CN118922209A - Cross-linked antibodies - Google Patents

Cross-linked antibodies Download PDF

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Publication number
CN118922209A
CN118922209A CN202280092390.2A CN202280092390A CN118922209A CN 118922209 A CN118922209 A CN 118922209A CN 202280092390 A CN202280092390 A CN 202280092390A CN 118922209 A CN118922209 A CN 118922209A
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conjugate
uaa
substituted
targeting domain
unsubstituted
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Inventor
赛尔吉奥·杜龙
詹森·罗兰
杰罗德·普塔钦
阿纳利亚·海特
刘君
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Enzraza Treatment Co
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Enzraza Treatment Co
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Priority claimed from PCT/US2022/082277 external-priority patent/WO2023122753A1/en
Publication of CN118922209A publication Critical patent/CN118922209A/en
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Abstract

Provided herein are compositions and methods for cross-linking single domain antibodies to targets with conjugates. Further provided herein are conjugates comprising a targeting domain, wherein the targeting domain comprises an unnatural amino acid. Further provided herein are methods of treating diseases with cross-linked single domain antibodies and multispecific antibodies.

Description

Cross-linked antibodies
Cross reference
The present application claims U.S. provisional patent application No. 63/293,025 filed on day 22 of 12 in 2021; U.S. provisional patent application Ser. No. 63/346,799, filed 5/27 at 2022; and U.S. provisional patent application No. 63/388,072, filed on 7.11 at 2022, which is incorporated herein by reference in its entirety.
Background
Proteins use primarily non-covalent interactions within or between proteins because amino acid side chains of proteins generally do not form covalent bonds with each other, except for cysteines, which produce relatively weak reversible disulfide bonds. Thus, a potentially bioactive Unnatural Amino Acid (UAA) that is capable of specifically reacting with multiple natural amino acid residues on a target protein would expand the diversity of proteins that can be covalently bonded in vivo, which can enhance existing protein properties or evolve new functions by utilizing new covalent bonds. Furthermore, covalent bonds between proteins will allow irreversible capture of protein-protein interactions in vivo, which can be used for protein identification, drug discovery, irreversible antagonists and payload delivery.
Disclosure of Invention
Provided herein are conjugates comprising a targeting domain and a payload, wherein the targeting domain comprises at least one Unnatural Amino Acid (UAA) residue, wherein the targeting domain is configured to bind to a target, and wherein the UAA residues are sufficiently close to form a covalent bond with the target when the targeting domain binds. Further provided herein are conjugates in which the payload is attached to an amino acid at position n+x relative to the UAA residue, where n is the position of the amino acid and x is at least 1. Further provided herein are conjugates in which the payload is attached to an amino acid at position n-x relative to the UAA residue, where n is the position of the amino acid and x is at least 1. Further provided herein are conjugates in which the UAA residues are within 5-20 angstroms of the target when the targeting domain binds to the target. Further provided herein are conjugates in which the targeting domain binds to a cell surface molecule. Further provided herein are conjugates in which the UAA residue is configured to form a covalent bond with a histidine, lysine, or tyrosine residue of the target. Further provided herein are conjugates in which the UAA residue comprises a fluorosulfate moiety. Further provided herein are conjugates in which the UAA residue comprises an aryl fluorosulfate moiety. Further provided herein are those wherein the UAA residue comprises formula I: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b). Further provided herein are those wherein the UAA residue comprises formula II: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b). Further provided herein are those wherein the UAA residue comprises formula III: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b).
Further provided herein are conjugates in which the UAA of the UAA residue has the structure of formula (IA):
wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is-O-or-NR-, m is 1; when Y is-n=m is 2.
Further provided herein are wherein the UAA of formula (IA) has formula (IA-a): is a conjugate of the structure of (a). Further provided herein are wherein the UAA of formula (IA) has formula (IA-b): is a conjugate of the structure of (a). Further provided herein are wherein UAA of formula (IA) has formula (IB): Is a conjugate of the structure of (a). Further provided herein are wherein UAA of formula (IA) has formula (IC): is a conjugate of the structure of (a).
