in bulk. Cell lines were thawed and kept in culture up to ¨passage 25 in RPM] 1640 supplemented with 10% FBS.
Co-culture assays: On Day 0 2,500 A2780-mCh-NLS or Kuramochi-mCh-NLS cells were plated in a NUNC Edge 96-well plate and Niraparib (Free Base) was added ¨4 hours later at the appropriate concentration, all in RPMI 1640 10%
FBS. Approximately 20 hours later the medium was removed and replaced with the appropriate concentration of Niraparib (Free Base) and the appropriate number of T
cells, all in OpTmizer medium supplemented with cell serum replacement, L-Glutamine, GlutaMAX, and 1L-2, 1L-7, and 1L-15 cytokines. When A2780-mCh-NLS
cells were used, the supernatant was centrifuged and resuspended in T cell medium to preserve any suspension component of A2780 cells. Plates were immediately transferred to IncuCyte when appropriate.
Flow cytometry measurement of surface PS: A2780-mCh-N1S cells were treated with 1.56 or 25 11M Niraparib or with equivalent volume of DMSO
(control). 48 hours later samples were trypsinized and stained using a_Tim4-Fc followed by a tluorescently-labeled secondary antibody to the Tim4-Fc. Samples_were immediately analyzed via Flow cytometry.
As shown in FIGS. 9A-9B, T cells modified with chimeric Tim4 receptor pCTX247 (Tim4 binding domain-CD28 transmembrane CD28 signaling domain ¨ CD3.; signaling domain; SEQ ID NO:257) demonstrate in vitro anti-tumor activity in two ovarian cancer cell lines. As shown in FIG. 10, T cells modified with chimeric Tim4 receptor pCTX797 (Tim4 binding domain-CD28 transmembrane CD28 signaling domain ¨ CD3C, signaling domain; SEQ ID NO:258) demonstrate in vitro anti-tumor activity in A2780 ovarian cancer model.
As shown in FIG. 11A-11B, Phosphatidyl swine is present on the cell surface in response to Niraparib in two ovarian cancer models.
As shown in FIG. 12, chimeric Tim4 receptor pCTX797 (Tim4 binding domain-CD28 transmembrane ¨ CD28 signaling domain ¨ CD3C signaling domain;
SEQ ID NO:258) exhibits synergy with additional PARP inhibitors (rucarparib, olaparib, and niraparib) compared to truncated EGFR. (EGFRt) control (pCTX236).
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheetincluding but not limited to, U.S Provisional Patent Application No.
63/066,150, filed on August 14, 2020, U.S. Provisional Patent Application No. 63/087,049, filed on October 2, 2020, and U.S. Provisional Patent Application No. 63/226,712 filed on July 28, 2021, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled.
Accordingly, the claims arc not limited by the disclosure.
Table 9.
Construct loV
Mucin Transmembrane Primary Secondary No. Domain Domain Domain Signaling Signaling Domain Domain 19 SEQ ID SEQ ID i SEQ ID NO:8 SEQ SEQ
ID
NO:34 NO:39 g NO:47 NO:5 20 SEQ ID SEQ ID SEQ ID NO:8 SEQ ID SEQ
ID
NO:34 NO:39 NO:4 .N0:9 21 S:EQ 1D SEQ ID SEQ ID NO:7 SEQ ID SEQ
ID
NO:34 NO:39 NO:4 NO:9 in some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 8. In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 9.
In some embodiments, a chimeric Tim receptor of Construct 19 or Construct 19' comprises amino acids 25-628 of SEQ ID NO:67. In some embodiments, a chimeric Tim receptor of Construct 19 or Construct 19' comprises an amino acid sequence of SEQ ID NO:67.
In some embodiments, a chimeric Tim receptor of Construct 20 or Construct 20' comprises amino acids 25-416 of SEQ ID NO:68. In some embodiments, a chimeric Tim receptor of Construct 20 or Construct 20' comprises an amino acid sequence of SEQ ID .N0:68.
In some embodiments, a chimeric Tim receptor of Construct 21 or Construct 21 comprises amino acids 25-422 of SEQ ID NO:69. In some embodiments, a chimeric Tim receptor of Construct 21 or Construct 21' comprises an amino acid sequence of SEQ ID NO:69.
Further exemplary chimeric 'Tim receptors are described in Table 10. In some embodiments, a chimeric Tim receptor of the present disclosure comprises a construct of Table 10. In some embodiments, a chimeric Tim receptor of the present disclosure does not include a chimeric Tim receptor having the amino acid sequence of SEQ ID NO:227, 238, 249, 72, or 73. In some embodiments, a chimeric Tim receptor of the present disclosure does not include a chimeric Tim receptor having the combination of components as described for constructs SEQ ID NO:227, 238, 249, 72, or 73.
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:127 or the amino acid sequence of S:EQ ID NO:127 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO: 128 or the amino acid sequence of SEQ ED NO.128 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID
NO:129 or the amino acid sequence of SEQ ID NO:129 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:130 or the amino acid sequence of SEQ ID
NO: .130 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:131 or the amino acid sequence of SEQ ID NO:131 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:132 or the amino acid sequence of SEQ ID NO:132 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:133 or the amino acid sequence of SEQ ID NO:133 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:134 or the amino acid sequence of SEQ ID NO:134 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:135 or the amino acid sequence of SEQ ID NO:135 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:136 or the amino acid sequence of SEQ ID NO:136 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:137 or the amino acid sequence of SEQ ID NO:137 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:138 or the amino acid sequence of SEQ ID NO:138 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:139 or the amino acid sequence of SEQ ID NO:139 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:140 or the amino acid sequence of SEQ ID NO:140 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:141 or the amino acid sequence of SEQ ID NO:141 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:142 or the amino acid sequence of SEQ ID NO:142 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:143 or the amino acid sequence of SEQ ID NO: 143 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:144 or the amino acid sequence of SEQ ID NO:144 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:145 or the amino acid sequence of SEQ ID NO:145 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:146 or the amino acid sequence of SEQ ID NO: 146 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:147 or the amino acid sequence of SEQ ID NO:147 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ NO:148 or the amino acid sequence of SEQ ID NO:148 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:149 or the amino acid sequence of SEQ ID NO:149 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:150 or the amino acid sequence of SEQ ID NO:150 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:151 or the amino acid sequence of SEQ ID NO:151 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:152 or the amino acid sequence of SEQ ID NO:152 absent the signal sequence (amino acids 1-24).
In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:153 or the amino acid sequence of SEQ 1D NO:153 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:154 or the amino acid sequence of SEQ ID NO:154 absent the signal sequence (amino acids 1-24). In some embodiments, a chimeric Tim 4 receptor comprises the amino acid sequence of SEQ ID NO:155 or the amino acid sequence of SEQ ID NO:155 absent the signal sequence (amino acids 1-24).
Le ..
'0 ie ..
A
be Table 10.
c be Construct Construct Description Signal Extracellular Transmembrane Intracellular -- Intracellular --Intracellular -- re .....
o Number Peptide Domain Domain Domain #1 Domain #2 Domain N3 ce) ON
be pCTX1161 hTIM4-TIM4-CT)28tm- 117144 TIM4 CD28 C7D28 CD3z Ct CA
[SEQ ID CD281cd-CD3z1CD [SEQ ID [SEQ ID [SEQ ID [SEQ
ID NO:41 [SEQ ID NO:27]
N.0:227] NO:218] NO:219] NO:220]
.
.
pCD.C1.162 hTIM4-TIM4-CD28tm- IITIM4 TIM4 CD28 [SEQ ID CD28icd-TLR2ICD- [SEQ ID [SEQ ID [SEQ ID CD28 [SEQ ID CD3z NO:228] CD3zICD NO:218]
NO:219] NO:2201 [SEQ ID NO:4] NO:222] [SEQ ID NO:27]
pCIX1163 hTIM4-TIM4-CD28tm- hunt TIM4 . CD28 [SEQ ID CD28icd-CD3zICD- [SEQ ID [SEQ ID [SEQ ID CD28 CD3z [SEQ ID
NO:229] TLR2ICD NO:218]
NO:219] NO:2201 [SEQ ID NO:41 [SEQ ID NO:27] NO:2221 pCIX1164 hTIN14-TIM4-CD28tm- hum4 TIM4 . CD28 [SEQ ID CD28icd-TLR8ICD- [SEQ ID [SEQ ID [SEQ ID CD28 TLR8 CDly NO:230] CD3zICD NO:2181 NO:219] NO:2201 [SEQ ID NO:41 [SEQ ID NO:47] [SEQ ID NO:271 --) 00 pCTX1165 hTLM4-TLM4-CD28tm- IIIIM4 TLM4 CD28 [SEQ ID CD28ial-CD3zTCD- [SEQ ID [SEQ 1D [SEQ ED CD28 CD3z TLR8 NO:231] TI.R8ICD NO:218]
NO:219] NO:2201 [SEQ ID NO:4] [SEQ ID NO:27] [SEQ ID NO:471 pCTX1166 urnvu TIM4 CD28 TLR2 [SEQ ID IITIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID CD3z NO:232] -11.1121CD-CD3z1CD
NO:218] NO:219] NO:220] NO:222] [SEQ ID NO:27]
pCTX1167 bTIM4 TIM4 CD28 [SEQ ID liTIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID CD3z [SEQ ID
NO:233] CD3zICD-TLR21CD
NO:218] NO:219] NO:220] [SEQ ID
NO:27] NO:222] oci pCTX1168 IMM4 TIM4 CD28 n ; 3 [SEQ ID liTIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID TLR8 CD3z NO:234] TLR81CD-CD3zICD
NO:218] NO:219] NO:220] [SEQ ID
NO:47] [SEQ ID NO:27] b4 o b.) pCTX1169 Mil& MN CD28 O' [SEQ ID 1iTIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID CD3z TLR8 A
o NO:235] CD3zICD-TLR81CD
NO:218] NO:219] NO:220] [SEQ ID
NO:27] [SEQ ID NO:47] o I-.
Le ..-'0 ..-A
pCTX1170 hum4 TIM4 CD28 be [SEQ ID IfTIN,14-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ
ID TRAF6 CD3z o be re NO:236] TRAF6ICD-CD3zICD
NO:218] NO:219] NO:220] [SEQ ID
NO:46] [SEQ ID NO:27] -...
a .
ce) pCTX1171 IfI1M4 TThil4 - CD28 ON
b.) CIO
[SEQ ID hTIM4-TIM4-CD28tm- [SEQ ID [SEQ ID [SEQ ID
CD3z TRAF6 CA
NO:237] CD3z1CD-TRAF6ICD
NO:218] NO:219] NO:2201 [SEQ ID NO:27] [SEQ ID NO:46]
pCTX1172 hTIM4 TIM4 TIM4 [SEQ ID hTIM4-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ ID
CD28 CD3z NO:238] CD28ial-CD3zICD
NO:218] NO:219] NO:221] [SEQ ID NO:41 [SEQ ID NO:27]
pCTX1173 IITIM4-TIM4-TIM4tm- hTIM4 TIM4 TIM4 [SEQ ID CD28ial-TLR2ICD- [SEQ ID [SEQ ID [SEQ ID CD28 [SEQ ID CD3z NO:239] CD3zICD NO:218]
NO:219] NO:2211 [SEQ ID NO:41 NO:222] [SEQ ID NO:27]
pCTX1174 hTIM4-TIM4-TIM4tin- hTIM4 TIM4 TIM4 [SEQ ID CD28ied-CD3zICD- [SEQ ID [SEQ ID [SEQ ID CD28 CD3z [SEQ ID
NO:2401 Tut21c0 NO:2181 1 NO:2191 NO:2211 1SEQ ID NO:41 1SEQ ID NO:271 NO:2221 -..) pCTX1175 hTIM4-TIM4-TIM4un- hTIM4 TIM4 TIM4 kr) [SEQ ID CD28ic4-ILR8ICD- [SEQ ID [SEQ ID [SEQ ID CO28 TLR8 CD3z NO:241] CD3zICD NO:218]
NO:219] NO:221] [SEQ ID NO:41 [SEQ ID NO:47] [SEQ ID NO:27]
pCTX1176 hTIM4-TIM4-TIM4lin- hTIM4 TIM4 TIM4 [SEQ ID CD28icd-CD3zICD- [SEQ ID [SEQ ID [SEQ ID CD28 CD3z TLR8 NO:242] TLR8ICD NO:218]
NO:219] NO:221] [SEQ ID NO:4] [SEQ ID NO:27] [SEQ ID NO:471 ......
pCTX1177 hTEM4 TIM4 TIM4 TLR2 [SEQ ID hTD/14-TIM4-TIM4lm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID CD3z NO:243] TLR2ICD-CD3zICD
NO:218] NO:219] NO:221] NO:222] [SEQ ID NO:27]
pCTX1178 hTIM4 TIM4 TIM4 TLR2 oci n [SEQ ID hTIM4-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ ID
CD3z [SEQ ID
NO:244] CD3zICD-TLR2ICD
NO:218] NO:219] NO:221] [SEQ ID
NO:27] NO:222] ri2 I
be pCTXI179 hTIM4 TIM4 TIM4 =
b.) [SEQ ID hTIN14-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ
ID TLR8 CD3z =..
O' N.0:245] 1 ILR8TCD-C)3zICD NO:218] NO:219] NO:2211 [SEQ
ID NO:47] [SEQ ID NO:27] A
A
I.+
Le .-'0 .-A
pCTX1180 num4 TIM4 TIM4 be [SEQ ID hTLM4-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ ID
CD3z TLR8 o be re NO:246] CD3zICD-TLR8ICD
NO:2181 NO:2191 NO:221] [SEQ ID
NO:27] [SEQ ID NO:47] .....
a pCTX1181 IiIIM4 TLM4 - TIM4 ON
b.) CIO
[SEQ ID hTIM4-TIM4-TIM40n- [SEQ ID [SEQ ID [SEQ ID
TRAF6 CD3z CA
NO:247] TRARICD-CD3z1CD
NO:218] NO:219] NO:221] [SEQ ID NO:461 [SEQ ID NO:27]
pCTX1182 hTIM4 TIM4 TIM4 [SEQ ID hTIM4-TIM4-TIM4tm- [SEQ ID [SEQ ID [SEQ ID
CD3z TRAF6 NO:248] CD3zICD-TRAF6ICD
NO:218] NO:219] NO:221) [SEQ ID NO:271 [SEQ ID NO:46]
pCTX1183 hTIM4 TIM4 TIM4 1IM4 [SEQ ID liTIM4-TIM4-TINI4hn- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID
NO:249] TiM4ICD NO:218]
NO:219] NO:221] NO:2251 pCTX1184 hTIM4 TIM4 TIN% 1IM4 [SEQ ID linv14-TIM4-TINI4lin- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID TLR2 CD3/
NO:2501 TIM4ICD-TLR2-CD3z NO:2181 1 NO:2191 NO:2211 NO:2241 1SEQ ID NO:871 ISE) ID NO:271 oo pCTX1185 hTIM4 TIM4 TIM4 1IM4 o [SEQ ID INIM4-TIM4-TINI4tm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID CD3z [SEQ ID
NO:251] TIM4ICD-CD3z-TLR2 NO:218] NO:219] NO:221] NO:224] [SEQ ID NO:27] NO:222]
pCTX1186 hTIM4 TIM4 TIM4 TIM4 [SEQ ID hTIN14-TIM4-TIM4lm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID TLR8 CD3z NO:252] TIM4ICD-TLR8-CD3z NO:218] NO:219] NO:221] NO:224] [SEQ ID NO:47] [SEQ ID NO:27]
pCTX1187 hTEM4 TIM4 TIM4 TIM4 [SEQ ID hTIN14-TIN14-TIM4lm- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID CD3z TLR8 NO:253] TIM4ICD-CD3z.-TLR8 NO:218] NO:219] NO:221] NO:224] [SEQ ID NO:27] [SEQ ID NO:4'7]
pCTX1189 hTIM4 TIM4 TIM4 TLR2 oci n [SEQ ID IITIM4-TIM4-TIM40n- [SEQ ID [SEQ ID [SEQ
ID [SEQ ID
NO:72] TLR2ICD NO:218]
NO:219] NO:221] NO:222] ri2 I
be pCTX1190 hTIM4 TIM4 T1M4 b.) [SEQ ID hT1M4-TIM4-TIM4tin- [SEQ ID [SEQ ID [SEQ
ID TLR8 =..
O' NO:73] 1 ILR8TCD NO:218]
NO:219] NO:2211 [SEQ ID NO:47] A
A
I.+
Polynucleotides, Vectors, and Host Cells In certain aspects, the present disclosure provides nucleic acid molecules that encode any one or more of the chimeric Tim receptors described herein. A
nucleic acid may refer to a single- or double-stranded DNA, cDNA, or RNA, and may include a positive and a negative strand of the nucleic acid which complement one another, including antisense DNA, cDNA, and RNA. A nucleic acid may be naturally occurring or synthetic forms of DNA or RNA.. The nucleic acid sequences encodi rig a desired chimeric Tim receptor can be obtained or produced using recombinant methods known in the art using standard techniques, such as by screening libraries from cells expressing the desired sequence or a portion thereof, by deriving the sequence from a vector known to include the same, or by isolating the sequence or a portion thereof directly from cells or tissues containing the same as described in, for example, Sambrook etal.
(1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology, 2003). Alternatively, the sequence of interest can be produced synthetically, rather than being cloned.
Polynucleotides encoding the chimeric Tim receptor compositions provided herein may be derived from any animal, such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, or a combination thereof.
In certain embodiments, a polynucleotide encoding the chimeric Tim receptor is from the same animal species as the host cell into which the polynucleotide is inserted.
The polynucleotides encoding chimeric Tim receptors of the present disclosure may be operatively linked to expression control sequences.
Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak consensus sequences);
sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
In certain embodiments, a polynucleotide encoding a chimeric Tim receptor comprises a sequence encoding a signal peptide (also referred to as leader peptide or signal sequence) at the 5'-end for targeting of the precursor protein to the secretory pathway. The signal peptide is optionally cleaved from the N-terminus of the extracellular domain during cellular processing and localization of the chimeric Tim receptor to the host cell membrane. A polypeptide from which a signal peptide sequence has been cleaved or removed may also be called a mature polypeptide.
Examples of signal peptides that may be used in the chimeric Tim receptors of the present disclosure include signal peptides derived from endogenous secreted proteins, including, e.g., GM-CSF (amino acid sequence of SEQ ID NO:10), Timl (amino acid sequence of SEQ ID NO:40), or Tim4 (amino acid sequence of SEQ ID NO:11 or 25).
In certain embodiments, a polynucleotide sequence encodes a mature chimeric Tim receptor polypeptide, or a polypeptide sequence comprises a mature chimeric Tim receptor polypeptide. It is understood by persons of skill in the art that for sequences disclosed herein that include a signal peptide sequence, the signal peptide sequence may be replaced with another signal peptide that is capable of trafficking the encoded protein to the extracellular membrane.
In certain embodiments, a chimeric Tim receptor encoding polynucleotide of the present disclosure is codon optimized for efficient expression in a target host cell comprising the polynucleotide (see, e.g, Scholten et al., (Fin. Innnunol.
119:135-145 (2006)). As used herein, a "codon optimized" polynucleotide comprises a heterologous polynucleotide having codons modified with silent mutations corresponding to the abundances of tRNA in a host cell of interest.
A single polynucleotide molecule may encode one, two, or more chimeric Tim receptors according to any of the embodiments disclosed herein. A
polynucleotide encoding more than one transgene may comprise a sequence (e.g., IRES, viral 2A peptide) disposed between each gene for multicistronic expression.
Polynucleotides encoding at least two transgenes (e.g., a chimeric Tim receptor and CAR) provided in the present disclosure may be used to compose tandem expression cassettes. A tandem expression cassette refers to a component of a vector nucleic acid comprising at least two transgenes under the control of, or operatively linked to, the same set of regulatory sequences for tandem or co-expression of the at least two transgenes. Regulatory sequences that may be used in tandem expression cassettes of the present disclosure include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak consensus sequences);
sequences that enhance protein stability; sequences that enhance protein secretion, or any combination thereof.
In one aspect, the present disclosure provides a tandem expression cassette comprising a polynucleotide encoding a chimeric Tim receptor of the present disclosure and a polynucleotide encoding a cellular immunotherapy agent (e.g., a CAR, TCR, etc.).
In certain embodiments, a tandem expression cassette can be constructed to optimize spatial and temporal control. For example, a tandem expression cassette can include promoter elements to optimize spatial and temporal control. In some embodiments, a tandem expression cassette includes tissue specific promoters or enhancers that enable specific induction of a tandem expression cassette to an organ, a cell type (e.g., immune cell), or a pathologic rnicroenvironment, such as a tumor or infected tissue. An "enhancer" is an additional promoter element that can function either cooperatively or independently to activate transcription. In certain embodiments, a tandem expression cassette includes a constitutive promoter. An exemplary constitutive promoter for use in tandem expression cassettes of the present disclosure is an EF-la promoter. In certain embodiments, a tandem expression cassette includes an inducible promoter. In certain embodiments, a tandem expression cassette includes a tissue specific promoter.
The at least two transgenes contained within the tandem expression cassettes may be in any order. For example, a tandem expression cassette comprising a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a CAR
may be arranged from 5' to 3': chimeric Tim receptor-CAR, or CAR- chimeric Tim receptor.