Further provided herein are wherein UAA of formula (IA) has formula (ID): is a conjugate of the structure of (a),
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
Further provided herein are conjugates wherein the UAA of formula (IA) has the structure of formula (IE):
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
Further provided herein are conjugates wherein UAA of formula (IA) has the structure of formula (IIA):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
Further provided herein are conjugates wherein UAA of formula (IA) has the structure of formula (IIB):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
Further provided herein are UAA of formula (IV) wherein the UAA residue isIs a conjugate of the structure of (a),
Wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
Ring a is a 5 to 6 membered aryl or heteroaryl group;
Each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; each R X is optionally substituted alkyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
Further provided herein are conjugates in which the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent. Further provided herein are conjugates in which the cytotoxic moiety comprises a small molecule drug or a chemotherapeutic drug. Further provided herein are conjugates wherein the radioligand reagent is selected from 153Sm、177Lu、90Y、131I、149Tb、211At、212Pb/212Bi、213Bi、223Ra、225Ac and 227 Th. Further provided herein are conjugates in which the radioligand reagent further comprises a chelator. Further provided herein are conjugates wherein the radioligand reagent is selected from 99mTc、131I、201Tl、111 In and 67 Ga. Further provided herein are conjugates, wherein the payload is attached to the conjugate with a linker. Further provided herein are conjugates in which the linker comprises a polymer. Further provided herein are conjugates in which the linker is a cleavable or non-cleavable linker. Further provided herein are conjugates wherein the linker is from 0.01kDa to 2.5 kDa. Further provided herein are conjugates wherein the linker is from 0.01kDa to 2.5 kDa. Further provided herein are conjugates in which the linker is a linear, branched, multimeric, or dendrimer. Further provided herein are conjugates in which the linker is a bifunctional or polyfunctional linker or a bifunctional or polyfunctional polymer. Further provided herein are conjugates in which the linker comprises a water-soluble polymer. Further provided herein are conjugates in which the water-soluble polymer is polyethylene glycol (PEG). Further provided herein are conjugates wherein the molecular weight of PEG is between 0.1kDa and 2.5 kDa. Further provided herein are conjugates wherein PEG comprises 1-8 monomers. Further provided herein are conjugates wherein the targeting domain comprises an antibody, antibody fragment, or antigen binding domain. Further provided herein are conjugates wherein the targeting domain comprises an antigen binding domain, the conjugate comprises a CDR region, and at least one UAA residue is within or near the CDR region. Further provided herein are conjugates in which UAA residues are contained within the CDR regions. Further provided herein are conjugates in which the targeting domain comprises a single domain antibody (sdAb). Further provided herein are conjugates wherein the cell surface molecule is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.
Also provided herein are conjugates comprising (i) a payload and (ii) an engineered single domain antibody (sdAb) comprising an sdAb having a CDR region and at least one Unnatural Amino Acid (UAA) residue within or near the CDR region, where the sdAb comprises any one of SEQ ID NOs 1-4 or 16-64. Further provided herein are conjugates in which the UAA residue comprises a fluorosulfate moiety. Further provided herein are conjugates in which the UAA residue comprises an aryl fluorosulfate moiety. Further provided herein are those wherein the UAA residue comprises formula I: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b). Further provided herein are those wherein the UAA residue comprises formula II: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: Is a conjugate of (a) and (b). Further provided herein are wherein the UAA has the structure: is a conjugate of (a) and (b). Further provided herein are those wherein the UAA residue comprises formula III: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b). Further provided herein are UAA wherein the UAA residue has the structure: is a conjugate of (a) and (b).
Further provided herein are conjugates in which the UAA of the UAA residue has the structure of formula (IA):
wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is-O-or-NR-, m is 1; when Y is-n=m is 2.
Further provided herein are wherein the UAA of formula (IA) has formula (IA-a): is a conjugate of the structure of (a). Further provided herein are wherein the UAA of formula (IA) has formula (IA-b): is a conjugate of the structure of (a). Further provided herein are wherein UAA of formula (IA) has formula (IB): Is a conjugate of the structure of (a). Further provided herein are wherein UAA of formula (IA) has formula (IC): is a conjugate of the structure of (a).
Further provided herein are wherein UAA of formula (IA) has formula (ID): is a conjugate of the structure of (a),
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
Further provided herein are wherein UAA of formula (IA) has formula (IE): Is a conjugate of the structure of (a):
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
Further provided herein are engineered sdabs, wherein UAA of formula (IA) has formula (IIA): Is characterized in that the structure of the (c) is that,
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
Further provided herein are engineered sdabs, wherein UAA of formula (IA) has formula (IIB): Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
Further provided herein are UAA of formula (IV) wherein the UAA residue isIs a conjugate of the structure of (a),
Wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
Ring a is a 5 to 6 membered aryl or heteroaryl group;
Each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; each R X is optionally substituted alkyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
Further provided herein are conjugates, wherein the engineered sdAb comprises SEQ ID No. 1, and wherein the UAA residue is comprised in any of SEQ ID NOs 5,6, and 7. Further provided herein are conjugates, wherein the UAA residue is present at an amino acid position selected from 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115 relative to SEQ ID No. 1. Further provided herein are conjugates, wherein the engineered sdAb comprises SEQ ID No. 2, and wherein the UAA residue is comprised in any of SEQ ID nos. 8, 9, and 10. Further provided herein are conjugates wherein the UAA residue is present at an amino acid position selected from 50, 52, 53, 54, 56, 58, and 100 relative to SEQ ID No. 2. Further provided herein are conjugates, wherein the engineered sdAb comprises SEQ ID NO:3, and wherein the UAA residue is comprised in any of SEQ ID NOs: 11, 12, and 13. Further provided herein are conjugates wherein the UAA residue is present at an amino acid position selected from 58, 62, 101, 103, and 107 relative to SEQ ID No. 3. Further provided herein are conjugates, wherein the engineered sdAb comprises SEQ ID No. 16, and wherein UAA is present at amino acid position 109 relative to SEQ ID No. 16. Further provided herein are conjugates, wherein the engineered sdAb comprises SEQ ID No. 23, and wherein UAA is present at an amino acid position selected from 52, 53, 54, 55, 56, 58, 60, 62, and 64 relative to SEQ ID No. 23. Further provided herein are conjugates, wherein the engineered sdAb comprises SEQ ID No. 24, and wherein the UAA is present at an amino acid position selected from 53, 55, 56, 57, 58, 60, 64, and 67 relative to SEQ ID No. 24.
Further provided herein are conjugates in which the payloads are not linked via a UAA residue side chain. Further provided herein are conjugates in which the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent. Further provided herein are conjugates in which the cytotoxic moiety comprises a small molecule drug or a chemotherapeutic drug. Further provided herein are conjugates wherein the radioligand reagent is selected from 153Sm、177Lu、90Y、131I、149Tb、211At、212Pb/212Bi、213Bi、223Ra、225Ac and 227 Th. Further provided herein are conjugates in which the radioligand reagent further comprises a chelator. Further provided herein are conjugates wherein the imaging agent comprises a fluorophore or a radioligand agent selected from 99mTc、131I、201Tl、111 In and 67 Ga.
Provided herein are methods comprising administering a conjugate described herein, wherein the conjugate covalently binds to a target on the surface of a cell. Further provided herein are methods wherein the cells comprise tumor cells. Further provided herein are methods wherein the conjugate kills or inhibits the growth of tumor cells. Further provided herein are methods wherein the target is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. Provided herein are methods of treating a disease or condition comprising administering a conjugate described herein. Further provided herein are methods wherein the disease comprises one or more of PCa (prostate cancer), CRPCa (castration-resistant prostate cancer), solid tumor (neovasculature), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal cancer), GC (gastric cancer), CRC (colorectal cancer), SCLC (small cell lung cancer), MPM (mesothelioma), PDAC (ductal pancreatic adenocarcinoma), ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), MSI-high tumor, melanoma, DLBCL (diffuse large B-cell lymphoma), endometrial cancer, cervical cancer, bladder cancer, brCa (breast cancer), TNBC (triple negative breast cancer), NE-PCa (neuroendocrine prostate cancer), GBM (glioblastoma), and RCC (renal cell carcinoma).
Provided herein are methods of making conjugates described herein, comprising: generating a targeting domain comprising at least one unnatural amino acid; and optionally conjugating the targeting domain to the payload via a linker. Further provided herein are methods wherein a targeting domain comprising at least one unnatural amino acid is synthesized in vivo. Further provided herein are methods wherein generating comprises using an orthogonal tRNA synthetase/suppressor tRNA pair. Further provided herein are methods wherein a pair comprising an orthogonal tRNA synthetase/suppressor tRNA (derived from pyrrolysine tRNA synthetase/tRNA Pyl) is produced. Further provided herein are methods wherein the orthogonal tRNA synthetases comprise SEQ ID NOS 84, 87, 92 or variants thereof.
Provided herein are methods of delivering a cytotoxic payload to a cell comprising administering a conjugate described herein, wherein the conjugate covalently binds to a target on the surface of the cell, thereby delivering the cytotoxic payload. Further provided herein are methods wherein the cells are tumor cells. Further provided herein are methods wherein the cells are contained in a tumor microenvironment. Further provided herein are methods of comprising cells within a mammalian subject. Further provided herein are methods of comprising cells within a human subject. Further provided herein are methods wherein the conjugate kills or inhibits the growth of tumor cells. Further provided herein are methods wherein the target is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. Further provided herein are methods wherein the human subject has or is diagnosed with a disease or condition selected from PCa (prostate cancer), CRPCa (castration-resistant prostate cancer), solid tumor (neovasculature), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal cancer), GC (gastric cancer), CRC (colorectal cancer), SCLC (small cell lung cancer), MPM (mesothelioma), PDAC (ductal pancreatic adenocarcinoma), ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), MSI-high tumors, melanoma, DLBCL (diffuse large B-cell lymphoma), endometrial cancer, cervical cancer, bladder cancer, brCa (breast cancer), TNBC (triple negative breast cancer), NE-PCa (neuroendocrine prostate cancer), GBM (glioblastoma), and RCC (renal cell carcinoma).