In certain embodiments, receptors that comprise two or more polypeptide chains that associate to form a multimer or complex may be encoded by two or more polynucleotide molecules within a tandem expression construct.
Exemplary multimeric receptors contemplated for expression in tandem expression constructs of the present disclosure include multichain CARs, TCRs, TCR-CARs, and TRuCTm constructs. Accordingly, exemplary tandem expression cassette embodiments encoding a chimeric 'rim receptor and a TCR may comprise a polynucleotide encoding a chimeric Tim receptor, a polynucleotide encoding a TCRa chain polypeptide, and a polynucleotide encoding a TCRI3 chain polypeptide.
In certain embodiments, tandem expression cassettes of the present disclosure may comprise an internal ribosome entry site (IRES) or peptide cleavage site such as a furin cleavage site or viral 2A peptide, disposed between each polynucleotide contained within the tandem expression cassette to allow for co-expression of multiple proteins from a single mRNA. For example, an :ERES, furin cleavage site, or viral 2A
peptide may be disposed between a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a CAR within a tandem expression cassette. In another example, an IRES, furin cleavage site, or viral 2A peptide may be disposed between each of a polynucleotide encoding a chimeric Tim receptor, a polynucleotide encoding a TCRa chain polypeptide, and a polynucleotide encoding a TCRI3 chain polypeptide.
In certain embodiments, a viral 2A peptide is a porcine teschovirus-1 (P2A), ihosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variant thereof. An exemplary T2A peptide comprises an amino acid sequence of any one of SEQ ID NOs:12, 28, 29, or 30. An exemplary P2A peptide comprises an amino acid sequence of SEQ ID NO: 13 or 31. An exemplary E2A peptide sequence comprises an amino acid sequence of SEQ ID NO: 14. An exemplary F2A peptide sequence comprises an amino acid sequence of SEQ ID NO:15.
Certain embodiments of tandem expression cassettes of the present disclosure comprise a polynucleotide encoding a CAR/or TCR specific for a target antigen (e.g., tumor antigen) and a polynucleotide encoding a chimeric Tim receptor of the present disclosure. Upon binding a target cell expressing the target antigen by the CAR/or TCR, a cell modified to express such a tandem expression cassette induces apoptosis of the target cell. Apoptosis induces exposure of pro-engulfment markers on the target cell, such as phosphatidylserine, which may then target the damaged or apoptotic cells for engulfment by the chimeric Tim receptor.
A polynucleotide encoding a desired chimeric Tim receptor can be inserted into an appropriate vector, e.g., a viral vector, non-viral plasmid vector, and non-viral vectors, such as lipid-based DNA vectors, modified mRNA (morIRNA), self-amplifying mRNA, CELiD, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty), for introduction into a host cell of interest (e.g., an immune cell).
Polynucleotides encoding a chimeric Tim receptor of the present disclosure can be cloned into any suitable vector, such as an expression vector, a replication vector, a probe generation vector, or a sequencing vector. In certain embodiments, a polynucleotide encoding the extracellular domain, a polynucleotide encoding the transmembmne domain, and a polynucleotide encoding the intracellular signaling domain are joined together into a single polynucleotide and then inserted into a vector.
In other embodiments, a polynucleotide encoding the extracellular domain, a polynucleotide encoding the transmembrane domain, and a polynucleotide encoding the intracellular signaling domain may be inserted separately into a vector such that the expressed amino acid sequence produces a functional chimeric Tim receptor. A
vector that encodes a chimeric Tim receptor is referred to herein as a "chimeric Tim receptor vector."
In certain embodiments, a vector comprises a polynucleotide encoding one chimeric Tim receptor. In certain embodiments, a vector comprises one polynucleotide encoding two or more chimeric Tim receptors. In certain embodiments, a single polynucleotide encoding two or more chimeric Tim receptors is cloned into a cloning site and expressed from a single promoter, with each chimeric Tim receptor sequence separated from each other by an internal ribosomal entry site (IRES), furin cleavage site, or viral 2A peptide to allow for co-expression of multiple genes from a single open reading frame (e.g., a multi ci stronic vector). In certain embodiments, a viral 2A peptide is a porcine teschovirus-1 (P2A), Thosea asigncr virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variant thereof. An exemplary T2A peptide comprises an amino acid sequence of SEQ DD NO:12, 28, 29, or 30. An exemplary P2A peptide comprises an amino acid sequence of SEQ ID
NO:13 or 31. An exemplary E2A peptide sequence comprises an amino acid sequence of SEQ
ID NO:14. An exemplary F2A peptide sequence comprises an amino acid sequence of SEQ ID NO:15.
In certain embodiments, a vector comprises two Or more polynucleotides, each polynucleotide encoding a chimeric Tim receptor. The two or more polynucleotides encoding chimeric Tim receptors may be cloned sequentially into a vector at different cloning sites, with each chimeric Tim receptor expressed under the regulation of different promoters. In certain embodiments, vectors that allow long-term integration of a transgene and propagation to daughter cells are utilized.
Examples include viral vectors such as, adenovirus, adeno-associated virus, vaccinia virus, herpes viruses, cytomegalovirus, pox virus, or retroviral vectors, such as lentiviral vectors.
Vectors derived from lentivirus can be used to achieve long-term gene transfer and have added advantages over vectors including the ability to transduce non-proliferating cells, such as hepatocytes, and low immunogenicity.
A vector that encodes a core virus is referred to herein as a "viral vector." There are a large number of available viral vectors suitable for use with the compositions of the instant disclosure, including those identified for human gene therapy applications (see Pfeifer and \Terme, Ann. Rev. Genomics Hum. Genet.
2:177, 2001). Suitable viral vectors include vectors based on RNA viruses, such as retrovirus-derived vectors, e.g., Maloney murine leukemia virus (MI,V)-derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, Fly, equine infectious anemia virus, S1V, and Maedi-Visna virus (ovine lentivirus). M:ethods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing chimeric receptor transgencs are known in the art and have been previous described, for example, in U.S. Patent 8,119,772; Walehli et al., PLoS One 6:327930, 2011;
Zhao et at, J. lmmunol. /74:4415, 2005; Engels et al., Hum. Gene filer. 14:1155, 2003;
Frecha etal., Mot iher. 18:1748, 2010; Verhoeyen et al., Methods Mol. Biol. 506:97, 2009.
Retroviral and lenti viral vector constructs and expression systems are also commercially available.
In certain embodiments, a viral vector is used to introduce a non-endogenous polynucleotide encoding a chimeric Tim receptor to a host cell. A
viral vector may be a retroviral vector or a lentiviral vector. A viral vector may also include a nucleic acid sequence encoding a marker for transduction. Transduction markers for viral vectors are known in the art and include selection markers, which may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. In particular embodiments, a viral vector further comprises a gene marker for transduction comprising a fluorescent protein (e.g., green, yellow), an extracellular domain of human CD2, or a truncated human EGFR (EGFRt or tEGFR; see Wang etal., Blood 118:1255, 2011). An exemplary tEGFR comprises an amino acid sequence of SEQ ID NO:16. When a viral vector genome comprises a plurality of genes to be expressed in a host cell as separate proteins from a single transcript, the viral vector may also comprise additional sequences between the two (or more) genes allowing for multicistronic expression.
Examples of such sequences used in viral vectors include internal ribosome entry sites (RES), furin cleavage sites, viral 2A peptides (e.g., T2A, P2A, E2A, F2A), or any combination thereof.
Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Iher. 5: 1517, 1998).
Other viral vectors recently developed for gene therapy uses can also be used with the compositions and methods of this disclosure. Such vectors include those derived from baculoviruses and a-viruses. (Jolly, D J. 1999. Emerging Viral Vectors.
pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).
In certain embodiments, a chimeric Tim receptor vector can be constructed to optimize spatial and temporal control. For example, a chimeric Tim receptor vector can include promoter elements to optimize spatial and temporal control.
In some embodiments, a chimeric Tim receptor vector includes tissue specific promoters or enhancers that enable specific induction of a chimeric Tim receptor to an organ, a cell type (e.g., immune cell), or a pathologic microenvironment, such as a tumor or infected tissue. An "enhancer" is an additional promoter element that can function either cooperatively or independently to activate transcription. In certain embodiments, a chimeric Tim receptor vector includes a constitutive promoter.
In certain embodiments, a chimeric Tim receptor vector includes an inducible promoter.
In certain embodiments, a chimeric Tim receptor vector includes a tissue specific promoter.
In certain embodiments, a chimeric Tim receptor vector can include a gene encoding a homing receptor, such as CCR4 or CXCR4, to improve homing and antitumor activity in viiv.
Where temporal control is desired, a chimeric Tim receptor vector may include an element that allows for inducible depletion of transduced cells.
For example, such a vector may include an inducible suicide gene. A suicide gene may be an apoptotic gene or a gene that confers sensitivity to an agent (e.g., a drug).
Exemplary suicide genes include chemically inducible caspase 9 (iCASP9) (U.S. Patent Publication No. 2013/0071414), chemically inducible Fas, or Herpes simplex virus thymidine kinase (HSV-TK), which confers sensitivity to ganciclovir. In further embodiments, a chimeric Tim receptor vector can be designed to express a known cell surface antigen that, upon infusion of an associated antibody, enables depletion of transduced cells.
Examples of cell surface antigens and their associated antibodies that may be used for depletion of transduced cells include CD20 and Rituximab, RQR.8 (combined CD34 and CD20 cpitopes, allowing CD34 selection and anti-CD20 deletion) and Rituximab, and EGFR and Cetuximab.
Inducible vector systems, such as the tetracycline (Tet)-On vector system which activates transgene expression with doxycycline (Heinz et al., Hum. Gene Ther.
2011, 22:166-76) may also be used for inducible chimeric Tim receptor expression.
Inducible chimeric Tim receptor expression may be also accomplished via retention using a selective hook (RUSH) system based on streptavidin anchored to the membrane of the endoplasmic reticulum through a hook and a streptavidin binding protein introduced into the chimeric Tim receptor structure, where addition of biotin to the system leads to the release of the chimeric Tim receptor from the endoplasmic reticulum (Agaugue et al., 2015, Mot. Ther. 23(Suppl. 1):S88).
In certain embodiments, a cell, such as an immune cell, obtained from a subject may be engineered into a non-natural or recombinant cell (e.g., a non-natural or recombinant immune cell) by introducing a polynucleotide that encodes a chimeric Tim receptor as described herein, whereby the cell expresses a cell surface localized chimeric Tim receptor. In certain embodiments, a host cell is an immune cell, such as a myeloid progenitor cell or a lymphoid progenitor cell. Exemplary immune cells that may be modified to comprise a polynucleotide encoding a chimeric Tim receptor or a vector comptising a polynucleotide encoding a chimeric Tim receptor include a T cell, a natural killer cell, a B cell, a lymphoid precursor cell, an antigen presenting cell, a dendritic cell, a Langerhans cell, a myeloid precursor cell, a mature myeloid cell, a monocyte, or a macrophage.
In certain embodiments, a B cell is genetically modified to express one or more chimeric Tim receptors. B cells possess certain properties that may be advantageous as host cells, including: trafficking to sites of inflammation, capable of internalizing and presenting antigen, capable of costimulating T cells, highly proliferative, and self-renewing (persist for life). In certain embodiments, a chimeric Tim receptor modified B cell is capable of digesting an engulfed target cell or engulfed target particle into smaller peptides and presenting them to T cells via an MHC
molecule. Antigen presentation by a chimeric Tim receptor modified B cell may contribute to antigen spreading of the immune response to non-targeted antigens. B
cells include progenitor or precursor cells committed to the B cell lineage (e.g., pre-pro-B cells, pro-B cells, and pre--B cells); immature and inactivated B cells; or mature and functional or activated B cells. In certain embodiments, B cells may be naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cell, plasmablast cell, memory B cells, or any combination thereof. Memory B cells may be distinguished from naive B cells by expression of CD27, which is absent on naive B cells. In certain embodiments, the B cells can be primary cells or cell lines derived from human, mouse, rat, or other mammals.
B cell lines are well known in the art. If obtained from a mammal, a B cell can be obtained from numerous sources, including blood, bone marrow, spleen, lymph node, or other tissues or fluids. A B cell composition may be enriched or purified.
In certain embodiments, a T cell is genetically modified to express one or more chimeric Tim receptors. Exemplary T cells include CD4' helper, CDS+
effector (cytotoxic), naive (CD45 RA+, CCR7+, CD62L+, CD27+, CD45R0-), central memory (CD45R0+, CD62L+, CD8+), effector memory (CD45RA+, CD45R0-, CCR7-, CD62L-, CD27-), T memory stem, regulatory, mucosal-associated invariant (MNIT), y5 (gd), tissue resident T cells, natural killer T cells, or any combination thereof. In certain embodiments, the 'I' cells can be primary cells or cell lines derived from human, mouse, rat, or other mammals. If obtained from a mammal, a T cell can be obtained from numerous sources, including blood, bone marrow, lymph node, thymus, or other tissues or fluids. A T cell composition may be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In certain embodiments, the T cells lack endogenous expression of a TCRa gene, TC1113 gene, or both. Such T cells may naturally lack endogenous expression of 'TCRa and 13 chains, or may have been modified to block expression (e.g., T cells from a transgenic mouse that does not express TCR a and 13 chains or cells that have been manipulated to inhibit expression of TCR a and 13 chains) or to knockout a TCRa chain, a TCI213 chain, or both genes.
In certain embodiments, host cells expressing a chimeric Tim protein of this disclosure on the cell surface arc not T cells or cells of a T cell lineage, but cells that are progenitor cells, stem cells or cells that have been modified to express cell surface anti-CD3.
In certain embodiments, gene editing methods are used to modify the host cell genome to comprise a polynucleotide encoding a chimeric Tim receptor of the present disclosure. Gene editing, or genome editing, is a method of genetic engineering wherein DNA is inserted, replaced, or removed from a host cell's genome using genetically engineered endonucleases. The nucleases create specific double-stranded breaks at targeted loci in the genome. The host cell's endogenous DNA repair pathways then repair the induced break(s), e.g., by non-homologous ending joining (NEIEJ) and homologous recombination. Exemplary endonucleases useful for gene editing include a zinc finger nuclease (ZEN), a transcription activator-like effector (TALE) nuclease, a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system (e.g., CRISPR-Cas9), a meganuclease, or combinations thereof. Methods of disrupting or knocking out genes or gene expression in immune cells including :B cells and T cells, using gene editing endonucleases are known in the art and described, for example, in PCT Publication Nos. WO 2015/066262;
WO 2013/074916; WO 2014/059173; Cheong et al., Nat. COMM. 2016 7:10934; Chu et al., Proc. Natl. Acad. Sci. USA. 2016 113:12514-12519; methods from. each of which are incorporated herein by reference in their entirety.
In certain embodiments, expression of an endogenous gene of the host cell is inhibited, knocked down, or knocked out. Examples of endogenous genes that may be inhibited, knocked down, or knocked out in a B cell include IGH, lGiç
ICA, or any combination thereof. Examples of endogenous genes that may be inhibited, knocked down, or knocked out in a T cell include a TCR gene (TRA or TRB), an HLA
gene (HLA class I gene or HLA class II gene), an immune checkpoint molecule (PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, .A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, or PVRIG/CD1.12R), or any combination thereof. Expression of an endogenous gene may be inhibited, knocked down, or knocked out at the gene level, transcriptional level, translational level, or a combination thereof. Methods of inhibiting, knocking down, or knocking out an endogenous gene may be accomplished, for example, by an RNA interference agent (e.g., siRNA, shRNA, miRNA, etc.) or an engineered endonuclease (e.g., CRISPR/Cas nuclease system, a zinc finger nuclease (ZFN), a Transcription Activator Like Effector nuclease (TALEN), a meganuclease), or any combination thereof. in certain embodiments, an endogenous B cell gene (e.g., IGH, IGK, or 10.) is knocked out by insertion of a polynucleotide encoding a chimeric Tim receptor of the present disclosure into the locus of the endogenous B cell gene, such as via an engineered endonuclease.
In certain embodiments, an endogenous T cell gene (e.g., a TCR gene, an HLA
gene, or an immune checkpoint molecule gene) is knocked out by insertion of a polynucleotide encoding a chimeric Tim receptor of the present disclosure into the locus of the endogenous 1' cell gene, such as via an engineered endonuclease.
In certain embodiments, a host cell may be genetically modified to express one type of chimeric Tim receptor. In other embodiments, a host cell may express at least two or more different chimeric Tim receptors.
The present disclosure also provides a composition comprising a population of chimeric Tim receptor modified host cells. In certain embodiments, the population of chimeric Tim receptor modified host cells may be a population of B cells, a population of T cells, a population of natural killer cells, a population of lymphoid precursor cells, a population of antigen presenting cells, a population of dendritic cells, a population of Langerhans cells, a population of myeloid precursor cells, a population of mature myeloid cells, or any combination thereof. Furthermore, a population of chimeric Tim receptor modified host cells of a particular cell type may be composed of one or more subtypes. For example, a population of B cells may be composed of chimeric Tim receptor modified naive B cells, plasma cells, regulatory B
cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablast cells, memory B
cells, or any combination thereof. In another example, a population of T cells may be composed of chimeric Tim receptor modified CD4+ helper T cells, CD8 effector (cytotoxic) T cells, naive (CD45 RA+, CCR7+, CD62L+, CD27+, CD45R0-) T cells, central memory (CD45R0+, CD62L+, CDS+) T cells, effector memory (CD45RA -I-, CD45R0-, CCR7-, CD62L-, CD27-) T cells, T memory stem cells, regulatory T
cells, mucosal-associated invariant T cells (MAIT), T6 (gd) cells, tissue resident T
cells, natural killer T cells, or any combination thereof.
In certain embodiments, a population of host cells is composed of cells that each expresses the same chimeric Tim receptor(s). In other embodiments, a population of host cells is composed of a mixture of two or more subpopulation of host cells, wherein each subpopulation expresses a different chimeric Tim receptor or set of chimeric Tim receptors.
In certain embodiments, when preparing chimeric Tim receptor modified host cells, e.g., B cells or T cells, one or more growth factor cytokines that promotes proliferation of the host cells, e.g., B cells or T cells, may be added to the cell culture.
The cytokines may be human or non-human. Exemplary growth factor cytokines that may be used to promote T cell proliferation include 1L-2, 1L-15, or the like.
Exemplary growth factor cytokines that may be used to promote B cell proliferation include CD4OL, 1L-2, IL-4, IL-15, 1L-21, BAIT, or the like.
Prior to genetic modification of the host cells with a chimeric Tim receptor vector, a source of host cells (e.g., T cells, B cells, natural killer cells, etc.) is obtained from a subject (e.g., whole blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue), from which host cells are isolated using methods known in the art. Specific host cell subsets can be collected in accordance with known techniques and enriched or depleted by known techniques, such as affinity binding to antibodies, flow cytometry and/or immunomagnetic selection. After enrichment and/or depletion steps and introduction of a chimeric Tim receptor, in vitro expansion of the desired modified host cells can be carried out in accordance with known techniques, or variations thereof that will be apparent those skilled in the art.
Chimeric Tim receptors of the present disclosure confer cytotoxic activity to host cells expressing the chimeric Tim receptors that is specific for phosphatidylserine. Thus, upon binding phosphatidylscrine exposed on the surface of a target cell, a host cell expressing a chimeric Tim receptor is capable of inducing apoptosis of the target cell. In certain embodiments, the host cell expressing the chimeric Tim receptor induces apoptosis of the target cell via: release of granzymes, perforins, granulysin, or any combination thereof; Fas ligand-Fas interaction;
or both.
In further embodiments, the chimeric Tim receptor further confers phosphatidylserine specific engulfment activity to host cells expressing the chimeric Tim receptor. in yet further embodiments, the host cell does not naturally exhibit an engulfment phenotype prior to modification with the chimeric Tim receptor.
Chimeric Tim receptors of the present disclosure may also be capable of costimulating T cells via at least one signaling pathway. In certain embodiments, chimeric Tim receptors provide costimulatory signals to T cells via at least two distinct signaling pathways (e.g., via the selected costimulatory signaling domain(s) in the chimeric Tim receptor). For example, a chimeiic Tim receptor comprising a CD28 costimulatory signaling domain may be capable of providing a costimulatory signal via CD28 and Tim 1. In certain embodiments, host immune cells expressing the chimeric Tim receptors exhibit reduction or inhibition of immune cell exhaustion. In certain embodiments, the host immune cell is a T cell or NK cells. In certain embodiments, exhausted T cells exhibit; (a) increased expression of PD-1, LAG3, TIM:3, or any combination thereof; (b) decreased production of IFN-y, TNE-a, or any combination thereof; or both (a) and (b). In certain embodiments, exhausted NK
cells exhibit; (a) increased expression of PD-1, NKG2A, TIM3, or any combination thereof;
(b) decreased production of IFN-,y, TN. F-a, or both; or both (a) and (h).
In certain embodiments, host cells expressing the chimeric Tim receptors exhibit an enhanced effector response (e.g., tumor specific). In certain embodiments, the effector response is enhanced T cell proliferation, cytokine production (e.g., IFN-y, :IL-2, TNF-a), cytotoxic activity, persistence, or any combination thereof.
In certain embodiments host cells expressing the chimeric Tim receptors exhibit a reduced immunosuppressive response to phosphatidylserine.