Provided herein are conjugates comprising (i) a first targeting domain, (ii) a second targeting domain, and (iii) a payload, wherein the first targeting domain comprises at least one first Unnatural Amino Acid (UAA), whereby the first targeting domain is capable of covalently binding to a first target at the site of the UAA, and the second targeting domain is configured to bind to a second target. In some embodiments, the first target and the second target are located on the same cell. In some embodiments, the first targeting domain comprises an antibody, antibody fragment, or antigen binding domain. In some embodiments, the first targeting domain comprises a single domain antibody (sdAb). In some embodiments, the first UAA is contained within or near a region of the first targeting domain that interfaces with the first target. In some embodiments, the second targeting domain comprises an antibody, antibody fragment, or antigen binding domain. In some embodiments, the second targeting domain comprises a single domain antibody (sdAb). In some embodiments, the first targeting domain and the second targeting domain are linked to form a fusion protein. In some embodiments, the first targeting domain and the second targeting domain are linked by chemical conjugation. In some embodiments, the first targeting domain and the second domain are linked by a linker. In some embodiments, the first targeting domain and the second targeting domain bind to the same target. In some embodiments, the first targeting domain and the second targeting domain bind to different epitopes of the same target. In some embodiments, the first targeting domain and the second targeting domain bind to the same epitope of the same target. In some embodiments, the same target is a monomer. In some embodiments, the same target is a multimeric molecule. In some embodiments, the first targeting domain and the second targeting domain bind different targets. In some embodiments, the first target is a first cell surface molecule. In some embodiments, the second target is a second cell surface molecule. In some embodiments, at least one first UAA comprises a fluorosulfate moiety. In some embodiments, at least one UAA comprises an aryl fluorosulfate moiety. In some embodiments, at least one first UAA comprises formula I: In some embodiments, at least one first UAA has the structure: In some embodiments, at least one first UAA has the structure: (FSY). In some embodiments, at least one first UAA comprises formula II: In some embodiments, at least one first UAA has the structure: In some embodiments, at least one first UAA has the structure: In some embodiments, at least one first UAA comprises formula III: In some embodiments, at least one first UAA has the structure: In some embodiments, at least one first UAA has the structure: In some embodiments, at least one first UAA has formula (IA): Wherein each X is independently O or NR'; y is a bond, -O-, -NR-, or-N=; a is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4, each R and R', when present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl, R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6, and wherein m is 1 when Y is-O-or-NR-, and m is 2 when Y is-n=in some embodiments, at least one first UAA has the formula (IA-a): is a structure of (a). In some embodiments, at least one first UAA has the formula (IA-b): is a structure of (a). In some embodiments, at least one first UAA has formula (IB): Is a structure of (a). In some embodiments, at least one first UAA has the formula (IC): is a structure of (a). In some embodiments, at least one first UAA has the formula (ID): Wherein: each X is independently O or NR'; y is a bond, -O-, -NR-, or-N=; a is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4, each R and R', when present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl, R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein when Y is a bond, -O-or-NR-, m is 1, when Y is-n=m is 2, in some embodiments at least one first UAA has the formula (IE): Wherein: each X is independently O or NR'; y is a bond, -O-, -NR-, or-N=; a is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4, each R and R', when present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl, R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein when Y is a bond, -O-or-NR-, m is 1, and when Y is-n=m is 2, in some embodiments at least one first UAA has formula (IIA): Wherein: x is independently O or NR'; and when present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, at least one first UAA has formula (IIB): Wherein: x is independently O or NR'; and when present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, at least one first UAA has formula (IV) Wherein each X is independently O or NR'; y is a bond, -O-, -NR-, or-N=; a is a bond OR- (CH 2)n -; m is 1 OR 2;n is an integer from 1 to 4; each R and R' is independently, when present, hydrogen, substituted OR unsubstituted alkyl, substituted OR unsubstituted heteroalkyl, substituted OR unsubstituted aryl, substituted OR unsubstituted heteroaryl, OR substituted OR unsubstituted heterocycloalkyl; ring A is a 5 to 6 membered aryl OR heteroaryl; each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; p is 0,1, 2, 3 or 4; each R X is optionally substituted alkyl; l is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6, and wherein when Y is a bond, -O-, or-NR-, m is 1; When Y is-n=m is 2. In some embodiments, the first cell surface molecule is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some embodiments, the second cell surface molecule is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some embodiments, the second domain comprises a second UAA, whereby the second domain is capable of covalently binding to the second target at the site of the second UAA. In some embodiments, the second UAA is different from at least one UAA contained in the first targeting domain. In some embodiments, the second UAA is the same as at least one UAA contained in the first targeting domain. In some embodiments, the second UAA comprises a fluorosulfate moiety. In some embodiments, the second UAA comprises an aryl fluorosulfate moiety. In some embodiments, the second UAA comprises formula I: in some embodiments, the second UAA has the structure: in some embodiments, the second UAA has the structure: in some embodiments, the second UAA comprises formula II: in some embodiments, the second UAA has the structure: in some embodiments, the second UAA has the structure: In some embodiments, the second UAA comprises formula III: in some embodiments, the second UAA has the structure: in some embodiments, the second UAA has the structure: in some embodiments, the second UAA has formula (IA): Wherein each X is independently O or NR'; y is a bond, -O-, -NR-, or-N=; a is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4, each R and R', when present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl, R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6, and wherein m is 1 when Y is-O-or-NR-, and m is 2 when Y