Phosphatidylserine is one of the primary apoptotic cell ligands that signal "eat me" to phagocytes. The removal of apoptotic cells by phagocytes generally reduces or prevents an inflammatory response via secretion of anti-inflammatory cytokines and TGF-f3 and the decrease of secretion of inflammatory cytokines TNF-a, IL-Ifl, and IL-12. Thus, phosphatidylserine may act as an immunosuppressive signal during the clearance of apoptotic cells. In certain embodiments, upon binding phosphatidylserine, a chimeric Tim receptor modified host cell exhibits increased antigen-specific cytokine production (e.g., IFN-y,1L-2, TNF-a), thereby reducing the immunosuppressive response to phosphatidylserine.
The expression of chimeric Tim receptors on host cells may be functionally characterized according to any of a large number of art-accepted methodologies for assaying host cell (e.g.. T cell) activity, including determination of T cell binding, activation or induction and also including determination of T
cell responses that are antigen-specific. Examples include determination of T cell proliferation, T cell cytokine release, antigen-specific 1' cell stimulation, (717.L activity (e.g., by detecting 51Cr or Europium release from pre-loaded target cells), changes in T cell phenotypic marker expression, and other measures of T cell functions.
Procedures for performing these and similar assays are may be found, for example, in Lefkovits (Immunology Methods. Manual: The Comprehensive Sourcebook of Techniques, 1998). See, also, Current! Protocols in Immunology; Weir, Handbook of Experimental Immunology, Black-well. Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA. (1979); Green and Reed, Science 281:1309 (1998) and references cited therein. Cytokine levels may be determined according to methods known in the art, including for example, ELISA, ELISPOT, intracellular cytokine staining, flow cytometry, and any combination thereof (e.g., intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion resulting from an antigen-specific elicitation or stimulation of an immune response may be determined by isolating lymphocytes, such as circulating lymphocytes in samples of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and/or cell viability, such as by incorporation of vitiated thymidinc or non-radioactive assays, such as MTT assays and the like.
In certain embodiments, a chimeric Tim receptor modified host cell has a phagocytic index of about 20 to about 1,500 for a target cell. A "phagocytic index" is a measure of phagocytic activity of the transduced host cell as determined by counting the number of target cells or particles ingested per chimeric Tim receptor modified host cell during a set period of incubation of a suspension of target cells or particles and chimeric Tim receptor modified host cells in media. Phagocyfic index may be calculated by multiplying [total number of engulfed target cells/total number of counted chimeric Tim receptor modified cells (e.g., phagocytic frequency)] x [average area of target cell or particle staining per chimeric Tim receptor + host cell x 100 (e.g., hybrid capture)] or [total number of engulfed particles/total number of counted chimeric Tim receptor modified host cells] x [number of chimeric Tim receptor modified host cells containing engulfed particles/ total number of counted chimeric Tim receptor cells] x 100. In certain embodiments, a chimeric Tim receptor modified cell has a phagocytic index of about 30 to about 1,500; about 40 to about 1,500; about 50 to about 1,500;
about 75 to about 1,500; about 100 to about 1,500; about 200 to about 1,500;
about 300 to about 1,500; about 400 to about 1,500; about 500 to about 1,500; about 20 to about 1,400; about 30 to about 1,400; about 40 to about 1,400; about 50 to about 1,400; about 100 to about 1,400; about 200 to about 1,400; about 300 to about 1,400; about 400 to about 1,400; about 500 to about 1,400; about 20 to about 1,300; about 30 to about 1,300; about 40 to about 1,300; about 50 to about 1,300; about 100 to about 1,300;
about 200 to about 1,300; about 300 to about 1,300; about 400 to about 1,300;
about 500 to about 1,300; about 20 to about 1,200; about 30 to about 1,200; about 40 to about 1,200; about 50 to about 1,200; about 100 to about 1,200; about 200 to about 1,200;
about 300 to about 1,200; about 400 to about 1,200; about 500 to about 1,200;
about 20 to about 1,100; about 30 to about 1,100; about 40 to about 1,100; about 50 to about 1,100; about 100 to about 1,100; about 200 to about 1,100; about 300 to about 1,100;
about 400 to about 1,100; or about 500 to about 1,100; about 20 to about 1,000; about to about 1,000; about 40 to about 1,000; about 50 to about 1,000; about 100 to about 1,000; about 200 to about 1,000; about 300 to about 1,000; about 400 to about 1,000; or 30 about 500 to about 1,000; about 20 to about 750; about 30 to about 750;
about 40 to about 750; about 50 to about 750; about 100 to about 750; about 200 to about 750;
about 300 to about 750; about 400 to about 750; or about 500 to about 750;
about 20 to about 500; about 30 to about 500; about 40 to about 500; about 50 to about 500; about 100 to about 500; about 200 to about 500; or about 300 to about 500. In further embodiments, the incubation time is from about 2 hours to about 4 hours, about 2 hours, about 3 hours, or about 4 hours. In yet further embodiments, a chimeric Tim receptor modified cell exhibits phagocytic index that is statistically significantly higher than a cell transduced with truncated EGET. control. Phagocytic index may be calculated using methods known in the art and as further described in the Examples and PCT
Application No. PCT/US2017/053553 (incorporated herein by reference in its entirety), including quantification by flow cytometry or fluorescence microscopy.
Host cells may be from an animal, such as a human, primate, cow, horse, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, or a combination thereof In a preferred embodiment, the animal is a human. Host cells may be obtained from a healthy subject or a subject having a disease associated with expression or overexpressi on of an antigen.
PARP Inhibitors Any suitable PARP inhibitor may be used in the compositions and methods of the disclosure. Exemplary PARP inhibitors include talazoparib, niraparib, rucaparib, olaparib (AZ 2281, KU59436), veliparib (ABT 888), CEP 9722, E7016, AG014699, MK4827, BMN-673, and Painiparib (BGB-290).
In a particular embodiment, the PARP inhibitor comprises niraparib. In a particular embodiment, the PARP inhibitor comprises talazoparib. In a particular embodiment, the PARP inhibitor comprises rucaparib. In a particular embodiment, the PARP inhibitor comprises olaparib. In a particular embodiment, the PARP
inhibitor comprises veliparib. In a particular embodiment, the PARP inhibitor comprises CEP
9722. In a particular embodiment, the PARP inhibitor comprises E7016. In a particular embodiment, the PARP inhibitor comprises AG014699. In a particular embodiment, the PARP inhibitor comprises MK4827. In a particular embodiment, the PARP
inhibitor comprises BMN-673. In a particular embodiment, the PARP inhibitor comprises Pamiparib.
Methods of Treatment A chimeric Tim receptor, a polynucleotides encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor, in combination with a PARP inhibitor according to any of the embodiments provided herein may be used in a method of treating a subject suffering from a disease, disorder or undesired condition. Embodiments of these methods include administering to a subject (i) a therapeutically effective amount of a pharmaceutical composition including one or more chimeric Tim receptors, polynucleotides encoding one or more chimefic Tim receptors, vectors comprising polynucleotides encoding one or more chimeric Tim receptors, or a population of host cells genetically modified to express one or more chimeric Tim receptors according to the present description; and (ii) a therapeutically effective amount of a pharmaceutical composition comprising a PARP
inhibitor.
The chimeric Tim receptor compositions as described herein may be administered before PARP inhibitor therapy (e.g., 1 day to 30 days or more before the PARP inhibitor therapy), concurrently with PARP inhibitor therapy (on the same day), or after PARP inhibitor therapy (e.g., 1 day ¨ 30 days or more after the PARP
inhibitor therapy). In certain embodiments, the chimeric Tim receptor modified cells are administered after administration of the one or more additional therapies. In further embodiments, the chimeric Tim receptor modified cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration of the PARP inhibitor. In still further embodiments, the chimeric Tim receptor modified cells are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week after administration of the PARP inhibitor therapy.
Where the PARP inhibitor therapy involves multiple doses, the chimeric Tim receptor modified cells may be administered after the initial dose of the one or more additional therapies, after the final dose of the one or more additional therapies, or in between multiple doses of the one or more additional therapies.
Diseases that may be treated with cells expressing a chimeric Tim receptor as described in the present disclosure include cancer. Adoptive immune and gene therapies are promising treatments for various types of cancer (Morgan etal., Science 314:126, 2006; Schmitt et al., Hum. Gene They. 20:1240, 2009; June, J.
Clin.
Invest. 117:1466, 2007) and infectious disease (Kitchen etal., PLoS One 4:38208, 2009; Rossi et al., Nat. Biatechnol. 25:1444, 2007; Zhang et al., PLoS Palhog.
6:el 001018, 2010; Luo et al., J. A/161. Med 89:903, 2011).
A wide variety of cancers, including solid tumors and leukemias are amenable to the compositions and methods disclosed herein. Exemplary cancers that may be treated using the receptors, modified host cells, and composition described herein include adenocarcinoma of the breast, prostate, and colon; all forms of bronchogenic carcinoma of the lung; myeloid leukemia; melanoma; hepatoma;
neuroblastoma; papilloma; apudoma; choristoma; branchiorna; malignant carcinoid syndrome; carcinoid heart disease; and carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, :Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell). Additional types of cancers that may be treated using the receptors, modified host cells, and composition described herein include histiocytic disorders; malignant histiocytosis; leukemia; Hodgkin's disease;
immunoproliferative small; non-Hodgkin's lymphoma; plasmacytoma; multiple myelorna; chronic myeloid leukemia (CML); acute myeloid leukemia (AML); plasrnacytoma;
reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosareoma;
fibroma; fibrosarcoma; giant cell tumors; histiocytoma; lipoma; liposarcoma;
mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma;
craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma;
myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymorna;
trophoblastic tumor. Further, the following types of cancers arc also contemplated as amenable to treatment using the receptors, modified host cells, and composition described herein: adenoma; cholangioma; cholesteatoma; cyclindroma;
cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma;
hepatoma;
hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma; myoblastoma; myomma; myosarcoma;
rhabdomyoma.; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma;
medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma;
neurofibroma; neuroma; paraganglioma; paraganglioma nonehromaffin. The types of cancers that may be treated also include angiokeratoma; angiolymphoid hyperplasia with eosi nophili a; angioma sclerosing; angiornatosis; glomangioma;
hemangioendothelioma; hemangiorna; hernangiopericytoma; hernangiosarcorna;
lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; careinosarcoma;
chondrosarcorna; cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma;
leiomyosarcoma; leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma;
myxosarcoma; ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasms;
nerofibromatosis; cervical dysplasia; and peritoneal cancer.
In some embodiments, compositions and methods of the present disclosure are useful to treat solid tumors. In some embodiments, the solid tumor cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian cancer, peritoneal cancer, or prostate cancer. In some embodiments, the breast cancer is triple negative breast cancer. In some embodiments, the ovarian cancer is advanced ovarian cancer. In some embodiments, the prostate cancer is advanced prostate cancer. In some embodiments, the solid tumor cancer is melanoma. In some embodiments, the solid tumor cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer.
In additional embodiments, the cancer is a Breast cancer (BRCA) mutated cancer. In particular embodiments, the cancer is a BRCA1 mutated cancer, a BRCA2 mutated cancer, or both.
A chimeric Tim receptor of the present disclosure may be administered to a subject in cell-bound form (e.g., gene therapy of target cell population). Thus, for example, a chimeric Tim receptor of the present disclosure may be administered to a subject expressed on the surface of T cells, Natural Killer Cells, Natural Killer T cells, B cells, lymphoid precursor cells, antigen presenting cells, dendritic cells, Langerhans cells, myeloid precursor cells, mature myeloid cells, including subsets thereof, or any combination thereof. In certain embodiments, methods of treating a subject comprise administering an effective amount of chimeric Tim receptor modified cells (i.e., recombinant cells that express one or more chimeric Tim receptors). The chimeric Tim receptor modified cells may be xenogeneic, syngeneic, allogeneic, or autologous to the subject.
Chimeric Tim receptor modified cells and PARP inhibitors may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as the condition of the patient, size, weight, body surface area, age, sex, type and severity of the disease, particular therapy to be administered, particular form of the active ingredient, time and the method of administration, and other drugs being administered concurrently. The present disclosure provides pharmaceutical compositions comprising chimeric Tim receptor modified cells and a pharmaceutically acceptable carrier, diluent, or excipient. Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. Other suitable infusion medium can be any isotonic medium formulation, including saline, Normosol R (Abbott), Plasina-Lyte A (Baxter), 5% dextrose in water, or Ringer's lactate.
A treatment effective amount of cells in a pharmaceutical composition is at least one cell (for example, one chimeiic Tim receptor modified T cell) or is more typically greater than 102 cells, for example, up to 106, up to 10, up to 108 cells, up to 109 cells, up to 10' cells, or up to 101' cells or more. In certain embodiments, the cells are administered in a range from about 106 to about 10" cells/m2, preferably in a range of about 10 to about 109 cells/m2. The number of cells will depend upon the ultimate use for which the composition is intended as well the type of cells included therein. For example, a composition comprising cells modified to contain a chimeric Tim receptor will comprise a cell population containing from about 5% to about 95% or more of such cells. in certain embodiments, a composition comprising chimeric Tim receptor modified cells comprises a cell population comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%
or more of such cells. For uses provided herein, the cells are generally in a volume of a liter or less, 500 mls or less, 250 mls or less, or 100 mls or less. Hence the density of the desired cells is typically greater than 104 cells/ml and generally is greater than 10' cells/ml, generally 108 cells/m1 or greater. The cells may be administered as a single infusion or in multiple infusions over a range of time. Repeated infusions of chimeric Tim receptor modified cells may be separated by days, weeks, months, or even years if relapses of disease or disease activity are present. A clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 106, 107, 108, 109, 1010, or 1011 cells. A preferred dose for administration of a host cell comprising a recombinant expression vector as described herein is about 107 cells/m2, about 5 x 107 cells/m2, about 108 cells/m2, about 5 x 108 cells/m2, about 109 cells/m2, about 5 x 109 cells/m2, about 1010 cells/m2, about 5 x 101 cells/m2, or about 10" cells/m2.
Chimeric Tim receptors and PARP inhibitors as described herein may be administered intravenously, intraperitoneally, intranasally, intratumorly, into the bone marrow, into the lymph node, and /or into cerebrospinal fluid.
In one aspect, methods of the present disclosure comprise conferring or enhancing phosphatidylserine-specific cytotoxic activity of a cell comprising introducing into a host cell a nucleic acid molecule encoding at least one chimeric Tim receptor or a chimeric Tim receptor vector according to any of the embodiments described herein; and expressing the at least one chimeric Tim receptor in the host cell, wherein the at least one chimeric Tim receptor enhances the phosphatidylserine-specific cytotoxic activity of the host cell as compared to a the host cell prior to modification to express a chimeric Tim receptor. In certain embodiments, the cytotoxic activity of the host cell is increased at least about 10%, 15%, 201?6, 25%, 30%, 35 /0, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more as compared to thc host cell prior to modification with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector. In some embodiments, the host cell is an immune cell.
In some embodiments, the host cell is a T cell or an NK cell. Methods of measuring cytotoxic activity of host cells, particularly immune cells such as T cells and NK cells, include a chromium (51Cr)-release assay, a or firefly luciferase release assay, flow cytometric methods of mediating target cell death and effector cell activity (see, e.g., Expert Rev. Vaccines, 2010, 9:601-616).
In certain embodiments, methods of the present disclosure comprise conferring or enhancing phosphatidylserine-specific cytotoxic activity of a cell further comprise conferring or enhancing phosphatidylserine-specific engulfment activity of the host cell expressing the at least one chimeric Tim receptor. In certain such embodiments, the host cell does not naturally exhibit an engulfment phenotype prior to modification with the chimeric Tim receptor. For example in certain such embodiments, the engulfment activity of the host cell is increased at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50 4, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more as compared to the host cell prior to modification to express the chimeric Tim receptor vector. In certain embodiments, the host cell does not naturally possess engulfment activity. In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell or an NK cell. Methods of measuring engulfment activity of host cells include methods as described in PCT/US2017/053553 (incorporated herein by reference in its entirety).
In another aspect, a chitneric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor according to any of the embodiments provided herein may be used in a method of enhancing effector function of the host cell. In certain embodiments, enhanced effector function comprises increased cytotoxic activity, increased antigen specific cytokine production (e.g., IFN-1, IL-2, TNF-a, or any combination thereof), increased anti-apoptotic signaling, increased persistence, increased expansion, increased proliferation, or any combination thereof. In certain embodiments, the effector function of the host cell is enhanced at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 1600/0, 170%, 180%, 190%, 200% or more as compared to a host cell that is not modified with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector. In some embodiments, the host cell is an immune cell. In certain embodiments, the host cell is a T cell or an NK cell.
In another aspect, host cells modified with chimeric Tim receptors of the present disclosure can be used in methods for inhibiting or reducing immune cell exhaustion. In some embodiments, the immune cell is a T cell or NK cell. In certain embodiments, reduced exhaustion in T cells comprises; (a) decreased expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof in T cells; (b) increased production of1FN-y, 1L-2, TNF-a, or any combination thereof in T cells; or both (a) and (b). In certain embodiments, reduced exhaustion in NK cells comprises; (a) decreased expression of PD-1, NKG2A, TI1043, or any combination thereof in NK cells; (b) increased production of IFN-y, TNF-a, or both in NK cells; or both (a) and (b). In certain embodiments, the expression of an immune checkpoint molecule is decreased at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a host immune cell expressing the chimeric 'rim receptor as compared to a host immune cell that is not modified with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector.
In certain embodiments, the expression of the cytokine is increased at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%
or more in a host immune cell expressing the chimeric Tim receptor as compared to a host immune cell that is not modified with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector.
In another aspect, a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor according to any of the embodiments provided herein may be used in a method of reducing an immunosuppressive response to phosphatidylserine in a host cell. In certain embodiments, the immunosuppressive response comprises secretion of anti-inflammatory cytokines (e.g., IL-10, TGF-0, or both), the decrease in secretion of inflammatory cytokines (e.g., TNI7-a, IL-113, and IL-12), or both. In certain embodiments, the immunosuppressive response of the host cell to phosphatidylserine is decreased at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% as compared to a host cell that is not modified with a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector. In some embodiments, the host cell is an immune cell. In certain embodiments, the host cell is a T cell or an NK cell.
In yet another aspect, a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor according to any of the embodiments provided herein may be used in methods for eliminating target cells bearing surface exposed phosphatidylserine, e.g., for the elimination of cancer cells bearing surface presented phosphatidylserine. In certain embodiments, the target cells are damaged, stressed, apoptotic, necrotic cells (e.g., tumor cells) bearing surface exposed phosphatidylserine In certain embodiments, host cells expressing chimeric Tim receptors clear damaged, stressed, apoptotic, or necrotic target cells bearing surface exposed phosphatidylserine via inducing apoptosis, or both inducing apoptosis and engulfment. Host cells expressing chimeric Tim receptors may be administered to a subject alone, or in combination with other therapeutic agents, including for example CAR-T cells, TCRs, antibodies, radiation therapy, chemotherapy, small molecules, oncolytic viruses, electropulse therapy, etc.
In another aspect, a chimeric Tim receptor, a polynucleotides encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell that expresses a chimeric Tim receptor according to any of the embodiments provided herein may be used in methods to enhance the effect of a therapeutic agent that induces cellular stress, damage, necrosis, or apoptosis. Certain therapies, such as chemotherapy, radiation therapy, UV light therapy, clectropulse therapy, adoptive cellular immunotherapy (e.g., CAR-T cells, TCRs) and oncolytic viral therapy, can induce cell damage or death to tumor cells, diseased cells, and cells in their surrounding environment. Cells expressing chimeric Tim receptors can be administered in combination with the cell damaging/cytotoxic therapy to bind to the phosphatidylserine moieties exposed on the outer leaflet of targeted cells and clear stressed, damaged, diseased, apoptotic, necrotic cells.
Chimeric Tim receptors and PARP inhibitors may be administered to a subject in combination with one or more additional therapeutic agents.
Examples of therapeutic agents that may be administered in combination with a chimeric Tim compositions according to the present description include radiation therapy, adoptive cellular immunotherapy agent (e.g., recombinant TCR, enhanced affinity TCR, CAR, TCR-CAR, scTCR fusion protein, dendritic cell vaccine), antibody therapy, immune checkpoint molecule inhibitor therapy, UV light therapy, electric pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy, or a pharmaceutical therapy, such as a chemotherapeutic agent, a therapeutic peptide, a hormone, an aptamer, antibiotic, anti-viral agent, anti-fungal agent, anti-inflammatory agent, a small molecule therapy, or any combination thereof In certain embodiments, the chimeric Tim receptor modified host cells may clear stressed, damaged, apoptotic, necrotic, infected, dead cells displaying surface phosphatidylserine induced by the one or more additional therapeutic agents.
In certain embodiments, a vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapy agent (e.g., chimeric antigen receptor, recombinant TCR, etc.). In certain embodiments, a single polynucleotide encoding the chimeric Tim receptor and cellular immunotherapy agent (e.g., CAR) is cloned into a cloning site and expressed from a single promoter, with the chimeric Tim receptor sequence and cellular immunotherapy agent (e.g., CAR) sequence separated from each other by an internal ribosomal entry site (IRES), furin cleavage site, or viral 2A peptide to allow for co-expression of multiple genes from a single open reading frame (e.g., a multicistronic vector). In certain embodiments, a viral 2A peptide is a porcine teschovirus-1 (P2A), 7hosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (172A), or variant thereof. An exemplary T2A peptide comprises an amino acid sequence of SEQ ID NO:12, 28, 29, or 30. An exemplary P2A peptide comprises an amino acid sequence of SEQ ID
NO:13 or 31. An exemplary E2A peptide sequence comprises an amino acid sequence of SEQ
ID NO:14. An exemplary F2A peptide sequence comprises an amino acid sequence of SEQ ID NO:15.
In certain embodiments, a polynucleotide encoding the chimeric Tim receptor and a polynucleotide encoding the cellular immunotherapy agent (e.g., CAR) binding protein are joined together into a single polynucleotide and then inserted into a vector. In other embodiments, a polynucleotide encoding the CER, and a polynucleotide encoding the CAR or TCR binding protein may be inserted separately into a vector in the same or different cloning sites, such that the expressed amino acid sequence produces a functional CER and CAR/or TCR. A vector that encodes a tandem expression cassette is referred to herein as a "tandem expression vector."
In certain embodiments, a vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapy agent (e.g., CAR). The pol ynucleotides encoding the chimeric Tim receptor and cellular immunotherapy agent (e.g., CAR) may be cloned sequentially into a vector at different cloning sites, with the chimeric Tim receptor and cellular immunotherapy agent (e.g., CAR) expressed under the regulation of different promoters.
In certain embodiments, a chimeric Tim receptor modified host cell may also be modified to co-express one or more small GTPases. Rho GTPases, a family of small (-21 k Da) signaling G proteins and also a subfamily of the Ras superfamily, regulate actin cytoskeleton organization in various cell types and promote pseudopod extension and phagosome closure during phagocytosis (see, e.g., Castellano et al., 2000, J. Cell Sci. 113:2955-2961). Engulfment requires F-actin recruitment beneath tethered cells or particles, and F.-actin rearrangement to allow membrane extension resulting in cell or particle internalization. RhoGTPases include RhoA, Racl, Rac2, RhoG, and CDC42. Other small GTPases, such as Rap I , is involved in regulation of complement mediated phagocytosis. Co-expression of a small GTPasc with the chimeric Tim receptor may promote target cell or particle internalization and/or phagosome formation by the host cell. In some embodiments, a recombinant nucleic acid molecule encoding a GTPase is encoded on a separate vector than the chimeric Tim receptor-containing vector. In other embodiments, a recombinant nucleic acid molecule encoding a GTPase is encoded on the same vector as the chimeric Tim receptor. The GTPase and chimeric Tim receptor may be expressed under the regulation of different promoters on the same vector (e.g., at different multiple cloning sites). Alternatively, the chimeric Tim receptor and GTPase may be expressed under the regulation of one promoter in a tnulticistronic vector. The polynucleotide sequence encoding the chimeric Tim receptor and the polynucleotide sequence encoding the small GTPase(s) may be separated from each other by an IRES or viral 2A. peptide in a multicistronic vector.
Exemplary 2A
peptides include T2A (SEQ ID NO:12), P2A (SEQ ID NO:13), E2A (SEQ ID NO:14), F2A (SEQ ID NO:15). Examples of GTPases that may be co-expressed with a chimeric Tim receptor include Racl, Rac2, Rab5 (also referred to as Rab5a), Rab7, R.apl, RhoA., RhoG, CDC42, or any combination thereof. In specific embodiments, the GTPase comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to a Racl amino acid sequence of SEQ ID NO:17, a Rab5 amino acid sequence of SEQ ID NO:18, a Rab7 amino acid sequence of SEQ ID
NO:19, a Rapl amino acid sequence of SEQ ID NO:20, a R.hoA amino acid sequence of SEQ ID NO:21, a CDC42 amino acid sequence of SEQ ED NO:22, or any combination thereof.
In certain embodiments, a chimeric Tim receptor modified host cell may also be modified to co-express a cellular immunotherapy agent (e.g., CAR, TCR, etc.).
In some embodiments, the cellular immtmotherapy agent comprises a chimeric antigen receptor (CAR). CARs are recombinant receptors that generally comprise: an extracellular domain comprising a binding domain that binds to a target antigen; an intracellular signaling domain (e.g., comprising an ITAM containing intracellular signaling domain and optionally an intracellular costimulatory domain), and a transmembrane domain positioned between and connecting the extraccllular domain and the intracellular signaling domain.
Binding domains suitable for use in CARs of the present disclosure include any antigen-binding polypeptide. A binding domain may comprise an antibody or antigen binding fragment thereof, including for example, a full length heavy chain, Fab fragment, Fab', F(ab')2, sFv, VH domain, VL domain, dAb, VI-111, CDR, and scFv.
In certain embodiments, a CAR binding domain is murine, chimeric, human, or humanized.
In certain embodiments, the binding domain of the CAR targets a cancer or tumor antigen. Exemplary antigens that a CAR may target include CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, LICAM, CD22, CDI9, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR, GD-3, }WV E6, HPV E7, MUC-1, HER2, folate receptor a, CD97, CD171, CD179a, CD44v6, WTI, VEGF-a, VEGFR1, IL-13Ral, 1L-13Ra2, It-11Ra, PSA, FcRII5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A
antigen, Lewis Y antigen, MAGE, MAGE-Al, RAGE-1, folate receptor 0, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM I, GM:3, o-acetyl-GD2, and In certain embodiments, the extracellular domain of CARs provided in the present disclosure optionally comprises an extracellular, non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain away from the host cell suiface to further enable proper cell to cell contact, binding, and activation. An extracellular spacer domain is generally located between the extracellular binding domain and the transmembrane domain of the CAR.
The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest et al., J. Immwtother. 28:203-11, 2005;
PCT
Publication No. WO 2014/031687). In certain embodiments, an extracellular spacer domain is an immunoglobulin hinge region (e.g., IgGl, IgG2, IgG3, I8G4, IgA, IgD).
An immunoglobulin hinge region may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region. An altered IgG4 hinge region is described in PCT Publication No. WO 2014/031687, which hinge region is incorporated herein by reference in its entirety. In a particular embodiment, an extracellular spacer domain comprises a modified IgG4 hinge region having an amino acid sequence of SEQ ID NO:3.
Other examples of hinge regions that may be used in the CARs described herein include the hinge region from the extracellular regions of type I
membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof. In a particular embodiment, an extracellular spacer domain comprises a CD8a hinge region having an amino acid sequence of SEQ ED NO:70. In another particular embodiment, an extracellular spacer domain comprises a CD28 hinge region having an amino acid sequence of SEQ ID NO:32. In further embodiments, an extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from: a CH1 domain, a CH2 domain, a CI-13 domain, or combinations thereof (see, e.g., PCT Publication W02014/031687, which spacers are incorporated herein by reference in their entirety). In yet further embodiments, an extracellular spacer domain may comprise a stalk region of a type El C-lectin (the extracellular domain located between the C-typelectin domain and the transmembrane domain). Type 11 C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.
CARs of the present disclosure comprise a transmembrane domain that connects and is positioned between the extracellular domain and the intracellular signaling domain. The transmembrane domain ranges in length from about 15 amino acids to about 30 amino acids. The transmembrane domain is a hydrophobic alpha helix that transverses the host cell membrane and anchors the CAR in the host cell membrane. The transmembrane domain may be directly fused to the binding domain or to the extracellular spacer domain if present. In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g, receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, or the like). The transmembrane domain can be selected from the same molecule as the extracellular domain or the intracellular signaling domain (e.g., a CAR
comprises a CD28 costimulatory signaling domain and a CD28 transmembrane domain). In certain embodiments, the transmembrane domain and the extracellular domain are each selected from different molecules. In other embodiments, the transmembrane domain and the intracellular signaling domain are each selected from different molecules. In yet other embodiments, the transmembrane domain, the extracellular domain, and the intracellular signaling domain are each selected from different molecules.
Exemplary transmembrane domains for use in CARs of the present disclosure include a CD28, CD2, CD4, CD8a, CD5, CD38, CDR.), CD3, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD7911, CD80, CD86, CD95 (Fas), CD134 (0X40), CD137 (4-IBB), CD150 (SLAMF1), CD152 (CTLA4), CD154 (CD4OL), CD200R, CD223 (LAG3), CD270 (BTLA), CD273 (PD-L2), CD274 (PD-Li), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRct, FcR13, FcRy, Fyn, GAL9, KIR, Lek, LAT, LRP, N.KG2D, NOTCH1, NOTCH2, NoTcH3, NOTCH4, P`FCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCR13, TIM3, TRIM, LPA5, and Zap70 transmembrane domain.
An exemplary CD28 transmembrane domain comprises an amino acid sequence of SEQ
113 .N0:7. In a particular embodiment, a transmembrane domain comprises a CD8a transmembrane domain having an amino acid sequence of SEQ ID NO:33.
The intracellular signaling domain of a CAR is an intracellular effector domain and is capable of transmitting functional signals to a cell in response to binding of the extracellular domain of the CAR to a target molecule (e.g., cancer antigen) and activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. In some embodiments, the CAR
induces a function of a T cell such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. The intracellular signaling domain may be any portion of an intracellular signaling molecule that retains sufficient signaling activity.
In some embodiments, the intracellular signaling domain is obtained from an antigen receptor component (e.g., TCR) or costimulatory molecule. In some embodiments, a full length intracellular signaling domain of an antigen receptor or costimulatory molecule is used.
In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor or costimulatory molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In further embodiments, an intracellular signaling domain is a variant of a full length or truncated portion of an intracellular signaling domain of an antigen receptor co stimulatory molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).
In certain embodiments, the intracellular signaling domain of a CAR
comprises an immunoreceptor tyrosine-based activation motif (ITAM) containing signaling domain. An ITAM containing signaling domain generally contains at least one (one, two, three, four, or more) ITAM:s, which refer to a conserved motif of YXXL/I-X6-8-YXXL/I. An ITAM containing signaling domain may initiate T cell activation signaling following antigen binding or ligand engagement. ITAM-signaling domains include, for example, intracellular signaling domains of CD37, CD38, CD3e, CD3C,, CD5, CD22, CD79a, CD278 (ICOS), DAP12, FcRy, and CD66d. Exemplary CD3C, signaling domains that may be used in CARs of the present disclosure comprise an amino acid sequence of SEQ ID NO:27 or 5.
CAR intracellular signaling domains optionally comprise a costimulatory signaling domain, which, when activated in conjunction with a primary or classic (e.g., 'TAM-driven) activation signal, promotes or enhances T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or combinations thereof. Costimulatory signaling domains for use in CARs include, for example, CD27, CD28, CD4OL, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAPIO, LAT, LFA-I , LIGHT, NKG2C, S1 ,P76, TRIM, ZAP70, or any combination thereof In a particular embodiment, the costimulatory signaling domain comprises a 0X40, CD2, CD27, CD28, LFA-1 (CD1 la/CD18), ICOS (CD278), or 4-1BB (CD137) signaling domain. Exemplary CD28 costimulatory signaling domains that may be used in CARs of the present disclosure comprise an amino acid sequence of SEQ ID NO:26 or 4. An exemplary 4-1BB costimulatory signaling domain comprises an amino acid sequence of SEQ ID NO: i22. In certain embodiments, a CAR comprises one, two, or more costimulatory signaling domains.
In some embodiments, CARs are recombinant receptors composed of an scFv binding domain derived from an antibody, a transmembrane domain, and an intracellular signaling domain(s). In some embodiments, the intracellular signaling domain(s) are derived from a TCR.
In certain embodiments, a chimeric antigen receptor comprises an amino acid sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof In certain embodiments, chimeric antigen receptor is murine, chimeric, human, or humanized.
In certain embodiments, a CAR is a first generation CAR, a second generation CAR, or a third generation CAR. A first generation CAR generally has an intracellular signaling domain comprising an intracellular signaling domain of CD3C, FcleRI, or other ITAM-containing activating domain to provide a T cell activation signal. Second generation CARs further comprise a costimulatory signaling domain (e.g., a costimulatory signaling domain from an endogenous T cell costimulatory receptor, such as CD28, 4-1I3B, or TCOS). Third generation CARs comprise an TTAM-containing activating domain, a first costimulatory signaling domain and a second costimulatory signaling domain.
In some embodiments, one or more of the extracellular domain, the binding domain, the linker, the transmembrane domain, the intracellular signaling domain, or the costimulatory domain comprises junction amino acids. "Junction amino acids" or "junction amino acid residues" refer to one or more (e.g., about 2-20) amino acid residues between two adjacent domains, motifs, regions, modules, or fragments of a protein, such as between a binding domain and an adjacent linker, between a transmembrane domain and an adjacent extracellular or intracellular domain, or on one or both ends of a linker that links two domains, motifs, regions, modules, or fragments (e.g., between a linker and an adjacent binding domain or between a linker and an adjacent hinge). Junction amino acids may result from the construct design of a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site or self-cleaving peptide sequences during the construction of a polynucleotide encoding a fusion protein). For example, a transmembrane domain of a fusion protein may have one or more junction amino acids at the amino-terminal end, carboxy -terminal end, or both.
Exemplary binding domain, extracellular spacer, transmembrane, and intracellular signaling domain sequences for use in CAR.s of the present disclosure are set forth in Table 11.
Table H.
Name Amino Acid Sequence SEQ ID
NO:
Flexible linker GGGGSGGGGSGGGGS
Flexible linker GTGG'SGGTGSGTGIS
1gG4 hinge .ESK.YGPPC.PPC.P
region (short) CD8a hinge TT.TPAPRPP-rpAPTIASQPLSLRITACRPAAGGAVErrit region GLDFACD
CD28 hinge IEVMYPPPYLDNEKSNGTI1HVKGKHLCPSPLFPGPSKP
region CD8a IYIWAPLAartGVLLLSLAIrrLYC
transmembrane region transmembrane FW VLVVVGGVLACYSLLVTVAHIFWV
region wildtype CD3 R'VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD
signaling ICRRGRDPEMGGKPQRRICNPQEGLYNELQKDKMAEA
domain YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM
QALPPR
variant CD3C, R'VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD
signaling 1CRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY
domain SE1GMKGERRRGKGHDGLYQGLsTATKDTYDALH1Q
ALPPR
wild.type CD28 RSICRSRLLHSDYNLNMTPRRPGPTRICHYOPYAPPRDFA
costimulatory AYRS
signaling domain variant CD28 RSICRSRGGHSDYNLNMTPRRPGPTRICHYQPYAPPRDF
CoStimulatory AAYRS
signaling domain with substitutions with positions in reference to full length protein costimulatory GGCEI.:
signaling domain C1MCSFR_Sign MLLVTSLLLCELPHPAFLLIP
al Peptide In certain embodiments, a chimeric Tim receptor modified host cell co-expresses a recombinant TCR. Recombinant TCR proteins include "traditional"
TCRs composed of a heterodimer of a chain polypeptide and chain polypeptide or a heterodimer of a y chain polypeptide and a 5 chain polypeptide, binding fragments and fusion proteins thereof, including for example, single chain TCRs, single domain TCRs, soluble TCR fusion TCR proteins, and TCR fusion constructs (TRuCTm). In certain embodiments, a tandem expression cassette comprises a polynucleotide encoding a recombinant TCR beta chain comprising a TCR beta variable region and a TCR
beta constant region, and a polynucleotide encoding a recombinant TCR alpha chain comprising a TCR. alpha variable region and a TCR. alpha constant region. In certain embodiments, a recombinant TCR is an enhanced affinity TCR. In one embodiment, a recombinant TCR is an enhanced affinity TCR.
In certain embodiments, a recombinant TCR binding protein is a single chain TCR (scTCR) comprising a Va joined to a vi3 by a flexible linker. In some embodiments, a scTCR comprises a Va-linker-V13 polypeptide. In other embodiments, a scTCR comprises a V134inker-Va polypeptide.
in certain embodiments, a chimeric Tim receptor modified host cell may also be modified to co-express a single chain TCR (scTCR) fusion protein. A
scTCR
fusion protein comprises a binding domain comprising a scTCR (a TcR Va domain linked to a TCR V13 domain), an optional extracellular spacer, a transmembrane domain, and an intracellular component comprising a single intracellular signaling domain providing an T cell activation signal (e.g., a CD31; ITAM-containing activating domain) and optionally a costimulatory signaling domain (see, Aggen etal., 2012, Gene Ther. 19:365-374; Stone etal., Cancer Immunol. Immunother. 2014, 63:1163-76).
In certain embodiments, a chimeric Tim receptor modified host cell may also be modified to co-express a T cell receptor-based chimeric antigen receptor (TCR-CAR). A TCR-CAR is a heterodimeric fusion protein generally comprising a soluble TCR (a polypeptide chain comprising a Vu domain and Ca domain and a polypeptide chain comprising a V13 domain and a C13 domain) wherein the v3c:13 polypeptide chain is linked to a transmembrane domain and an intracellular signaling component (e.g., an ITAM-containing activating domain and optionally a costimulatory signaling domain) (see, e.g., Wa1seng et al., 2017 Scientific Reports 7:10713).
In certain embodiments, an engineered host cell that co-expresses a chimeric Tim receptor and a cellular immunotherapy agent (e.g., CAR., 'FCR, etc.) comprises a recombinant nucleic acid encoding the chimeric Tim receptor and a recombinant nucleic acid molecule encoding the cellular immunotherapy agent on separate vectors within the engineered host cell.
In some embodiments, an engineered host cell that co-expresses a chimeric Tim receptor and a cellular immunotherapy agent (e.g., CAR., TeR, etc.) comprises a recombinant nucleic acid encoding the chimeric Tim receptor and a recombinant nucleic acid molecule encoding the cellular immunotherapy agent on the same vector as the chimeric Tim receptor within an engineered host cell. The chimeric Tim receptor and cellular immunotherapy agent may be expressed under the regulation of different promoters on the same vector (e.g., at different multiple cloning sites).
Alternatively, the chimeric Tim receptor and cellular immunotherapy agent may be expressed under the regulation of one promoter in a multicistronic vector (e.g., tandem expression vector). The polynucleotide sequence encoding the chimeric Tim receptor and the polynucleotide sequence encoding the cellular immunotherapy agent may be separated by an IRES or viral 2A peptide in a multicistronic vector.
Tandem expression cassettes, tandem expression vectors, and engineered host cells comprising the same are described in International Application Publication No. W02019/191339, which is incorporated herein by reference in its entirety.
Host cells expressing chimeric Tim receptors may be administered to a subject alone, or in combination with other therapeutic agents, including for example CAR-T cells, TCRs, antibodies, radiation therapy, chemotherapies, small molecules, oncolytic viruses, electropulse therapy, etc.
In certain embodiments, the chimeric Tim receptor and adoptive cellular immunotherapy agent (e.g., a CAR, TCR-CAR, TCR, etc. described above) are administered to the subject in the same host cell or different host cells. In certain embodiments, the chimeric Tim receptor and adoptive cellular immunotherapy agent are expressed in the same host cell from the same vector or from separate vectors. In certain embodiments, the chimeric Tim receptor and adoptive cellular immunotherapy agent are expressed in the same host cell from a multicistronic vector. In certain embodiments, the chimeric Tim receptor is expressed in the same host cell type as the adoptive cellular immunotherapy agent (e.g., the chimeric Tim receptor is expressed CD4 T cells and the CAR/or TCR is expressed in CD4 T cells, or the chimeric Tim receptor is expressed CD8 T cells and the CAR/or TCR is expressed in CD8 T
cells).
In other embodiments, the chimeric Tim receptor is expressed in a different host cell type as the adoptive immunotherapy agent (e.g., the chimeric Tim receptor is expressed CD4 T cells and the CAR/or TCR is expressed in CD8 T cells). Cellular immunotherapy compositions comprising a combination of immune cells or cellular subsets engineered with chimeric Tim receptors and a cellular immunotherapy agent (e.g., CAR, TCR, etc.), methods of making, and methods of use are described in PCT
International Publication No. W02019/191340, which is incorporated herein by reference in its entirety.
Exemplary antigens that a recombinant TCR, enhanced affinity TCR, CAR, TCR-CAR, or sc*TCR. fusion protein may target include WT-1, mesothelin, MART-1, NY-ESO-1, MAGE-A3, HPV E7, survivin, a Fetoprotein, and a tumor-specific neoantigen.
CARs of the present disclosure may target a variety of antigens, including a viral antigen, bacterial antigen, fungal antigen, parasitic antimn, tumor antigen, autoimnnune disease antigen. Exemplary antigens that a CAR may target include CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, M1JC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA.125, CD56, c-Met, EGFR, GD-3, HPV E6, IIPV E7, MUC-1, HER2, folate receptor a, CD97, CD171, CD179a, CD44v6, WTI, VEGF-a, VEGFR1, IL-13Ral, IL-13R2, IL-11R.a, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-Al, RAGE-1, folate receptor p, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GI\41, GM3, o-acetyl-GD2, and GD2.
Radiation therapy includes external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiation, 3-dimensional conformal radiation therapy, intensity-modulated radiation therapy, volumetric modulated arc therapy, particle therapy, proton therapy, and auger therapy), brachytherapy, systemic radioisotope therapy, intraoperative radiotherapy, or any combination thereof.
Exemplary antibodies for use in conjunction with the chimeric Tim compositions described herein include rituxmab, pertuzumab, trastuzumab, alenituzumab, Ibrituniornab tiuxetan, Brentuximab vedotin, cetuximab, bevacizurnab, abciximab, adalimumab, alefacept, basilizimab, belitnumab, bezlotoxumab, canakinumab, certolizumab pegol, daclizumab, denosumab, efalizumab, golimumab, olaratumab, palivizumab, panitumumab, and tocilizumab.