is-n=in some embodiments, the second UAA has the formula (IA-a): is a structure of (a). In some embodiments, the second UAA has formula (IA-b): Is a structure of (a). In some embodiments, the second UAA has formula (IB): is a structure of (a). In some embodiments, the second UAA has the formula (IC): is a structure of (a). In some embodiments, the second UAA has the formula (ID): Wherein: each X is independently O or NR'; y is a bond, -O-, -NR-, or-N=; a is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4, each R and R', when present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl, R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein when Y is a bond, -O-or-NR-, m is 1, and when Y is-n=m is 2, in some embodiments, the second UAA has the formula (IE): wherein: each X is independently O or NR'; y is a bond, -O-, -NR-, or-N=; a is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4, each R and R', when present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl, R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein when Y is a bond, -O-or-NR-, m is 1, and when Y is-n=m is 2, in some embodiments, the second UAA has formula (IIA): Wherein: x is independently O or NR'; and when present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, the second UAA has formula (IIB): Wherein: x is independently O or NR'; and when present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, the second UAA is of formula (IV) Wherein each X is independently O or NR'; y is a bond, -O-, -NR-, or-N=; a is a bond OR- (CH 2)n -; m is 1 OR 2;n is an integer from 1 to 4; each R and R' is independently, when present, hydrogen, substituted OR unsubstituted alkyl, substituted OR unsubstituted heteroalkyl, substituted OR unsubstituted aryl, substituted OR unsubstituted heteroaryl, OR substituted OR unsubstituted heterocycloalkyl; ring A is a 5 to 6 membered aryl OR heteroaryl; each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; p is 0,1, 2, 3 or 4; each R X is optionally substituted alkyl; l is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6, and wherein when Y is a bond, -O-, or-NR-, m is 1; When Y is-n=m is 2. In some embodiments, the first targeting domain comprises any one of SEQ ID NOs 1-4 or 16-64. In some embodiments, the first targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs 1-4 or 16-64. In some embodiments, the second targeting domain comprises any one of SEQ ID NOs 1-4 or 16-64. In some embodiments, the second targeting domain comprises a sequence having at least 70% identity to any one of SEQ ID NOs 1-4 or 16-64. in some embodiments, the conjugate comprises SEQ ID NO 66-72. In some embodiments, the conjugate comprises a sequence having at least 70% sequence identity to SEQ ID NOS 66-72. In some embodiments, the conjugate comprises any one of SEQ ID NOs 1-4 or 16-64. In some embodiments, the conjugate comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs 1-4 or 16-64. In some embodiments, the conjugate comprises a sequence having at least 70% sequence identity to SEQ ID NOS 66-72. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO. 1. In some embodiments, UAA is present at an amino acid position selected from 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115 relative to SEQ ID NO. 1. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO. 2. In some embodiments, UAA is present at an amino acid position selected from 50, 52, 53, 54, 56, 58, and 100 relative to SEQ ID NO. 2. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO 3. In some embodiments, UAA is present at an amino acid position selected from the group consisting of 58, 62, 101, 103, and 107 relative to SEQ ID NO. 3. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO 16. In some embodiments, at least one of the first targeting domain and the second targeting domain comprising SEQ ID NO. 16 further comprises an unnatural amino acid at position 109 relative to SEQ ID NO. 16. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO 18. In some embodiments, the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent. In some embodiments, the cytotoxic moiety comprises a small molecule drug or a chemotherapeutic drug. In some embodiments, the radioligand reagent is selected from 153Sm、177Lu、90Y、131I、149Tb、211At、212Pb/212Bi、213Bi、223Ra、225Ac and 227 Th. In some embodiments, the radioligand reagent further comprises a chelator. In some embodiments, the radioligand reagent is selected from 99mTc、131I、201Tl、111 In and 67 Ga. In some embodiments, the payload is attached to the conjugate with a linker. In another aspect, provided herein are methods comprising administering the conjugates provided herein, wherein the conjugates covalently bind to a first target on the surface of a first cell. In some embodiments, the conjugate binds to a second target on the surface of the first cell. In some embodiments, the first targeting domain and the second targeting domain bind to the same target. In some embodiments, the first targeting domain and the second targeting domain bind to the same epitope of the same target. In some embodiments, the first targeting domain and the second targeting domain bind to different epitopes of the same target. In some embodiments, the first targeting domain and the second targeting domain bind to different targets on the surface of the first cell. In some embodiments, the second domain comprises a second UAA, and wherein the second UAA is covalently bound to a second target. In some embodiments, the first cell is a tumor cell. In some embodiments, the conjugate kills or inhibits growth of tumor cells when bound to the first target. In some embodiments, the conjugate kills or inhibits the growth of tumor cells when bound to the second target. In some embodiments, the conjugate kills or inhibits the growth of tumor cells when bound to the first target and the second target. In some embodiments, the first target is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some embodiments, the second target is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some embodiments, the payload is a radiolabeling agent or a cytotoxic agent. In some embodiments, the payload is an imaging agent. In some embodiments, the method images or identifies the first cell when the conjugate binds to the first target and the second target on the first cell. In still another aspect, provided herein is a method of making a conjugate provided herein, comprising: (a) Synthesizing in vivo a first targeting domain comprising at least one unnatural amino acid; and (b) conjugating the payload to the first targeting domain or the second targeting domain, optionally via a linker. In some embodiments, the method further comprises synthesizing the second targeting domain in vivo as a fusion protein with the first targeting domain. In some embodiments, the synthesis includes the use of an orthogonal tRNA synthetase/suppressor tRNA pair. In some embodiments, the synthesis includes an orthogonal tRNA synthetase/suppressor tRNA pair that is derived from a pyrrolysine tRNA synthetase/tRNA Pyl.