Exemplary inhibitors of immune checkpoint molecules that may be for use in conjunction with the chimeric Tim compositions described herein include checkpoint inhibitors targeting PD-I,1, PD-1.2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CI:LA-4, BTLA, KIR, LAG3, T1M3, A2alt, CD244/2B4, CD160, mu, LAIR-1, PVRIG/CD112R, or any combination thereof. In certain embodiments, an immune checkpoint inhibitor may be an antibody, a peptide, an RNAi agent, or a small molecule. An antibody specific for CTLA-4 may be ipilimumab or tremelimumab.
An antibody specific for PD-1 may be pidilizumab, nivolumab, or pcmbrolizumab. An antibody specific for PD-Li may be durvalumab, atezolizumab, or avelumab.
Exemplary chemotherapeutics for use in conjunction with the chimeric Tim receptor compositions described herein may include an alkylating agent, a platinum based agent, a cytotoxic agent, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor.
A chemotherapeutic includes non-specific cytotoxic agents that inhibit mitosis or cell division, as well as molecularly targeted therapy that blocks the growth and spread of cancer cells by targeting specific molecules that are involved in tumor growth, progression, and metastasis (e.g., oncogenes). Exemplary non-specific chemotherapeutics for use in conjunction with the expression cassette compositions described herein may include an alkylating agent, a platinum based agent, a cytotoxic agent, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as fol ate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA
synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), hypomethylating agent, and a DNA repair inhibitor.
Examples of chemotherapeutic agents considered for use in combination therapies contemplated herein include vemurafenib, dabrafenib, trametinib, cobimetinib, anastrozole (Arimidexe), bicalutamide (Casodex0), bleomycin sulfate (Blenoxanee), busulfan (Myleran0), busulfan injection (Busulfex0), capecitabine (Xeloda6), N4-pentoxycarbony1-5-deox y-5-fluorocyti dine, carboplatin (Paraplatine), camiustine (BiCNUO), chlorambucil (Leukeran ), cisplatin (Platinole), cladribine (Leustatin0), cyclophosphamide (Cytoxan or NeosarS), cytarabine, cytosine arabinoside (Cytosar-U0), cytarabine liposome injection (DepoCyte), dacarbazine (DTIC-Dome ), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidinee), daunorubicin citrate liposome injection (DaunoXomee), dexamethasonc, docctaxcl (Taxoterce), doxorubicin hydrochloride (Adriamycine, Rubex ), etoposi de (Vepesida), fludarabine phosphate (FludaraV), 5-fluorouracil (Adrucile, Efudexe), flutamide (Eulexine), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea (HydreaC), Idarubicin (Idamycine), ifosfamide (IFEM.)), irinotecan (Camptosare), L-asparaginase (ELSPARO), leucovorin calcium, melphalan (Alkeran0), 6-mercaptopurine (Purinethole), methotrexate (Folexe), mitoxantrone (Novantrone ,), myiotarg, paclitaxel (Taxo10), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadele),fdabra tamoxifen citrate (Nolvadex0), teniposide (Vumone), 6-thioguanine, thiotepa, tirapazamine (Tirazone0), topotecan hydrochloride for injection (Ilycamptine), vinblastine (Velban0), vincristine (Oncovine), ibrutinib, venetociax, crizotinib, aiectinib, brigatinib, ceritinib, and vinorelbine (Navelbine0).
Exemplary alkylating agents for use in combination therapies contemplated herein include nitrogen mustards, ethyleni mine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard , Chlorethaminacile, Demethyldopan , Desmethyldopan , Haemanthamine , Nordopan , lifracil nitrogen Mustard , Uracilloste, Uracilmostaza , Uramustin , Uramustinee), chiormethine (Mustargene), cycl ophosphami de (Cytoxane, Neosar , Clafene, Endoxane, :Procytox , Revi rninuneTm), ifosfamitle (Mitoxana0), melphalan (Alkeran0), Chloramtbucil (Leukerane), pipobroman (Amedel , Vercyte ), triethylenemelamine (Hemel , Hexa.lene, Hexastate), triethylenethiophosphoramine, Temozolomide (Tem od a re), thiotepa (Thiopl ex ), busulfan (Busilvex , Mylerang), carmustine (BiCNUO), lomustine (CeeNUO), streptozocin (Zanosare), and Dacarbazine (DTIC:-Dome ). Additional exemplary alkylating agents for use in combination therapies contemplated herein include, without limitation, Oxaliplatin (Eloxatine); Temozolomide (Temodare and Temodale); Dactinomycin (also known as actinomycin-D, Cosmegen0); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran0); Altretamine (also known as hexamethylmelamine (11M114), Hexalen0); Carmustine (BiCNUID); Bendamustine (Treandae); Busulfan (Busulfex and Mylerame); Carboplatin (Paraplatine); Lomustine (also known as CCNU, CecNUO); Cisplatin (also known as CDDP, Platinol and Platinol -AQ);
Chiorambucil (LeukeranS); Cycl ophosphami de (Cytoxan and Neosar0);
Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-DomeS); Altretamine (also known as hexamethylmelamine (IIMM), Hexalen8); Ifosfamide (Ifex0);
Prednumustine; Procarbazine (Matulane6); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen0); Streptozocin (Zanosar0); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex )); Cyclophosphamide (Endoxan , CytoxanCiD, Neosar , Procytox , Revimmunee); and I3endamustine HCl (Treanda0).
Exemplary platinum based agents for use in combination therapies contemplated herein include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthriplatin, and triplatin tetranitrate.
Exemplary hypomethylating agents for use in combination therapies include azacitidine and decitabine.
Exemplary molecularly targeted inhibitors for use in conjunction with the chimeric Tim receptor compositions described herein include small molecules that target molecules involved in cancer cell growth and survival, including for example, receptor tyrosine kinase inhibitors, RAF inhibitors, BCL-2 inhibitors, ABL
inhibitors, TRK inhibitors, c-K:IT inhibitors, c-MET inhibitors, CDK4/6 inhibitors, FAK
inhibitors, FGFR inhibitors, FLT3 inhibitors, IDIII inhibitors, IDF12 inhibitors, PDGFRA inhibitors, and RE!' inhibitors Exemplary molecularly targeted therapy includes hormone antagonists, signal transduction inhibitors, gene expression inhibitors (e.g., translation inhibitors), apoptosis inducers, angiogenesis inhibitors (e.g., a VEGF pathway inhibitor), tyrosine kinase inhibitors (e.g., an EGF/EGFR pathway inhibitor), growth factor inhibitors, GTPase inhibitors, serine/threonine kinase inhibitors, transcription factor inhibitors, inhibitors of driver mutations associated with cancer, B-Raf inhibitors, RAF
inhibitors, a MEK inhibitors, mTOR inhibitors, adenosine pathway inhibitors, EGFR
inhibitors, PI3K inhibitors, BCL2 inhibitors, VEGFR inhibitors, MET inhibitors, MYC
inhibitors, BCR-ABL inhibitors, ABL inhibitors, HER2 inhibitors, IT-RAS inhibitors, K-RAS
inhibitors, PDGFR inhibitors, ALK inhibitors, ROSI inhibitors, BTK inhibitors, TRK
inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4/6 inhibitors, FAK
inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PARP
inhibitors, PARP inhibitors, PDGFRA inhibitors, and RET inhibitors. In certain embodiments, use of molecularly targeted therapy comprises administering a molecularly targeted therapy specific for the molecular target to a subject identified as having a tumor that possesses the molecular target (e.g., driver oncogene). in certain embodiments, the molecular target has an activating mutation. In certain embodiments, use of chimeric Tim receptor modified cells in combination with a molecularly targeted inhibitor increases the magnitude of anti-tumor response, the durability of anti-tumor response, or both. In certain embodiments, a lower than typical dose of molecularly targeted therapy is used in combination with chimeric Tim receptor modified cells.
Exemplary angiogenesis inhibitors include, without limitation A6 (Angstrom Pharmaceuticals), ABT-510 (Abbott Laboratories), AB`17-627 (Atrasentan) (Abbott Laboratories/Xinlay), ABT-869 (Abbott Laboratories), Actimid (CC4047, Pomalidomide) (Celgene Corporation), AdGVPEDF.11D (GeriVec), ADH-1 (Exherin) (Adherex Technologies), AEE788 (Novartis), AG-013736 (Axitinib) (Pfizer), (Prinomastat) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX5 I
(AngioGenex), ALN-VSP (ALN-VSP 02) (Alnylaxn Pharmaceuticals), AMG 386 (Amgen), AMG706 (Amgen), Apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573 (Ridaforolimus/MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ
197 (ArQule), ASA404 (Novartis/Antisoma), Ati pri mod (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals), Avastin (Bev acizumab) (Genentech), AZD2171 (Cediranib/Recentin) (AstraZeneca), BAY 57-9352 (Telati nib) (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim Pharmaceuticals), 13113W 2992 (Boehringer Ingelheim Pharmaceuticals), :BMS-275291 (Bristol-Myers Squibb), BM:S-582664 (Brivanib) (Bristol-Myers Squibb), BMS-690514 (Bristol-Myers Squibb), Calcitriol, CCI-779 (Torisel) (Wyeth), CDP-791 (ImClone Systems), Ceflatonin (Homoharringtonine/HHT) (ChemGenex Therapeutics), Celebrex (Celecoxib) (Pfizer), CEP-7055 (Ccphalon/Sanofi), CHIR-265 (Chiron Corporation), NGR-TNF, COL-3 (Mctastat) (Collagenex Pharmaceuticals), Combretastatin (Oxigene), CP-751,871(Figitumumab) (Pfizer), CP-547,632 (Pfizer), CS-7017 (Daiichi Sankyo Pharma), CT-322 (Angiocept) (Adnexus), Curcumin, Dalteparin (Fragmin) (Pfizer), Disulfiram (Antabuse), (Eisai Limited), E7080 (Eisai Limited), EMD 121974 (Cilengitide) (EMD
Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), Endostar (Simcere), Erbitux (linClone/I3ristol-Myers Squibb), EZN-2208 (Enzon Pharmaceuticals), EZN-2968 (Enzon Pharmaceuticals), GC1008 (Genzyme), Genistein, GSK1363089 (Foretinib) (GlaxoSmithKline), GW786034 (Pazopanib) (GlaxoSmithKline), GT-111 (Vascular Biogenics Ltd.), IMC-112113 (Ramucirumab) (imClone Systems), IMC-18F1 (imClone Systems), IMC-3G3 (ImClone LLC), INCB007839 (Incyte Corporation), INGN 241 (Introgen Therapeutics), Iressa (ZD1839/Gefitinib), LBH589 (Faridak/Panobinostst) (Novartis), Lucentis (Ranibizumab) (Genentech/Novartis), LY317615 (Enzastaurin) (Eli Lilly and Company), Macugen (Pegaptanib) (Pfizer), MEDI522 (Abegrin) (MedImmune), MLN518 (Tandutinib) (Millennium), Neovastat (AE941/Benefin) (Aeterna Zentaris), Nexavar (Bayer/Onyx), NM-3 (Genzyme Corporation), Noscapine (Cougar Biotechnology), NPI-2358 (Nereus Pharmaceuticals), OSI-930 (051), Pal amid 529 (Paloma Pharmaceuticals, Inc.), Parizein Capsules (2ME2) (EntreMed), Panzem NCD
(2ME2) (EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen lndustries/Medigen Biotechnology), PKC412 (Novartis), Polyphenon E (Green Tea Extract) (Polypheno E International, Inc.), PPI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (Vatalanib) (Novartis), PXD101 (Belinostat) (CuraGen Corporation), ltADOO I (Everolimus) (Novartis), RAF265 (Novartis), Regorafenib (BAY73-4506) (Bayer), Revlimid (Celgene), Retaane (Alcon Research), SN38 (Liposomal) (Neopharm), SNS-032 (BMS-387032) (Sunesis), S0M230 (Pasireotide) (Novartis), Squalamine (Genaera), Suramin, Sutent (Pfizer), Tarceva (Genentech), TB-403 (Thrombogenics), Tempostatin (Collard Biopharmaceuticals), Tetrathiomolybdate (Sigma-Aldrich), TG100801 (TargeGen), Thalidomide (Celgene Corporation), Tinzaparin Sodium, TKI258 (Novartis), IRC093 (Tracon Pharmaceuticals Inc.), VEGF Trap (Aflibercept) (Regencron Pharmaceuticals), VEGF
Trap-Eye (It egeneron Pharmaceuticals), Veglin (VasGene Therapeutics), Bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), Vorinostat (Merck), and ZSTK474.
Exemplary B-Raf inhibitors include vemurafenib, dabrafenib, and encorafenib.
Exemplary MEK inhibitors include binimetinib, cobimetinib, refameti nib, selumetinib, and trameti nib.
Exemplary BTK inhibitors include ibrutinib. Loxo-305, tirabrutinib, GDC-0853, acalabrutinib, ONO-4059, spebrutinib, BGB-3111, 1-1M71224, and M7583.
Exemplary TRK inhibitors include entrectinib, larotrectinib, CH7057288, ONO-7579, LOX0-101, lestaurtinib, and LOX0-195.
Exemplary c-KIT inhibitors include imatinb, sunitinb, and ponatinib.
Exemplary c-MET inhibitors include capmatinib, crizotinib, tivantinib, onartuzumab, INCB28060, AMG-458, savolitinib, and tepotinib.
Exemplary CDK4/6 inhibitors include palbociclib, ribociclib, abemaciclib, and trilaciclib Exemplary FAK inhibitors include defactinib, GSK2256098, B1853520, and PF-00562271.
Exemplary FGFR inhibitors include erdafitinib, pemigatinib, infigratinib, rogaratinib, AZD4547, BG.1398, FP-1039, and ARQ 087.
Exemplary FLT-3 inhibitors include quizartinib, crenolanib, gilteritinib, midostaurin, and lestaurtinib.
Exemplary I1)111 inhibitors include ivosidenib, BAY-1436032, and AGI-5198.
An exemplary IDH2 inhibitor includes enasidenib.
Exemplary PDGFRA inhibitors include imatinib, regorafenib, crenolanib, and olaratumab.
Exemplary pan-RAF inhibitors include belvarafenib, LX11254, LY3009120, INU-152, and HM95573.
Exemplary RET inhibitors include lenvatinib, alectinib, vandetanib, cabozantinib, BLU-667, and LOX0-292.
Exemplary ROS1 inhibitors include ceritinib, lorlatinib, entrectinib, crizotinib, TPX-0005, and DS-6051b.
Exemplary Vascular Endothelial Growth Factor (VEGF) receptor inhibitors include, but are not limited to, Bevacizumab (Avasting), axitinib (Inlyta0);
Brivanib alaninate (BMS-582664, (S) ......... ((R)-1-(4-(4-Fluoro-2-methy1-1H-indo1-5-yloxy)-5-methylpyrrolo[2,1-][1,2,4]triazin-6-yloxy)propan-2-y1)2-aminopropanoate);
Sorafenib (Nexavar0); Pazopanib (Votriente); Sunitinib malate (Sutent0);
Cediranib (AZD2171, CAS 288383-20-1); Vargatef (BIBF1120, CAS 928326-83-4); :Foretinib (GSK1363089); Telatinib (BAY57-9352, CAS 332012-40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib (Gleevece); Ponatinib (AP24534, CAS 943319-70-8);
Tivozanib (AV951, CAS 475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Vatalanib dihydrochloride (PTK787, CAS 212141-51-0); Brivanib (BMS-540215, CAS 649735-46-6); Vandetanib (Caprelsa or AZD6474); M:otesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethy1-1H-indo1-6-y1)-2-[(4-pyridinylmethyl)arnino]-3-pyridinecarboxamide, described in PCT Publication No. WO
02/066470); Dovitinib dilactic acid (TK1258, CAS 852433-84-2); Linfanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 849217-68-1); Lestaurtinib (CAS
111358-88-4); N15-M5-(1, 1-Di methylethyl)-2-oxazolylimethyl ]thio]-2-thiazoly1]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-Amino-1-044(3-methoxyphenyl)am in o)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)pi peridin-3-ol (BMS690514); N-(3,4-Di chloro-2-11 uoroph en yl )-6-methoxy-7-[[(3aa,513,6aa)-octahydro-2-methylcyclopenta[c]pyrrol-5-Amethoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); 4-Methy1-34[1-methyl-6-(3-pyridiny1)-1H-pyrazolo[3,4-d]pyrimidin-4-yliamino]-N-[3-(trifluoromethypphenyl]-benzamide (BHG712, CAS
940310-85-0); and Aflibercept (Eylea0).
Exemplary EGF pathway inhibitors include, without limitation tyrphostin 46, EKB-569, crlotinib (Tarccvaa), gcfitinib (Ircssa0), crbitux, ninnotuzumab, lapatinib (Tykerbe), cetuximab (anti-EGFR mAb), 188Re-1abe1ed nimotuzumab (anti-EGFR mAb), and those compounds that are generically and specifically disclosed in WO 97/02266, EP 0 564 409, WO 99/03854, EP 0 520 722, EP
0 566 226, EP 0 787 722, EP 0 837 063, U.S. Pat. No. 5,747,498, WO 98/10767, WO
97/30034, WO 97/49688, WO 97/38983 and WO 96/33980. Exemplary EGFR
antibodies include, but are not limited to, Cetuximab (Erbitux ); Panitumumab (Vectibix ); Matuzumab (EMD-72000); Trastuzumab (Herceptin0); Nimotuzumab (fiR3); Zalutumumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1). Exemplary Epidermal growth factor receptor (EGFR) inhibitors include, hut not limited to, Erlotinib hydrochloride (Tarceva0);
ceritinib;
brigatinib; osimeritinib; icotinib; Gefitnib (Iressa0); N44-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3"S")-tetrahydro-3-furanyfloxy]-6-quinazoliny1]-4(diinethylamino)-2-butenainide, 17ovoke); Vandetanib (Caprelsa0); Lapatinib (Tykerb ); (3R,4R)-4-Amino-1-04-((3-methoxyphenypamino)pyrrolo[2.,1-f][1,2,4]triazin-5-yOmethyl)piperidin-3-ol (BMS690514); Canertinib dihydrochloride (C1-1033); 6-[4-[(4-Ethyl-1-pi perazinypmethyl ]pheny1]-N-[(1R)-1-pheny I
ethyl]-7H-Pyrrolo[2,3-d]pyrimidin-4-amine (AF:E788, CAS 497839-62-0); Mubritinib (TAK165);
Pelitinib (EKB569); Afatinib (BIEW2992); Netatinib (HK1-272); N-[44[1-[(3-Fluorophenyl)methy1]-1H-indazol-5-yflaminoj-5-methylpyrrolo[2,1-1][1,2,4]triazin-6-y1]-carbamic acid, (3S)-3-morpholinylmethyl ester (B1\4S599626); N-(3,4-Dichloro-2-fluoropheny1)-6-methoxy-7-[[(3aa,50,6aa)-octahydro-2-methylcyclopenta[c]pyrrol-yllmethoxyl-4-quinazolinamine (X1,647, CAS 781613-23-8); 4-[44(1R)-1-Phenylethyliamino]-7H-pyrrolo[2,3-d]pyrimidin-6-y1Fphenol (PK1166, CAS 187724-61-4); rocelitinib Exemplary mTOR inhibitors include, without limitation, rapamycin (RaparnuneS), and analogs and derivatives thereof; SDZ-RAD; Temsirolimus (Torisele; also known as CCI-779); Ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,95,12S,15R,16E,18R,19Rõ21R,23 S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hcxamethy1-2,3,10,14,20-pcntaoxo-11,36-dioxa-azatri cycl o[30 .3.1 . 04'9]hex atri aconta-16,24,26,28-tetraen-12-yl]propy1]-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); Everolimus (Afinitor or RAD001); Rapamycin (AY22989, SirolimusS); Simapimod (CAS 164301-51-3); (5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-y1}-2-methoxyphenyl)methanol (AZD8055); 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridiny1)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N241,4-dioxot[4-(4-oxo-8-pheny1-4H-1-benzopyran-2-yOmorpholinium-4-yl]methoxyibuty1]-1..-arginylglycyl-L-a-aspartylL-serine-, inner salt (SF1126, CAS 936487-67-1).