Incorporation by reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Drawings
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and drawings in which illustrative embodiments that utilize the principles of the invention are set forth, in which:
FIGS. 1A-1E show various SDS-PAGE analysis of cross-linking between FSY-modified sdabs with FSY at different positions and PSMA at 37℃with a molar ratio of sdAb to PSMA of about 7:1 (final concentration of PSMA 0.125mg/mL,1.25 uM). Fig. 1A depicts a gel showing a C1 construct comprising FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA and C1-FSY are labeled. Fig. 1B depicts a gel showing a C1 construct comprising FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA and C1-FSY are labeled. Fig. 1C depicts a gel showing a C1 construct comprising FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA and C1-FSY are labeled. Fig. 1D depicts a gel showing a C1 construct comprising FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA and C1-FSY are labeled. Fig. 1E depicts a gel showing a C1 construct comprising FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA and C1-FSY are labeled.
FIGS. 2A-2C show various SDS-PAGE analysis of cross-linking between C2 (PSMA-specific sdAb) and PSMA at 37℃with a molar ratio of sdAb to PSMA of 8:1. FIG. 2A depicts a gel showing a C2 construct comprising FSY at positions 50-61 in the presence of target PSMA and wild-type C2-CDR2 library controls. The thick arrows indicate the presence of crosslinked product. PSMA and C2 are marked with thin arrows. Fig. 2B depicts a gel showing a C2 construct comprising FSY in the presence of target PSMA. FIG. 2C depicts a gel showing a C2 construct comprising FSY in the presence of target PSMA as well as a wild-type C2-CDR3 library control. The thick arrows indicate the presence of crosslinked product. PSMA and C2-FSY are marked with thin arrows.
FIG. 3A shows SDS-PAGE analysis of cross-linking kinetics between FSY-modified sdAb (FSY at position 28 or 102 of C1) and PSMA at 37℃for 180 minutes, wherein the molar ratio of sdAb to PSMA is 5:1 (final concentration of PSMA 0.125mg/mL,1.25 uM).
Figure 3B shows a graph of the kinetics of percent PSMA cross-linking versus time for a cross-linking experiment between FSY modified sdAb and PSMA. The y-axis is labeled cross-linked PSMA (0 to 60, 10 units apart) and the x-axis is labeled time (min, 0 to 180, 30min apart). The constructs tested were C1-28FSY (circles), C1-102FSY (squares), C1-112FSY (triangles), C1-113FSY (inverted triangles).
FIG. 4 shows a diagram of a monomeric C1-related sdAb construct. C1 (wild-type sequence comprising two cysteine disulfide bonds), C39 (C1-C101A/C104A, one cysteine disulfide bond replaced with two alanine, thereby removing the disulfide bond), C1-102FSY (construct C1, histidine 102 replaced with unnatural amino acid FSY), and C39-102FSY (C1-C101A/C104A/H102 (FSY), both C101 and C104 cysteines replaced with alanine, histidine 102 replaced with FSY).
FIG. 5A shows SDS-PAGE analysis of the cross-linking kinetics of the double paratope construct C40-102FSY (generated by ligating the C39-102FSY sequence with a C39 copy without FSY) or the single paratope construct of C39-102FSY with PSMA at 37℃in 180 minutes. The bands corresponding to C39-102FSY, C40-102FSY, receptor and crosslinking species are labeled. The rate of coupling of the biparatopic construct is increased compared to the single paratope construct.
FIG. 5B shows a graph of PSMA cross-linking versus time for cross-linking between the double paratope construct C40-102FSY or the single paratope construct C39-102FSY and PSMA.
FIGS. 6A and 6B show SDS-PAGE analysis of cross-linking between various FSY modified C3 constructs. FIG. 6A shows the results for C3 constructs with FSY at positions 26-35 and 50-60; the crosslinked product of construct C3-58FSY is indicated by asterisks. FIG. 6B shows the results for C3 constructs with FSY at positions 61-66 and 99-113); cross-linked products were observed for at least constructs C3-62FSY, C3-101FSY, C3-103FSY, C3-107 FSY.
FIG. 7A shows SDS-PAGE analysis of cross-links between various pooled FSK modified C8 constructs and FAP receptors. FIG. 7B shows SDS-PAGE analysis of cross-linking between various individual FSK modified C8 constructs and FAP receptors.