Exemplary Phosphoinositide 3-kinase (PI3K) inhibitors include, but are not limited to, duvelisib, idelalisib, 442-(1H-Indazol-4-y1)-64[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-ylimorpholine (also known as GDC 0941 and described in PCT Publication Nos. WO 09/036082 and WO
09/055730); 2-Methy1-244-[3-methyl-2-oxo-8-(quinolin-3-y1)-2,3-dihydroimidazo[4,5-clquinolin-l-yl]phenylipropionitrile (also known as BEZ 235 or NVP-BEZ 235, and described in PCT Publication No. WO 06/122806); 4-(trifluoromethyl)-5-(2,6-ditnorpholinopyritnidin-4-yl)pridin-2-amine (also known as BKM120 or N VP-BKM120, and described in PCT Publication No. W02007/084786); Tozasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)-54[4-(4-Pyridiny1)-6-quinolinyl]methyleneF
2,4-thiazolidinedi one (GSK1059615, CAS 958852-01-2); (1E,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-14(di-2-propenylamino)methylenei-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(tnethoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtlio[1,2-c]pyran-2,7,10(11-1)-trione (PX866, CAS 502632-66-8); and 8-Pheny1-2-(morpholin-4-y1)-chromen-4-one (LY294002, CAS 154447-36-6). Exemplary Protein Kinase B (PKB) or AKT inhibitors include, but are not limited to. 8-[4-(1-Aminocyclobutyl)pheny1]-9-pheny1-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one (MK-2206, CAS 1032349-1); Perifosine (KRX0401); 4-Dodecyl-N-1,3,4-thiadiazol-2-y1 -benzenesulfonamide (PHT-427, CAS 1191951-57-1); 442-(4-Amino-1,2,5-oxadiazo1-3-y1)-1-ethy1-7-[(3S)-3-piperidinylmethoxy]-1H-imidazo[4,5-c]pyridin-4-y1]-2-mothyl-3-butyn-2-ol (GSK690693, CAS 937174-76-0); 8-(1-Hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6-one (palomid 529, P529, or SG-00529); Tricirbine (6-Amino-4-methy1-8-(13-D-ribofuranosyl)-4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine), (ctS)-a-[[[5-(3-Methyl-1H-indazol-5-yl)-3-pyridinyl]oxylmethyl:1-benzeneethanamine (A674563, CAS 552325-73-2); 4-[(4-Chlorophenyl)methy1]-147H-pyrrolo[2,3-d]pyrimidin-4-y1)-4-piperidinamine (CCT128930, CAS 885499-61-6); 4-(4-Chloropheny1)-444-(1H pyrazol-4-yl)pheny1]-piperidine (AT7867, CAS 857531-00-1); and Archexin (RX-0201, CAS 663232-27-7).
In certain embodiments, a tyrosine kinase inhibitor used in combination with chimeric Tim receptor modified cells is an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK inhibitors include crizotinib, ceritinib, alectinib, bnigatinib, dalantercept, entrectinib, and lorlatinib.
In certain embodiments where chimeric 'I'im receptor modified cells are administered in combination with one or more additional therapies, the one or more additional therapies may be administered at a dose that might otherwise be considered subtherapeutic if administered as a monotherapy. In such embodiments, the chimeric Tim receptor composition may provide an additive or synergistic effect such that the one or more additional therapies can be administered at a lower dose.
Combination therapy includes administration of a chimeric Tim receptor compositions as described herein before an additional therapy (e.g., 1 day to 30 days or more before the additional therapy), concurrently with an additional therapy (on the same day), or after an additional therapy (e.g., 1 day - 30 days or more after the additional therapy). In certain embodiments, the chimeric Tim receptor modified cells are administered after administration of the one or more additional therapies. In further embodiments, the chimeric Tim receptor modified cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration of the one or more additional therapies. In still further embodiments, the chimeric Tim receptor modified cells are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week after administration of the one or more additional therapies. Where the one or more additional therapies involves multiple doses, the chimeric Tim receptor modified cells may be administered after the initial dose of the one or more additional therapies, after the final dose of the one or more additional therapies, or in between multiple doses of the one or more additional therapies.
In certain embodiments, methods of the present disclosure include a depletion step. A depletion step to remove chimeric Tim receptors from the subject may occur after a sufficient amount of time for therapeutic benefit in order to mitigate toxicity to a subject. In such embodiments, the chimeric Tim receptor vector may include an inducible suicide gene, such as 1CASP9, inducible Fas, or HSV-TK.
Similarly, a chimeric Tim receptor vector may be designed for expression of a known cell surface antigen such as CD20 or truncated EGFR (SEQ TD NC):16) that facilitates depletion of transduced cells through infusion of an associated monoclonal antibody (mAb), for example, Rituximab for CD20 or Cetuximab for EGFR. Alemtuzumab, which targets CD52 present on the surface of mature lymphocytes, may also be used to deplete transduced B cells, T cells, or natural killer cells.
Subjects that can be treated by the compositions and methods of the present disclosure include animals, such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs The subject may be male or female, and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled.
Accordingly, the claims are not limited by the disclosure.
EXAMPLES
EVALUATING TIM-CER-T CELLS FOR SYNTHETIC LETHAL PARP INHIBITOR SYNERGISMS
IN OVARIAN CANCER
Kuramochi ovarian cancer cells expressing an mCherry-NLS plasmid were incubated with 0.78 to 100 uM of the PARP inhibitor Niraparib. The number of Kuramochi cells / well was tracked via imaging. At t-50, the number of Kuramochi cells in culture decreases monotonically with increasing Niraparib concentration. The results are shown in FIG. IA.
Similar experiments were performed at various doses of the PARP
inhibitors Niraparib, Olaparib, Talazoparib, Veliperib, and Rucaparib, and the number of Kuramochi cells was plotted at t=50 h. Talazoparib and Niraparib exhibit the most sensitive EC50 values, followed by Rucaparib and Olaparib. Veliparib has the least impact on the number of detected Kuramochi cells of the five PARP inhibitors tested.
Niraparib and Rucaparib almost eliminate the Kuramochi culture by t=50h. The results are shown in FIG. 1B.
PAM' INHIBITOR UFREGULATES SURFACE PHOSPHATIDYLSERINE AND HAS NEGLIGIBLE
Kuramochi cells expressing a mCherry-NLS plasmid were incubated with various concentrations of Niraparib, a Tim4-Fc chimera, and an anti-mouse IgG2a antibody conjugated to Alexa488. The number of Tim4+ Kuramochi cells (i.e., green objects) increases significantly above the IgG only control in response to incubation with even 1.56 pivI Niraparib (FIGS. 2A-2B). The number of Kuramochi cells increases in response to 1.56-12.5 l.LM Niraparib (FIG. 2C), indicating that PARP
inhibition induces surface phosphatidylserine without killing Kuramochi cells.
Co-cultures of primary human CD4+ and CD8+ T cells were incubated with various concentrations of Niraparib and T cell viability, diameter, and density were measured using a ViCell cell counter on days 7, 8, and 9 after activation with TransAct, corresponding to days 1, 2, and 3 after incubation with Niraparib. T cell viability, average diameter, and expansion from day 7 to day 9 exhibit negligible changes as a function of Niraparib concentration (FIG. 3).
CHIMERIC TIM RECEPTOR/ NIRAPARM SYNERGISTICALLY KILL K.URAMOCHI CELLS /N
HMO AT DIFFERENT DOSES
Kuramochi cells expressing a mCherry-NLS plasmid were incubated for hours with 25, 12.5, or 6.25 M. Niraparib. The next day the Niraparib concentration was reduced to 0.521.IM and co-cultures of primary human CD4+ and CD8+ T cells expressing chimeric Tim receptor construct 13A (TIM4 binding domain-CD28 15 transmembrane domain ¨ CD28 costimulatory domain- CD3t; signaling domain) (SEQ
ID NO:162) were added to the Kuramochi culture. T cell cultures expressing chimeric Tim receptor construct 13A significantly reduce the number of Kuramochi cells in culture below the mock transduction control (control-T), depending on the experimental conditions. The number of Kuramochi cells expressing mCherry-NLS was monitored 20 via IncuCyte. The results are shown in FIG. 4. Combination therapy with chimeric Tim receptor Construct 13A and niraparib synergistically kill Kuramochi cells at different doses.
Kuramochi cells expressing a mCherry-NLS plasmid were incubated for ¨20 hours with 1.56 p,M Niraparib. The next day, media were removed from cells and replaced with fresh media with 0 1.1.M Niraparib, and primary human CD4+ and CD8+ T
cells expressing chimeric Tim receptor CTX140 (Tim4 binding domain-CD28 hinge-CD28 transmembrane-CD28 costimulatory domain-DAP12)(SEQ ID NO:195) or CTX156 (tEGFR control) were added to the Kuramochi culture. The number of Kuramochi cells expressing mCherry-NLS was monitored via IncuCyte. The results are shown in FIG. 5. T cell cultures expressing chimeric Tim receptor construct significantly reduce the number of Kuramochi cells in culture below the mock transduction control.
2,500 Kuramochi ovarian cancer cells expressing an mCherry-NLS
plasmid were plated on day 0. 25, 12.5, or 6.25 !AM Niraparib were added to cell culture -4 hours later. -20 hours after initial Niraparib dose, primary human CD4+ and CD8-F- T cells expressing chimeric Tim receptor CTX137 (Tim4 binding domain-transmembrane-CD28 costirnulatory domain-DAP12) (SEQ ID NO:197) or CTX156 (tEGFR control) and maintenance dose of 0.52 pM Niraparib were added. The results for 25 M. Niraparib pre-treatment are shown in FIG. 6. The results for 25, 12.5, or 6.25 pM Niraparib pre-treatment with varying effector:target (E:T) ratios are shown in FIG.
7. T cell cultures expressing chimeric Tim receptor construct CTX137 significantly reduce the number of Kuramochi cells in culture below the mock. transduction control.
To induce Ptd-Ser exposure, BRCA-2 mutated Kuramochi cell line was treated with therapeutic doses of the PARP inhibitor Niraparib. Brief exposure to Niraparib elicits changes in membrane phospholipid symmetry, in a dose dependent manner, and is effective in inhibiting growth of Kuramochi cells (HG. 8A). The addition of chimeric Tim4 receptor pCTX133 (Tim4-TLR2-CD3z), at low effector:
target ratios (1:1), enhanced the potency of Niraparib in vitro compared to transduced-controls, demonstrating the ability of pCTX133 to elicit direct cytotoxic effects on target cells (HG. 8B).
T cell transduction and culturing: CD4 and CD8 T cells were isolated from frozen healthy donor PBMCs using Miltenyi CD4+ and CD8+ isolation kits.
and CD8 T cells were then mixed in a 1:1 ratio and incubated overnight in a flat bottom 24 well plate in OpTmizer medium supplemented with cell serum replacement, L-Glutamine, GlutaMAX, and IL-2, 1L-7, and IL-15 cytokincs, and TransAct diluted 1:50.
16-24 hours past activation with TransAct the T cells were transduced with the appropriate virus at an MO1¨ 10 with 81.1g/mL polybrene and 1:50 TransAct in a flat bottom 96 well plate. ¨24 hours after transduction the T cells were transferred to a 24 well GRex plate in OpTmizer medium supplemented with cell serum replacement, L-Glutamine, GlutaMAX, and 1L-2, 1L-7, and 1L-15 cytokines. T cells were counted and supplemented with fresh medium approximately every two days thereafter.
Cell line Engineering: A2780 (human ovarian cancer cell line) and Kuramochi (BRCA deificient human ovarian cancer cell line) cells were transduced with lentiviral vectors encoding a mCherry-Nuclear localization sequence (NLS) gene and then sorted according to mCherry expression. A2780-mCherry-NLS cell lines were clonally sorted, expanded, and banked, whereas Kuramochi-mCherry-NLS cells were expanded and banked in bulk. Cell lines were thawed and kept in culture up to ¨passage 25 in RPM] 1640 supplemented with 10% FBS.
Co-culture assays: On Day 0 2,500 A2780-mCh-NLS or Kuramochi-mCh-NLS cells were plated in a NUNC Edge 96-well plate and Niraparib (Free Base) was added ¨4 hours later at the appropriate concentration, all in RPMI 1640 10%
FBS. Approximately 20 hours later the medium was removed and replaced with the appropriate concentration of Niraparib (Free Base) and the appropriate number of T
cells, all in OpTmizer medium supplemented with cell serum replacement, L-Glutamine, GlutaMAX, and 1L-2, 1L-7, and 1L-15 cytokines. When A2780-mCh-NLS
cells were used, the supernatant was centrifuged and resuspended in T cell medium to preserve any suspension component of A2780 cells. Plates were immediately transferred to IncuCyte when appropriate.
Flow cytometry measurement of surface PS: A2780-mCh-N1S cells were treated with 1.56 or 25 11M Niraparib or with equivalent volume of DMSO
(control). 48 hours later samples were trypsinized and stained using a_Tim4-Fc followed by a tluorescently-labeled secondary antibody to the Tim4-Fc. Samples_were immediately analyzed via Flow cytometry.
As shown in FIGS. 9A-9B, T cells modified with chimeric Tim4 receptor pCTX247 (Tim4 binding domain-CD28 transmembrane CD28 signaling domain ¨ CD3.; signaling domain; SEQ ID NO:257) demonstrate in vitro anti-tumor activity in two ovarian cancer cell lines. As shown in FIG. 10, T cells modified with chimeric Tim4 receptor pCTX797 (Tim4 binding domain-CD28 transmembrane CD28 signaling domain ¨ CD3C, signaling domain; SEQ ID NO:258) demonstrate in vitro anti-tumor activity in A2780 ovarian cancer model.
As shown in FIG. 11A-11B, Phosphatidyl swine is present on the cell surface in response to Niraparib in two ovarian cancer models.
As shown in FIG. 12, chimeric Tim4 receptor pCTX797 (Tim4 binding domain-CD28 transmembrane ¨ CD28 signaling domain ¨ CD3C signaling domain;
SEQ ID NO:258) exhibits synergy with additional PARP inhibitors (rucarparib, olaparib, and niraparib) compared to truncated EGFR. (EGFRt) control (pCTX236).
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheetincluding but not limited to, U.S Provisional Patent Application No.
63/066,150, filed on August 14, 2020, U.S. Provisional Patent Application No. 63/087,049, filed on October 2, 2020, and U.S. Provisional Patent Application No. 63/226,712 filed on July 28, 2021, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled.
Accordingly, the claims arc not limited by the disclosure.
Claims (88)
1. A. method for treating a cancer, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian cancer, peritoneal cancer, prostate cancer, lung cancer, or melanoma, in a subject in need thereof, the method comprising administering to the subject an effective arnount of:
a chimeric T-cell immunoglobulin and mucin (Tim) receptor comprising a single chain chimeric protein, the single chain chimeric protein comprising:
(a) an extracellular doraain comprising a binding domain comprising:
(i) a Timl .10/ domain or a Tirn4 IgV domain; and (ii) a Timl mucin domain or a Tim4 mucin domain;
(b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) a transmembrane domain positioned between and connecting the extracellular domain and the intracellular signaling dornain; and a Poly (ADP-ribose) polymerase (PARP) inhibitor).
a chimeric T-cell immunoglobulin and mucin (Tim) receptor comprising a single chain chimeric protein, the single chain chimeric protein comprising:
(a) an extracellular doraain comprising a binding domain comprising:
(i) a Timl .10/ domain or a Tirn4 IgV domain; and (ii) a Timl mucin domain or a Tim4 mucin domain;
(b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) a transmembrane domain positioned between and connecting the extracellular domain and the intracellular signaling dornain; and a Poly (ADP-ribose) polymerase (PARP) inhibitor).
2. The method of claim 1, wherein the breast cancer is triple negative breast cancer.
3. The method of claim 1, wherein the ovarian cancer is advanced ovarian cancer.
4. The method of claim 1, wherein the prostate cancer is advanced prostate cancer.
5. The method of claim 1, wherein the lung cancer is non-small cell lung cancer.
6. The method of any one of claims 1-5, wherein the cancer is a BReast CAncer gene (BRCA) mutated cancer.
7. The method of any one of claims 6, wherein the cancer is a BRCAI rnutated cancer, a BRCA2 mutated cancer, or both.
8. The method of any one of claims 1-7, wherein the PARP inhibitor is talazoparib, niraparib, rucaparib, olaparib, veliparib, CEP 9722, E7016, AG014699, MK4827, BMN-673, Parniparib, or a combination thereof
9. The method of any one of claims 1-8, wherein the PARP inhibitor comprises ni rapari b .
10. The method of any one of claims 1-8, wherein the PARP inhibitor cornprises talazoparib.
11. The method of any one of claims 1-8, wherein the PARP inhibitor comprises rucaparib.
12. 'The method of any one of+Aaims 1-8, wherein the PARP inhibitor coinprises ol aparib.
13. The method of any one of claiins 1-8, wherein the PARP inhibitor comprises veliparib .
14. The method of any one of claims 1-8, wherein the PARP inhibitor comprises CEP
9722.
9722.
15. The method of any one of claims 1-8, wherein the PARP inhibitor comprises E7016.
16. The method of any one of claims 1-8, wherein the PARP inhibitor comprises AGO 1 4699.
17. The method of any one of claims 1-8, wherein the PARP inhibitor comprises MK4827.
18. The method of any one of claims 1-8, wherein the PARP inhibitor comprises BMN-673.
19. The method of any one of claims 1-8, wherein the PARP inhibitor comprises Pamiparib.
20. The method of any one of claims 1-19, further comprising administering an.
additional therapeutic agent.
additional therapeutic agent.
21.. The method of clairn 20, wherein the additional therapeutic agent comprises radiation, cellular immunotherapy, antibody, immune checkpoint molecule inhibitor, chemotherapy, hormone therapy, peptide, antibiotic, anti-viral agent, anti-fungal agent, anti-inflamrnatory agent, UV light therapy, electric pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy, a small molecule therapy, or a com.bination thereof.
22. The method of claim 21, wherein the cellular immunotherapy is a chimeric antigen receptor or T cell receptor.
23. The method of claim 20 or 21, wherein the additional therapeutic agent comprises an angiogenesis inhibitor (e.g., a VEGF pathway inhibitor), tyrosine kinase inhibitor (e.g., an EGF pathway inhibitor), receptor tyrosine kinase inhibitor, growth factor inhibitor, GTPase inhibitor, serine/threonine kinase inhibitor, transcription factor inhibitor, B-Raf inhibitor, RAF inhibitor, MEK inhibitor, mTOR inhibitor, EGFR
inhibitor, ALK inhibitor, ROS1 inhibitor, BCL-2 inhibitor, PI3K inhibitor, "VEGFR
inhibitor, BCR-ABL inhibitor, MET inhibitor, MYC inhibitor, ABL inhibitor, inhibitor, BTK inhibitor, H-RAS inhibitor, K-RAS inhibitor, PUG-FR inhibitor, TRK
inhibitor, c-KIT inhibitor, c-MET inhibitor, CDK4/6 inhibitor, FAK inhibitor, FGFR
inhibitor, FLT3 inhibitor, IDH1 inhibitor, 1DH2 inhibitor, PDGFRA inhibitor, or RET
nhi bi tor.
inhibitor, ALK inhibitor, ROS1 inhibitor, BCL-2 inhibitor, PI3K inhibitor, "VEGFR
inhibitor, BCR-ABL inhibitor, MET inhibitor, MYC inhibitor, ABL inhibitor, inhibitor, BTK inhibitor, H-RAS inhibitor, K-RAS inhibitor, PUG-FR inhibitor, TRK
inhibitor, c-KIT inhibitor, c-MET inhibitor, CDK4/6 inhibitor, FAK inhibitor, FGFR
inhibitor, FLT3 inhibitor, IDH1 inhibitor, 1DH2 inhibitor, PDGFRA inhibitor, or RET
nhi bi tor.
24. The method of any one of claims 1-23, wherein the binding domain comprises the Timl IgV domain and the Tirni mucin domain.
25. The method of any one of claims 1-23, wherein the binding domain comprises the Ti m4 IgV domain and the Tim4 mucin domain.
26. The method of any one of clairns 1-23, wherein the binding domain comprises the Tim1 1gV domain and the Tim4 mucin domain.
27. The method of any one of claims 1-23, wherein the binding domain comprises the Tim4 IgV domain and the Tirnl mucia domain.
28. The method of any one of claims 1-24 or 26, wherein the Timl IgV domain comprises the amino acid sequence set forth in SEQ ID NO:38.
29. The method of any one of claiins 1-24, 26, or 28, wherein the Tim1 IgV
domain is a modified Timl IgV domain comprising a R66G substitution in SEQ ID NO:38.
domain is a modified Timl IgV domain comprising a R66G substitution in SEQ ID NO:38.
30. The method of claim 29, wherein the modified Timl Tel domain comprises the amino acid sequence set forth in SEQ ID NO:41.
31. The method of any one of claims 1-25, or 27-30, wherein the Timi mucin domain comprises the amino acid sequence set forth in SEQ 1D NO:39.
32. The method of any one of claims 1-23, 25, or 27, wherein the Tim4 IgV
domain comprises the amino acid sequence set forth in SEQ ID NO:34
domain comprises the amino acid sequence set forth in SEQ ID NO:34
33. The method of any one of claims 1-23, 25, or 26, wherein the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35.
34. The method of any one of claims 1-33, wherein the transmembrane domain comprises a Tim4 transmembrane domain, Timl transmembrane domain, CD28 transmembrane domain, 4-1BB transmembrane domain, 0X40 transmembrane domain, CD27 transmembrane domain, ICOS transmembrane domain, CD2 transmembrane domain, LIFA-1 Vansmembrane domain, CD30 transmernbrane domain, CD40 transmembrane domain, PD-1 transmembrane domain, CD7 transmembrane domain, LIGHT transrnernbrane domain, NKG2C transmembrane domain, or B7-H3 transmembrane domain.