FIG. 7C shows SDS-PAGE analysis of cross-linking kinetics between various FSK modified C8 constructs and FAP receptor.
FIGS. 8A-8D show SDS-PAGE analysis of cross-linking between various FSY modified C9 constructs and Her3 receptor. FIG. 8A shows the results of libraries of C9 constructs with FSY at various positions with Her3 receptor. FIG. 8B shows the results for the C9 construct with FSY at positions 52-68 with Her3 receptor. FIG. 8C shows the results of C9 constructs with FSY at positions 53, 55, 56, 57, 58, 60, 64 or 67 with Her3 receptor. FIG. 8D shows SDS-PAGE analysis of the cross-linking kinetics between the FSY modified C9 construct (C9-55 FSY) and Her3 receptor.
FIG. 9 shows the results of binding assays for C2-54FSY and C2-54TYR sdabs to human prostate tumor cell lines LNCaP (PSMA+) and PC3 (PSMA-) using flow cytometry.
FIGS. 10A and 10B show SDS-PAGE analysis of FSY-modified sdabs cross-linked to cells. FIG. 10A shows Western blots using LNCaP cells incubated at different concentrations and time points and C2-54 FSY. The blots were analyzed using anti-PSMA antibodies (upper panel), anti-sdAb antibodies (middle panel) and anti-GAPDH (lower panel) as loading controls. FIG. 10B shows Western blotting using LNCaP cells and C2-54TYR, and shows no cross-linking with PSMA. The rightmost lane is a control showing a sample of C2-54FSY incubated with LNCaP cells for 9 hours at 1 uM. FIG. 10C shows a graph of the cross-linking kinetics for different concentrations of C2-54FSY at different time points.
FIG. 11A shows Western blot analysis of the cross-linking kinetics of C2-54FSY and C3-101FSY with PSMA at 1uM and 24nM in LNCaP cells at different times. The upper panel shows the blot of anti-PSMA antibodies, while the lower panel shows the anti-GAPDH control. FIG. 11B shows a graph of the cross-linking kinetics of C2-54FSY and C3-101FSY with PSMA on LNCaP cells.
Fig. 12A and 12B show the study design and results of in vivo crosslinking assays. FIG. 12A shows the study design and Western blot of LNCaP and PC3 tumor tissue samples from mice administered with C2-54 TYR and C2-54 FSY. The upper panel shows PSMA region of gel blotted with anti-VHH antibody, the middle panel shows free VHH region of gel blotted with anti-VHH antibody, and the lower panel shows anti-GAPDH blot. The left panel shows samples from LNCaP tumor bearing animals, while the right panel shows PC3 samples. Crosslinking was only observed in LNCaP (PSMA+) tumors and in the presence of C2-54 FSY. FIG. 12B shows a graph of plasma concentrations of C2-54 TYR and C2-54FSY after administration.
FIG. 13 shows SDS-PAGE analysis of cross-linking kinetics of C8-54FSY, C8-55FSY and C8-56FSY (left to right). The band positions representing crosslinks and C8-FSY are marked. Lanes represent (from left to right) 0, 15, 30, 60, 120, 180 minutes and ladders for each construct.
FIG. 14 shows SDS-PAGE analysis of cross-links of various C15-FSY-containing constructs. Left gel: lanes (left to right) represent positions: 2. constructs with FSY substitutions at 4, 5, 6, 7, 8, 9, 10, 11, 12, 33, 34, 35, ladder and Her 2. Right gel: lanes (left to right) represent positions: 36. constructs with FSY substitutions at 37, 66, 67, 68, 69, wt ladder and Her 2. The label indicates the crosslinked band position and the construct comprising FSY.
FIG. 15 shows SDS-PAGE analysis of the double paratope construct C38-FSY cross-linked with Her 2-Fc. Lanes (from left to right) are time 0, 0.5, 1.5, 2.5, 3.5, 4.5, 5.5 hours, her2 and ladder. The labels represent the band positions of the crosslinks, her2-Fc and C38-FSY.
FIG. 16 depicts fluorescence versus concentration for binding studies of constructs in A431 and Colo320DM cells via flow cytometry: a431-C23-TYR, solid squares; a431-C23-FSY, solid triangles; colo320DM-C23-TYR, the "x" symbol; and Colo320DM-C23-FSY, open circles. The y-axis is labeled AF680 GeoMean at intervals of 40,000 units from 0 to 200,000. The x-axis is marked as the concentration (M) from 10 -11 to 10 -5 on a 10-base logarithmic scale.
FIG. 17 shows the time-dependent manner of intratumoral free and EGFR cross-linked AF680 labeled compounds C23-TYR and C23-FSY in A431 (EGFR+) and COLO320DM (EGFR-) tumors. Free AF-680 labeled C23-TYR and C23-FSY were detected in the approximately 15kD region (bottom panel), and EGFR-crosslinked sdAb band was detected in the approximately 175kD region (top panel). Time-dependent cross-linking of EGFR with C23-FSY was observed in EGFR+A431 tumors, but no time-dependent cross-linking of EGFR with C23-TYR was observed. Neither free sdAb retention nor cross-linking was observed in EGFR-COLO320DM tumors.