35. The method of clairn 34, wherein the Tim4 transrnembrane dornain comprises or consists of an amino acid sequence of SEQ ID NO:144 or 23; the Timl transmembrane domain comprises or consists of the amino acid sequence of S:EQ ID NO:8; the transmernbrane domain comprises or consists of an amino acid sequence of SEQ
ID
NO:145; the 4-1BB transmernbrane domain comprises or consists of an amino acid sequence of SEQ ID NO:146; the 0X40 transmembrane domain comprises or consists of an arnino acid sequence of SEQ ID NO:147; the CD27 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:148; the 1COS
transmembrane domain comprises or consists of an amino acid sequence of SEQ ID
NO:149; the CD2 transrnembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:150; the LFA-1 transmembrane domain comprises or consists of an arnino acid sequence of SEQ ID NO:151; the CD30 transmembrane domain comprises or consists of an amino acid sequence of SEQ ..113 NO:152; the CD40 transmembrane domain comprises or consists of an amino acid sequence of SEQ 1D
NO:153; the PD-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:154; the CD7 transmernbrane domain comprises or consists of an amino acid sequence of SEQ ID NO:155; the LIGHT transmcmbranc domain comprises or consists of an amino acid sequence of SEQ =ID NO:156; the NKG2C
transmembrane domain comprises or consists of an amino acid sequence of SEQ ID
NO:157; or the B7-H3 transmembrane dornain comprises or consists of an amino acid sequence of SEQ ID =NO:158.
ID
NO:145; the 4-1BB transmernbrane domain comprises or consists of an amino acid sequence of SEQ ID NO:146; the 0X40 transmembrane domain comprises or consists of an arnino acid sequence of SEQ ID NO:147; the CD27 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:148; the 1COS
transmembrane domain comprises or consists of an amino acid sequence of SEQ ID
NO:149; the CD2 transrnembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:150; the LFA-1 transmembrane domain comprises or consists of an arnino acid sequence of SEQ ID NO:151; the CD30 transmembrane domain comprises or consists of an amino acid sequence of SEQ ..113 NO:152; the CD40 transmembrane domain comprises or consists of an amino acid sequence of SEQ 1D
NO:153; the PD-1 transmembrane domain comprises or consists of an amino acid sequence of SEQ ID NO:154; the CD7 transmernbrane domain comprises or consists of an amino acid sequence of SEQ ID NO:155; the LIGHT transmcmbranc domain comprises or consists of an amino acid sequence of SEQ =ID NO:156; the NKG2C
transmembrane domain comprises or consists of an amino acid sequence of SEQ ID
NO:157; or the B7-H3 transmembrane dornain comprises or consists of an amino acid sequence of SEQ ID =NO:158.
36. The method of any one of claims 1-34, wherein the transmembrane domain comprises a Timl transmembrane domain, Tim4 transmembrane domain, or CD28 transmembrane domain.
37. The method of claim 42, wherein the Timl transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:8, the Tirn4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:6 or 23, or the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:7.
38. The method of any one of claims 1-37, wherein the chimeric Tim receptor further comprises an extracellular spacer domain.
39. The method of claim 38, wherein the extracellular spacer domain comprises an IgG4 hinge region or a CD28 hinge region.
40. The method of claim 39, wherein the IgG4 hinge region comprises the amino acid sequence set forth in SEQ ID NO:3, or the CD28 hinge region comprises the amino acid sequence set forth in SEQ ID NO:32.
41. The method of any one of claims 1-40, wherein the primary signaling domain comprises a CD28 costimulatory signaling domain; a 4-1B:B costimulatory signaling domain; a CD27 costimulatory signaling dornain; an ICOS costimulatory signaling domain; a LFA-1 costimulatory signaling domain; an 0X40 costimulatory signaling domain; a CD2 costirnulatory signaling domain; or an ICAM-1 costimulatory signaling domain.
42. The method of claim 41, wherein the CD28 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:118 or 119; the 4-1BB
costimulatory signaling domain comprises the amino acid sequence of SEQ ID
NO:122;
the CD27 costimulatory signaling domain comprises the amino acid sequence of SEQ
ID NO:169; the ICOS costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:172; the LFA-1 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:171; the 0)(40 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:166; the CD2 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:167; or the ICAM-1 costimulatory signaling comprises the amino acid sequence of SEQ ID NO:170.
costimulatory signaling domain comprises the amino acid sequence of SEQ ID
NO:122;
the CD27 costimulatory signaling domain comprises the amino acid sequence of SEQ
ID NO:169; the ICOS costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:172; the LFA-1 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:171; the 0)(40 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:166; the CD2 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:167; or the ICAM-1 costimulatory signaling comprises the amino acid sequence of SEQ ID NO:170.
43. The method of any one of claims 1-42, wherein the secondary signaling domain comprises a CD:3C signaling domain.
44. The method of claim 43, wherein the CD3c signaling domain comprises the arnino acid sequence of SEQ ID NO:5 or SEQ ID NO:27.
45. The rnethod of any one of claims 1-40, wherein the primary signaling domain comprises a CD28 costimulatory signaling domain; a 4-1BB costimulatory signaling domain; a CD27 costimulatory signaling domain; a ICOS costimulatory signaling domain; a LFA-1 costimulatory signaling domain; an 0X40 costimulatory signaling domain; a CD2 costimulatory signaling domain; or an ICAM:- costimulatory signaling domain.
46. The method of claim 45, wherein the CD28 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:118 or 119; the 4-1BB
costimulatory signaling domain comprises the amino acid sequence of SEQ ID
NO:122;
the CD27 costirnulatory signaling domain comprises the amino acid sequence of SEQ
ID NO:169; thc ICOS costimulatory signaling domain comprises thc amino acid sequence of SEQ ID NO:172; the LF A-1 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:171; the 0X40 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:166; the CD2 costirnulatory signaling domain comprises the amino acid sequence of SEQ ID NO:167; or the ICAM-1 costimulatory signaling domain comprises the amino acid sequence of SEQ ID
NO:170.
costimulatory signaling domain comprises the amino acid sequence of SEQ ID
NO:122;
the CD27 costirnulatory signaling domain comprises the amino acid sequence of SEQ
ID NO:169; thc ICOS costimulatory signaling domain comprises thc amino acid sequence of SEQ ID NO:172; the LF A-1 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:171; the 0X40 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:166; the CD2 costirnulatory signaling domain comprises the amino acid sequence of SEQ ID NO:167; or the ICAM-1 costimulatory signaling domain comprises the amino acid sequence of SEQ ID
NO:170.
47. The method of any one of claims 1-42, 45, or 46, wherein the secondary signaling domain comprises a DAPI2 signaling domain.
48. The method of claim 47, wherein the DAP12 signaling domain comprises the amino acid sequence of SEQ ID NO:180.
49. The method of any one of claims 1-40, wherein the primary intracellular signaling domain comprises a Timl signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3( signaling domain. TI.,R2 signaling domain, or a TLR8 signaling domain.
50. The rnethod of claim 49, wherein the Timl signaling domain comprises the amino acid sequence set forth in SEQ ID NO:44, the Tim4 signaling domain coinprises the amino acid sequence set forth in SEQ ID NO:45, 224, or 225, the TRAF2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:48, the TRAF6 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:46, the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID
NO:4 or 26, the DAP12 signaling domain comprises the amino acid sequence set forth in SEQ
ID NO:9, the CD3( signaling domain comprises the amino acid sequence set forth in SEQ11) NO:5, the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:222;or the TLR8 signaling domain comprises the amino acid sequence sct forth in SEQ ID NO:47.
NO:4 or 26, the DAP12 signaling domain comprises the amino acid sequence set forth in SEQ
ID NO:9, the CD3( signaling domain comprises the amino acid sequence set forth in SEQ11) NO:5, the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:222;or the TLR8 signaling domain comprises the amino acid sequence sct forth in SEQ ID NO:47.
51. The method of any one of claims 1-42, 49, or 50, wherein the secondary intracellular signaling domain cornprises a Timl signaling dornain, a Tim4 signaling domain, a TRAF2 signaling dornain, a TRAF6 signaling dom.ain, a CD28 signaling domain, a DAP12 signaling domain, a CD3C signaling domain, TLR2 signaling domain, or a TLR8 signaling domain.
52. The method of claim 51, wherein the Timl signaling domain cornprises the amino acid sequence set forth in SEQ ID NO:44, the Tim4 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:45, 224, or 225, the TRAF2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:48, the TRAF6 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:46, the CD28 signaling doinain comprises the amino acid sequence set forth in SEQ ID
NO:4 or 26, the DAP12 signaling domain comprises the amino acid sequence set forth in SEQ
ID NO:9, the CD3C signaling domain comprises the amino acid sequence set forth in SEQ ID NO:5, the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:222; or the TI-118 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:47.
NO:4 or 26, the DAP12 signaling domain comprises the amino acid sequence set forth in SEQ
ID NO:9, the CD3C signaling domain comprises the amino acid sequence set forth in SEQ ID NO:5, the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:222; or the TI-118 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:47.
53. The method of any one of claims 1-52, wherein (i) the binding domain comprises the Tim4 IgV domain ami the Timl mucin domain, the primary intracellular signaling domain comprises a TLR8 signaling doinain, the secondary intracellular signaling domain comprises a CD3C
signaling domain, and the transmembrane domain comprises a Timl transmembrane domain;
(ii) the binding domain comprises the Tim4 IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprises a CD28 signaling domain, the secondary intracellular signaling domain comprises a DAP12 signaling domain, and the transmembrane domain comprises a Timl transmembrane domain; or (iii) the binding domain comprises the Tim4 IgV domain and the Timl rnucin domain; thc primary intraccllular signaling domain cornpriscs a CD28 signaling domain, the secondary intracellular signaling domain comprises a DAP12 signaling domain, and the transmernbrane domain comprises a CD28 transmernbrane domain.
signaling domain, and the transmembrane domain comprises a Timl transmembrane domain;
(ii) the binding domain comprises the Tim4 IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprises a CD28 signaling domain, the secondary intracellular signaling domain comprises a DAP12 signaling domain, and the transmembrane domain comprises a Timl transmembrane domain; or (iii) the binding domain comprises the Tim4 IgV domain and the Timl rnucin domain; thc primary intraccllular signaling domain cornpriscs a CD28 signaling domain, the secondary intracellular signaling domain comprises a DAP12 signaling domain, and the transmernbrane domain comprises a CD28 transmernbrane domain.
54. The method of claim 53, wherein:
(i) the Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO:34, the Timl Mucin domain comprises the amino acid sequence of SEQ ID NO:39, the TLR8 signaling domain comprises the amino acid sequence of SEQ ID NO:47, the CD31; signalin.g domain comprises the amino acid sequence of SEQ ID NO:5 or SEQ
ID NO:27; and the Timl transmembrane domain comprises the amino acid sequence of SEQ ID NO:8;
(ii) the Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO:34, the Timl Mucin domain comprises the amino acid sequence of SEQ ID NO:39, the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO:4, the DAP12 signaling domain comprises the amino acid sequence of SEQ ID NO:9; and the Timl transmembrane domain comprises the amino acid sequence of SEQ ID NO:8; or (i i) the Tim4 TgV domain comprises the amino acid sequence of SEQ ID
NO:34, the Tim1 Mucin dornain cornprises the arnino acid sequence of SEQ :ID
NO:39, the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO:4, the DAP12 signaling domain comprises the amino acid sequence of SEQ.113 NO:9; and the CD28 transmembrane domain compiises the amino acid sequence of SEQ ID NO:7.
(i) the Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO:34, the Timl Mucin domain comprises the amino acid sequence of SEQ ID NO:39, the TLR8 signaling domain comprises the amino acid sequence of SEQ ID NO:47, the CD31; signalin.g domain comprises the amino acid sequence of SEQ ID NO:5 or SEQ
ID NO:27; and the Timl transmembrane domain comprises the amino acid sequence of SEQ ID NO:8;
(ii) the Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO:34, the Timl Mucin domain comprises the amino acid sequence of SEQ ID NO:39, the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO:4, the DAP12 signaling domain comprises the amino acid sequence of SEQ ID NO:9; and the Timl transmembrane domain comprises the amino acid sequence of SEQ ID NO:8; or (i i) the Tim4 TgV domain comprises the amino acid sequence of SEQ ID
NO:34, the Tim1 Mucin dornain cornprises the arnino acid sequence of SEQ :ID
NO:39, the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO:4, the DAP12 signaling domain comprises the amino acid sequence of SEQ.113 NO:9; and the CD28 transmembrane domain compiises the amino acid sequence of SEQ ID NO:7.
55. 'The method of claim 53 or 54, wherein:
(i) the single chain chimeric protein comprises amino acids 25-628 of SEQ ID
NO:67;
(ii) the single chain chimeric protein comprises amino acids 25-416 of SEQ ID
NO:68; or (iii) the single chain chimeric protein comprises amino acids 25-422 of SEQ ID
NO:69.
(i) the single chain chimeric protein comprises amino acids 25-628 of SEQ ID
NO:67;
(ii) the single chain chimeric protein comprises amino acids 25-416 of SEQ ID
NO:68; or (iii) the single chain chimeric protein comprises amino acids 25-422 of SEQ ID
NO:69.
56. The method of any one of claims 53-55, wherein:
(i) the single chain chimeric protein comprises the amino acid sequence of SEQ
ID NO:67;
(ii) the single chain chimeric protein comprises the amino acid sequence of SEQ
ID NO:68; or (iii) the single chain chimeric protein comprises the amino acid sequence of SEQ ID NO:69.
(i) the single chain chimeric protein comprises the amino acid sequence of SEQ
ID NO:67;
(ii) the single chain chimeric protein comprises the amino acid sequence of SEQ
ID NO:68; or (iii) the single chain chimeric protein comprises the amino acid sequence of SEQ ID NO:69.
57. The method of of any one of claims 1-52, wherein the chimeric receptor comprises:
(i) the binding domain comprising the Tim1 IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a Timl signaling domain, the secondary intracellular signaling domain comprising a CD3i;
signaling domain, and the transmembrane domain comprising a Timl transrnembrane domain;
(ii) the binding domain comprising the Tintl IgV domain and the Timl mucin domain, the prirnary intracellular signaling domain comprising a Tim4 signaling domain, the secondary intracellular signaling domain comprising a CD3C, signaling domain; and the transmembrane domain comprising a Tim1 transmembrane domain;
(iii) the binding domain comprising the Timl. IgV domain and the Tim1 mucin domain; the primary intracellular sign.aling dom.ain comprising a CD28 signaling domain, and the transmembrane domain comprising a CD28 transmembrane domain;
(iv) the binding domain comprising the Timl IgV dornain and the Timl mucin domain; the primary intracellular signaling domain comprising a TRAF6 signaling domain, and the transmembrane domain comprising a Tirni transrnetnbrane domain;
(v) the binding domain comprising the Timl IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a TRAF6 signaling domain, and the transmembrane domain comprising a CD28 transmembrane domain;
(vi) the binding domain comprising the Timi .1gV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a TRAF2 signaling domain, and the transmembrane domain comprising a Timl transmembrane domain;
(vii) the binding domain comprising the Timl IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a TRAF2 signaling domain, and the transmembrane domain comprising a CD28 transmembrane dom.ain;
(viii) the binding domain comprising the Timl IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a TLR8 signaling domain, the secondary intracellular signaling domain comprising a CD3),;
signaling domain, and the transmembrane domain comprising a Timl transrnembrane domain;
(ix) the binding domain comprising the Tim.1 IgV domain and the Tim I mucin domain; the primary intracellular signaling domain comprising a CD28 signaling dornain, the secondary intracellular signaling domain comprising a DAP12 signaling domain, and the transmembrane domain comprising a CD28 transmembrane domain;
or (x) the binding dornain comprising the Timl IgV domain and the Timl inucin domain; the primary intracellular signaling domain comprising a CD28 signaling domain, the secondary intracellular signaling domain comprising a DAP12 signaling domain, and the transmembrane domain comprising a Timl transmembrane domain.
(i) the binding domain comprising the Tim1 IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a Timl signaling domain, the secondary intracellular signaling domain comprising a CD3i;
signaling domain, and the transmembrane domain comprising a Timl transrnembrane domain;
(ii) the binding domain comprising the Tintl IgV domain and the Timl mucin domain, the prirnary intracellular signaling domain comprising a Tim4 signaling domain, the secondary intracellular signaling domain comprising a CD3C, signaling domain; and the transmembrane domain comprising a Tim1 transmembrane domain;
(iii) the binding domain comprising the Timl. IgV domain and the Tim1 mucin domain; the primary intracellular sign.aling dom.ain comprising a CD28 signaling domain, and the transmembrane domain comprising a CD28 transmembrane domain;
(iv) the binding domain comprising the Timl IgV dornain and the Timl mucin domain; the primary intracellular signaling domain comprising a TRAF6 signaling domain, and the transmembrane domain comprising a Tirni transrnetnbrane domain;
(v) the binding domain comprising the Timl IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a TRAF6 signaling domain, and the transmembrane domain comprising a CD28 transmembrane domain;
(vi) the binding domain comprising the Timi .1gV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a TRAF2 signaling domain, and the transmembrane domain comprising a Timl transmembrane domain;
(vii) the binding domain comprising the Timl IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a TRAF2 signaling domain, and the transmembrane domain comprising a CD28 transmembrane dom.ain;
(viii) the binding domain comprising the Timl IgV domain and the Timl mucin domain; the primary intracellular signaling domain comprising a TLR8 signaling domain, the secondary intracellular signaling domain comprising a CD3),;
signaling domain, and the transmembrane domain comprising a Timl transrnembrane domain;
(ix) the binding domain comprising the Tim.1 IgV domain and the Tim I mucin domain; the primary intracellular signaling domain comprising a CD28 signaling dornain, the secondary intracellular signaling domain comprising a DAP12 signaling domain, and the transmembrane domain comprising a CD28 transmembrane domain;
or (x) the binding dornain comprising the Timl IgV domain and the Timl inucin domain; the primary intracellular signaling domain comprising a CD28 signaling domain, the secondary intracellular signaling domain comprising a DAP12 signaling domain, and the transmembrane domain comprising a Timl transmembrane domain.
58. The method of claim 57, wherein the chimeric Tirn4 receptor comprises:
(i) the Tim.1 IgV dom.ain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain comprising the amino acid sequence of SEQ ll) NO:39, the Timl signaling domain comprising the amino acid sequence of SEQ ID
NO:44, the CD3 signaling domain comprising the amino acid sequence of SEQ ID
NO:4, and the Tim" trap membrane domain comprising the amino acid sequence of SEQ ID NO:8;
(ii) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain comprising the amino aci.d sequence of SEQ ID
NO:39, the Tim4 signaling domain comprising the amino acid sequence of SEQ ID
NO:45, 224, or 225, the CDR', signaling domain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27; and the Tim.1 transrnembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(iii) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl inucin dornain comprising the arnino acid sequence of SEQ ID
NO:39, the CD28 signaling dornain comprising the arnino acid sequence of SEQ
ID
NO:4, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:7;
(iv) the Tirni IgV domain comprising the arnino acid sequence of SEQ ID
NO:38, the Timl mucin domain com.prising the arnino acid sequence of SEQ ID
NO:39, the Tim.1 mucin domain com.prising the amino acid sequence of SEQ. ID
NO:39, the TRAF6 signaling domain comprising the amino acid sequence of SEQ ID
NO:46, and the Tirnl transmernbrane dornain comprising the amino acid sequence of SEQ NO:8;
(v) the Tim]. IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain co.m.prising the arnino acid sequence of SEQ ID
NO:39, the TRAF6 signaling domain comprising the amino acid sequence of SEQ ED
NO:46, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:7;
(vi) the Thnl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Tim.1 mucin domain com.prising the arnino acid sequence of SEC) ID
NO:39, the TRAF2 signaling domain comprising the amino acid sequence of SEQ ID
NO:48, and the Timl transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(vii) the Timl IgV domain comprising the amino acid sequence of SEQ ED
NO:38, the Timl mucin domain comprising the amino acid sequence of SEQ ID
NO:39, the TRAF2 signaling domain comprising the amino acid sequence of SEQ ED
NO:48, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:7;
(viii) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Tim.1 mucin domain com.prising the arnino acid sequence of SEQ ID
NO:39, the TLR8 signaling domain comprising the amino acid sequence of SEQ ID
NO:47, the CD3c signaling domain comprising the amino acid sequence of SEQ ID
NO:5 or SEQ ID NO:27, and the Timl transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(ix) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain comprising the amino acid sequence of SEQ ID
NO:39, the CD28 signaling domain comprising the amino acid sequence of SEQ ID
NO:4, the DAP12 signaling domain 9, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:7; or (x) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl rnucin domain comprising the amino acid sequence of SEQ ID
NO:39, the CD28 signaling domain comprising the amino acid sequence of SEQ ID
NO:4, the DAP12 signaling domain comprising the amino acid sequence of SEQ ID
NO:9, and the Timi transtnembrane domain comprising the amino acid sequence of SEQ ID NO:8.
(i) the Tim.1 IgV dom.ain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain comprising the amino acid sequence of SEQ ll) NO:39, the Timl signaling domain comprising the amino acid sequence of SEQ ID
NO:44, the CD3 signaling domain comprising the amino acid sequence of SEQ ID
NO:4, and the Tim" trap membrane domain comprising the amino acid sequence of SEQ ID NO:8;
(ii) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain comprising the amino aci.d sequence of SEQ ID
NO:39, the Tim4 signaling domain comprising the amino acid sequence of SEQ ID
NO:45, 224, or 225, the CDR', signaling domain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27; and the Tim.1 transrnembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(iii) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl inucin dornain comprising the arnino acid sequence of SEQ ID
NO:39, the CD28 signaling dornain comprising the arnino acid sequence of SEQ
ID
NO:4, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:7;
(iv) the Tirni IgV domain comprising the arnino acid sequence of SEQ ID
NO:38, the Timl mucin domain com.prising the arnino acid sequence of SEQ ID
NO:39, the Tim.1 mucin domain com.prising the amino acid sequence of SEQ. ID
NO:39, the TRAF6 signaling domain comprising the amino acid sequence of SEQ ID
NO:46, and the Tirnl transmernbrane dornain comprising the amino acid sequence of SEQ NO:8;
(v) the Tim]. IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain co.m.prising the arnino acid sequence of SEQ ID
NO:39, the TRAF6 signaling domain comprising the amino acid sequence of SEQ ED
NO:46, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:7;
(vi) the Thnl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Tim.1 mucin domain com.prising the arnino acid sequence of SEC) ID
NO:39, the TRAF2 signaling domain comprising the amino acid sequence of SEQ ID
NO:48, and the Timl transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(vii) the Timl IgV domain comprising the amino acid sequence of SEQ ED
NO:38, the Timl mucin domain comprising the amino acid sequence of SEQ ID
NO:39, the TRAF2 signaling domain comprising the amino acid sequence of SEQ ED
NO:48, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:7;
(viii) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Tim.1 mucin domain com.prising the arnino acid sequence of SEQ ID
NO:39, the TLR8 signaling domain comprising the amino acid sequence of SEQ ID
NO:47, the CD3c signaling domain comprising the amino acid sequence of SEQ ID
NO:5 or SEQ ID NO:27, and the Timl transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(ix) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain comprising the amino acid sequence of SEQ ID
NO:39, the CD28 signaling domain comprising the amino acid sequence of SEQ ID
NO:4, the DAP12 signaling domain 9, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:7; or (x) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl rnucin domain comprising the amino acid sequence of SEQ ID
NO:39, the CD28 signaling domain comprising the amino acid sequence of SEQ ID
NO:4, the DAP12 signaling domain comprising the amino acid sequence of SEQ ID
NO:9, and the Timi transtnembrane domain comprising the amino acid sequence of SEQ ID NO:8.
59. The method of any one of claims 1-52, wherein the chimeric Tim receptor comprises:
(i) the single chain chirneric protein comprising amino acids 21-456 of SEQ ID
NO:49;
(ii) the single chain chimeric protein comprising amino acids 21-471 of SEQ ID
NO:50;
(iii) the single chain chimeric protein comprising amino acids 21-363 of SEQ
ID
NO:51;
(iv) the single chain chimeric protein comprising amino acids 21-590 of SEQ ID
NO:52;
(v) the single chain chimeric protein cornprising amino acids 21-596 of SEQ ID
NO:53;
(vi) the single chain chimeric protein comprising amino acids 21-619 of SEQ JD
NO:54;
(vii) the single chain chimeric protein comprising amino acids 21-625 of SEQ
ID NO:55;
(viii) the single chain chimeric protein comprising amino acids 21-621 of SEQ
ID NO:56;
(ix) the single chain chimeric protein comprising arnino acids 21-415 of SEQ
ID
NO:57; or (x) the single chain chimeric protein comprising amino acids 21409 of SEQ ID
NO:58.
(i) the single chain chirneric protein comprising amino acids 21-456 of SEQ ID
NO:49;
(ii) the single chain chimeric protein comprising amino acids 21-471 of SEQ ID
NO:50;
(iii) the single chain chimeric protein comprising amino acids 21-363 of SEQ
ID
NO:51;
(iv) the single chain chimeric protein comprising amino acids 21-590 of SEQ ID
NO:52;
(v) the single chain chimeric protein cornprising amino acids 21-596 of SEQ ID
NO:53;
(vi) the single chain chimeric protein comprising amino acids 21-619 of SEQ JD
NO:54;
(vii) the single chain chimeric protein comprising amino acids 21-625 of SEQ
ID NO:55;
(viii) the single chain chimeric protein comprising amino acids 21-621 of SEQ
ID NO:56;
(ix) the single chain chimeric protein comprising arnino acids 21-415 of SEQ
ID
NO:57; or (x) the single chain chimeric protein comprising amino acids 21409 of SEQ ID
NO:58.
60. The method of claim 59, wherein the chimeric Tim receptor comprises:
(i) the single chain chimeric protein comprising the amino acid sequence of SEQ
ID NO:49;
(ii) the single chain chimeric protein cornprising the arnino acid sequence of SEQ ID NO:50;
(iii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:51;
(iv) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:52;
(v) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:53;
(vi) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:54;
(vii) the single chain chimeric protein comprising the arnino acid sequence of SEQ 1D NO:55;
(viii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:56;
(ix) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:57; or (x) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:58.
(i) the single chain chimeric protein comprising the amino acid sequence of SEQ
ID NO:49;
(ii) the single chain chimeric protein cornprising the arnino acid sequence of SEQ ID NO:50;
(iii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:51;
(iv) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:52;
(v) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:53;
(vi) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:54;
(vii) the single chain chimeric protein comprising the arnino acid sequence of SEQ 1D NO:55;
(viii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:56;
(ix) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:57; or (x) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:58.
61. The method of any one of claims 1-52, wherein the chimeric Tim receptor comprises:
(i) the binding domain comprising the Timl IgV dom.ain and the Timl m.ucin domain; the primary intracellular signaling domain comprising a Tim 1 signaling domain, the secondary intracellular signaling domain comprising a CD3C
signaling domain, and the transmembrane domain cornprising a Timl transmembrane domain;
(ii) the binding domain comprising the Timl IgV domain a.nd the Timl mucin domain, the primary intracellular signaling domain com.prising a Tim4 signaling domain, the secondary intracellular signaling domain comprising a CD3C
signaling domain; and the transrnembrane domain comprising a Timi transmembrane domain;
(iii) the binding domain comprising the Tim4 IgV domain and the Tim4 mucin dornain; the primary intracellular signaling domain comprising a Tim4 signaling domain, the secondary intracellular signaling domain comprising a CD3C
signaling domain; and the transmembrane domain comprising a Tim4 transmembrane domain;
or (iv) the binding domain comprising the Tim4 IgV domain and the Tim4 mucin domain; the primary intracellular signaling domain comprising a Timl signaling domain, the secondary intracellular signaling domain comprising a CD3C
signaling domain; and the transm.embrane domain comprising a Tirn4 transmembrane domain.
(i) the binding domain comprising the Timl IgV dom.ain and the Timl m.ucin domain; the primary intracellular signaling domain comprising a Tim 1 signaling domain, the secondary intracellular signaling domain comprising a CD3C
signaling domain, and the transmembrane domain cornprising a Timl transmembrane domain;
(ii) the binding domain comprising the Timl IgV domain a.nd the Timl mucin domain, the primary intracellular signaling domain com.prising a Tim4 signaling domain, the secondary intracellular signaling domain comprising a CD3C
signaling domain; and the transrnembrane domain comprising a Timi transmembrane domain;
(iii) the binding domain comprising the Tim4 IgV domain and the Tim4 mucin dornain; the primary intracellular signaling domain comprising a Tim4 signaling domain, the secondary intracellular signaling domain comprising a CD3C
signaling domain; and the transmembrane domain comprising a Tim4 transmembrane domain;
or (iv) the binding domain comprising the Tim4 IgV domain and the Tim4 mucin domain; the primary intracellular signaling domain comprising a Timl signaling domain, the secondary intracellular signaling domain comprising a CD3C
signaling domain; and the transm.embrane domain comprising a Tirn4 transmembrane domain.
62. The method of claim 61, wherein the chimeric Tim receptor comprises:
(i) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain com.prising the arnino acid sequence of SEQ
NO:39, the Timl signaling domain comprising the amino acid sequence of SEQ ID
NO:44, the CD3C signaling domain comprising the amino acid sequence of SEQ ID
NO:5 or SEQ ID NO:27, and the Timl transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(ii) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Tim.1 mucin domain comprising the amino acid sequence of SEQ ID
NO:39, thc Tim4 signaling domain comprising the amino acid sequence of SEQ ID
NO:45, 224, or 225, the CD3C signaling domain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27; and the Timl transmembrane domain comprising the arnino acid sequence of SEQ ID NO:8;
(iii) the Tim4 IgV domain comprising the amino acid sequence of SEQ ID
NO:34, the Tim4 mucin domain comprising the amino acid sequence of SEQ ID
NO:35, the Tim4 signaling domain comprising the amino acid sequence of SEQ ID
NO:45, 224, or 225, the CD3t; signaling domain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27; and the Tim4 transrnembrane domain comprising the amino acid sequence of SEQ. ID NO:5; or (iv) the Titn4 IgV domain comprising the amino acid sequence of SEQ ID
NO:34, the Tim4 mucin domain cornprising the amino acid sequence of SEQ ID
NO:35, the Timi signaling domain comprising the amino acid sequence of SEQ ID
NO:44, the CD3c signaling domain comprising the amino acid sequence of SEQ ID
NO:5 or SEQ ID NO:27; and the Tim4 transrnembrane dornain comprising the amino acid sequence of SEQ ID NO:6.
(i) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Timl mucin domain com.prising the arnino acid sequence of SEQ
NO:39, the Timl signaling domain comprising the amino acid sequence of SEQ ID
NO:44, the CD3C signaling domain comprising the amino acid sequence of SEQ ID
NO:5 or SEQ ID NO:27, and the Timl transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(ii) the Timl IgV domain comprising the amino acid sequence of SEQ ID
NO:38, the Tim.1 mucin domain comprising the amino acid sequence of SEQ ID
NO:39, thc Tim4 signaling domain comprising the amino acid sequence of SEQ ID
NO:45, 224, or 225, the CD3C signaling domain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27; and the Timl transmembrane domain comprising the arnino acid sequence of SEQ ID NO:8;
(iii) the Tim4 IgV domain comprising the amino acid sequence of SEQ ID
NO:34, the Tim4 mucin domain comprising the amino acid sequence of SEQ ID
NO:35, the Tim4 signaling domain comprising the amino acid sequence of SEQ ID
NO:45, 224, or 225, the CD3t; signaling domain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27; and the Tim4 transrnembrane domain comprising the amino acid sequence of SEQ. ID NO:5; or (iv) the Titn4 IgV domain comprising the amino acid sequence of SEQ ID
NO:34, the Tim4 mucin domain cornprising the amino acid sequence of SEQ ID
NO:35, the Timi signaling domain comprising the amino acid sequence of SEQ ID
NO:44, the CD3c signaling domain comprising the amino acid sequence of SEQ ID
NO:5 or SEQ ID NO:27; and the Tim4 transrnembrane dornain comprising the amino acid sequence of SEQ ID NO:6.
63. The method of claim 61 or 62, wherein the chimeric Tim receptor comprises:
(i) the single chain chirneric protein cornprising amino acids 21-456 of SEQ
ID
NO:49;
(ii) the single chain chimeric protein comprising amino acids 21-471 of SEQ ID
NO:50;
(iii) the single chain chimeric protein comprising amino acids 25-490 of SEQ
ID
NO:59; or (iv) the single chain chimeric protein comprising amino acids 25-495 of SEQ
1E) NO:60.
(i) the single chain chirneric protein cornprising amino acids 21-456 of SEQ
ID
NO:49;
(ii) the single chain chimeric protein comprising amino acids 21-471 of SEQ ID
NO:50;
(iii) the single chain chimeric protein comprising amino acids 25-490 of SEQ
ID
NO:59; or (iv) the single chain chimeric protein comprising amino acids 25-495 of SEQ
1E) NO:60.
64. The method of any one of claims 61-63, wherein the chimeric Tim receptor comprises:
(i) the single chain chimeric protein comprising the amino acid sequence of SEQ
ID NO:49;
(ii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:50;
(iii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:59; or (iv) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:60.
(i) the single chain chimeric protein comprising the amino acid sequence of SEQ
ID NO:49;
(ii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:50;
(iii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:59; or (iv) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:60.
65. The method of any one of claims 1-52, wherein the chimeric Tim receptor comprises:
(i) the single chain chimeric protein comprising the amino acid sequence of SEQ
ID NO:195 or amino acids 25-473 of SEQ JD NO:195;
(ii) the single chain chimeric protein cornprising the amino acid sequence of SEQ ID NO:196 or amino acids 25-446 of SEQ ID NO:196;
(iii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:197 or amino acids 25-434 of SEQ1D NO:197; or (iv) the single chain chimeric protein comprising the amino acid sequence of SEQ 11) NO:198 or amino acids 25-428 of SEQ ID NO:198.
(i) the single chain chimeric protein comprising the amino acid sequence of SEQ
ID NO:195 or amino acids 25-473 of SEQ JD NO:195;
(ii) the single chain chimeric protein cornprising the amino acid sequence of SEQ ID NO:196 or amino acids 25-446 of SEQ ID NO:196;
(iii) the single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:197 or amino acids 25-434 of SEQ1D NO:197; or (iv) the single chain chimeric protein comprising the amino acid sequence of SEQ 11) NO:198 or amino acids 25-428 of SEQ ID NO:198.
66. The method of any one of claims 1-52, wherin the chimeric Tim receptor comprises:
(a) an extracellular domain coinprising a binding domain comprising: (i) a Tim4 IgV dornain and a Tim4 mucin domain;
(b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain selected from a CD28 signaling domain, a CD3 signaling domain, and a 4-1BB signaling domain, and a secondary intracellular signaling domain selected from a TLR2 signaling domain or TLR8 signaling domain; and (c) a transmembrane domain positioned between and connecting the extracellular domain and the intracellular si gnaling domain.
(a) an extracellular domain coinprising a binding domain comprising: (i) a Tim4 IgV dornain and a Tim4 mucin domain;
(b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain selected from a CD28 signaling domain, a CD3 signaling domain, and a 4-1BB signaling domain, and a secondary intracellular signaling domain selected from a TLR2 signaling domain or TLR8 signaling domain; and (c) a transmembrane domain positioned between and connecting the extracellular domain and the intracellular si gnaling domain.
67. The method of claim 66, wherein the Tim4 IgNi domain comprises the amino acid sequence set forth in SEQ ID NO:34, the Tirn4 mucin domain comprises the arnino acid sequence set forth in SEQ ID NO:35, or both.
68. The method of claim 67, wherein the binding domain comprises the amino acid sequence of SEQ JD NO:2 or 42.
69. The method of any one of claims 66-68, wherein the chimeric Tim receptor further comprises an extracellular spacer domain.
70. The method of claim 69, wherein the extracellular spacer domain comprises an 1gG4 hinge region or a CD28 hinge region.
71. The method of claim 70, wherein the IgG4 hinge region comprises the amino acid sequence set forth in SEQ ID NO:3, or the CD28 hinge region comprises the amino acid sequence set forth in SEQ ID NO:32.
72. The method of any one of claims 66-71, wherein the transmembrane domain comprises a Timl transmembrane domain, Tim4 transmembrane domain, or CD28 transmembrane domain.
73. The method of claim 72, wherein the Tim l transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:8, the Tim4 transmernbrane domain comprises the amino acid sequence set forth in SEQ ID NO:6 or 23, or the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:7.
74. The method of any one of claims 66-73, wherein the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:26, the CD.31; signaling domain comprises thc amino acid sequence set forth in SEQ ID
NO:5 or SEQ JD NO:27, or the 4-1BB signaling domain comprises the amino acid sequence set forth in SEQ ID NO:100.
NO:5 or SEQ JD NO:27, or the 4-1BB signaling domain comprises the amino acid sequence set forth in SEQ ID NO:100.
75. The method of any one of claims 66-74, wherein the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:222, or the TLR8 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:47.
76. The method of any one of claims 1-52, wherin the chimeric Tim receptor comprises:
(a) an extracellular domain comprising a binding domain comprising: (i) a Tim4 IgV domain and a Tirn4 mucin dornain;
(b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a prirnary intracellular signaling domain comprising an immunoreceptor tyrosine-based activation rnotif (ITAM) containing signaling domain; a secondary intracellular signaling domain comprising a costimulatory signaling domain, Timl signaling domain, or Tim4 signaling domain; and the tertiary intracellular signaling domain comprising a TI,R signaling domain; and (c) a transrnernbrane domain positioned between and connecting the extracellular domain and the intracellular si gnaling domain.
(a) an extracellular domain comprising a binding domain comprising: (i) a Tim4 IgV domain and a Tirn4 mucin dornain;
(b) an intracellular signaling domain, wherein the intracellular signaling domain comprises a prirnary intracellular signaling domain comprising an immunoreceptor tyrosine-based activation rnotif (ITAM) containing signaling domain; a secondary intracellular signaling domain comprising a costimulatory signaling domain, Timl signaling domain, or Tim4 signaling domain; and the tertiary intracellular signaling domain comprising a TI,R signaling domain; and (c) a transrnernbrane domain positioned between and connecting the extracellular domain and the intracellular si gnaling domain.
77. The chimeric Tim receptor of claim 76, wherein the ITAM containing signaling domain is a CD3C signaling domain or DAP12 signaling domain.
78. The chimeric Tim receptor or claim 76 or 77, wherein the costimulatory signaling domain is 4-1BB signaling domain or CD28 signaling domain.
79. The chimeric Tim receptor of any one of claims 76-78, wherein the TLR
signaling domain is a TLR2 signaling domain or TLR8 signaling domain.
signaling domain is a TLR2 signaling domain or TLR8 signaling domain.
80. The method of any one of claims 1-79, wherein the single chain protein comprises the amino acid sequence of set forth in any of Tables 1, 2, or 4-10.
81. The method of any one of claims 1-80, wherein the chimeric Tim receptor is administered as a polynucleotide encoding the single chain protein.
82. The method of claim 81, wherein the chimeric Tim receptor is administered as a vector comprising the polynucleotide.
83. The method of claim 82, wherein the chimeric Tim receptor is administered as an engineered cell comprising the chimeric Tim receptor, the polynucleotide, or the vector.
84. The method of claim 83, wherein the cell is an immune cell.
85. The method of claim 83 or 84, wherein the cell is a T cell.
86. The method of any one of claims 83-85, wherein the cell is a CD4+ T cell, a CD8+
T cell, or a CD4+/CD8 T cell.
T cell, or a CD4+/CD8 T cell.
87. 'The method of any one of claims 83-85, wherein the cell is a human cell.
88. The method of any one of claims 1-87, wherein the chimeric Tim receptor is adrninistered as a composition comprising the chimeric Tim receptor, the polynucleotide, the vector, or the engineered cell, and a pharmaceutically acceptable excipient.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202063066150P | 2020-08-14 | 2020-08-14 | |
US63/066,150 | 2020-08-14 | ||
US202063087049P | 2020-10-02 | 2020-10-02 | |
US63/087,049 | 2020-10-02 | ||
US202163226712P | 2021-07-28 | 2021-07-28 | |
US63/226,712 | 2021-07-28 | ||
PCT/US2021/046041 WO2022036285A1 (en) | 2020-08-14 | 2021-08-13 | Compositions and methods for treating cancer with chimeric tim receptors in combination with inhibitors of poly (adp-ribose) polymerase |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3189480A1 true CA3189480A1 (en) | 2022-02-17 |
Family
ID=85727270
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3189480A Pending CA3189480A1 (en) | 2020-08-14 | 2021-08-13 | Compositions and methods for treating cancer with chimeric tim receptors in combination with inhibitors of poly (adp-ribose) polymerase |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP4196151A1 (en) |
JP (1) | JP2023537762A (en) |
CA (1) | CA3189480A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024124080A1 (en) * | 2022-12-08 | 2024-06-13 | Recursion Pharmaceuticals, Inc. | Rna-binding motif protein 39 (rbm39) degraders for treatment of cancers |
-
2021
- 2021-08-13 EP EP21778257.2A patent/EP4196151A1/en active Pending
- 2021-08-13 CA CA3189480A patent/CA3189480A1/en active Pending
- 2021-08-13 JP JP2023510448A patent/JP2023537762A/en active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024124080A1 (en) * | 2022-12-08 | 2024-06-13 | Recursion Pharmaceuticals, Inc. | Rna-binding motif protein 39 (rbm39) degraders for treatment of cancers |
Also Published As
Publication number | Publication date |
---|---|
EP4196151A1 (en) | 2023-06-21 |
JP2023537762A (en) | 2023-09-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7549533B2 (en) | Expression vectors for chimeric phagocytic receptors, genetically modified host cells and uses thereof - Patents.com | |
JP7444781B2 (en) | Cellular immunotherapy compositions and uses thereof | |
US20240150788A1 (en) | Lymphocyte targeted lentiviral vectors | |
WO2019191334A1 (en) | Chimeric tim4 receptors and uses thereof | |
WO2022036265A1 (en) | Chimeric tim receptors and uses thereof | |
US20240058446A1 (en) | Chimeric tim4 receptors and uses thereof | |
WO2022036285A1 (en) | Compositions and methods for treating cancer with chimeric tim receptors in combination with inhibitors of poly (adp-ribose) polymerase | |
WO2022036287A1 (en) | Anti-cd72 chimeric receptors and uses thereof | |
US20210253696A1 (en) | Bivalent chimeric engulfment receptors and uses thereof | |
CA3189480A1 (en) | Compositions and methods for treating cancer with chimeric tim receptors in combination with inhibitors of poly (adp-ribose) polymerase | |
WO2023010097A1 (en) | Chimeric tim4 receptors and uses thereof | |
US20240285682A1 (en) | Chimeric tim receptors and uses thereof | |
US20240285684A1 (en) | Anti-cd72 chimeric receptors and uses thereof | |
CN118043067A (en) | Chimeric TIM4 receptors and uses thereof | |
CN117015396A (en) | Compositions and methods for treating cancer with chimeric TIM receptors in combination with poly (ADP-ribose) polymerase inhibitors |