FIG. 18A depicts a graph of photons/s/g tissue in three biological replicates of A431 tumors derived from animals administered C23-TYR or C23-FSY test preparations. Values from individual animals are shown, the center bar shows the mean intensity, and the error bars represent SEM (by paired t-test, × p=0.024, × p=0.002). At the time points of 8 hours and 24 hours, the level of C23-FSY present in A431 tumors was significantly higher compared to the C23-TYR protein without FSY. The y-axis is labeled at 0.5x10 10 intervals as photons/s/g of tissue of 0 to 1.5x10 10. The x-axis is labeled as 8 hours and 24 hours (hr) post-administration. Via paired t-test (double tail), asterisks indicate statistical significance (=p.ltoreq.0.05, =p.ltoreq.0.005).
FIG. 18B depicts a graph of quantitative ex vivo fluorescence intensity of A431 and COLO320DM tumors in animals administered C23-FSY 8 hours and 24 hours after dosing. The figure shows photons/s/g tissue in three biological replicates. Values from individual animals are shown, the center bar shows the average intensity, and the error bars represent SEM. At the 8 and 24 hour time points, the level of C23-FSY in A431 tumors was significantly higher than in EGFR-COLO320DM tumors, indicating the tumor locking specificity of the C23-FSY protein. The y-axis is labeled at 0.5X10 10 intervals as photons/s/g of tissue of 0 to 1X 10 10. For each set of histograms, the x-axis is labeled as time 8 and 24hr post-dose (hr). The left panel is model a431 tumors and the right is model Colo320DM tumors.
Figure 19 shows fluorescent images of SDS PAGE gels showing tumor-associated free sdAb and PSMA-crosslinked sdAb in a time-dependent manner. Mice bearing LNCaP tumors were administered in triplicate with either C30-TYR or C30-FSY. At designated time points after treatment, tumors were harvested and gel electrophoresed to detect fluorophore conjugated sdAb assay preparations. Free (non-crosslinked) sdAb-AF680 was detected in the approximately 20kD region (lower panel), and PSMA-crosslinked sdAb-AF680 species of C30-FSY migrated in the 100kD region (upper panel). Lanes marked with x show vehicle samples.
FIG. 20 shows quantitative analysis of tumor-associated free and PSMA-crosslinked test articles C30-TYR and C30-FSY. Fluorescence band intensities of free and PSMA-crosslinked species bands in the above gels were quantified via densitometry and compared to a standard curve. Total intratumoral test article concentration (free and PSMA cross-linked, pg/mg tumor tissue) versus time (h) was plotted. Data points expressed as x represent samples below the detection and quantification limit. The tumor exposure of C30-FSY increased to about 3X compared to non-covalent C30-TYR.
FIG. 21A depicts a comparative graph of cytotoxicity of C26-54TYR and C26-54FSY test preparations in PC3PIP (PSMA positive) and PC3flu (PSMA negative) cell lines showing concentration of sdAb conjugated to MMAE versus cell viability. The y-axis is marked at 20 unit intervals as% viability of 0 to 120. The x-axis is labeled as test article concentration (M) at a base 10 log interval from 10 -11 to 10 -6.
FIG. 21B depicts a graph comparing cytotoxicity of C28-101TYR and C28-101FSY test preparations in PC3PIP (PSMA positive) and PC3flu (PSMA negative) cell lines showing concentration of sdAb conjugated to MMAE versus cell viability. The y-axis is marked at 20 unit intervals as% viability of 0 to 120. The x-axis is labeled as test article concentration (M) at a base 10 log interval from 10 -11 to 10 -6.
FIG. 22 depicts SDS-PAGE analysis of FSY cross-linking kinetics, wherein the 52FSY and 54FSY variants of C17 are incubated with Her2 receptor and cross-linking efficiency is examined. Lanes 1-5 represent C17-52FSY at 0, 30, 60, 120 and 180 min; lanes 6-10 represent C17-54FSY at 0, 30, 60, 120 and 180 min; lanes 11 and 12 are FcHer and ladder, respectively. The band positions corresponding to C17-FSY, receptor and cross-linked product are marked.
FIG. 23A depicts a graph comparing cytotoxicity after 5 hours wash out using the C33-52TYR, C33-52FSY, C33-54TYR and C33-54FSY test preparations, showing concentration of sdAb conjugated to MMAE in BT474 cells versus cell viability. The y-axis is marked as% viability from-20 to 120 at 20 unit intervals. The x-axis is labeled as the test article concentration (nM) at a base 10 log interval from 10 -3 to 10 3.
Figure 23B depicts a cytotoxicity comparison graph using consecutive 6 days of exposure to C33 test preparations, showing concentration of sdAb conjugated to MMAE in BT474 cells versus cell viability. The y-axis is marked as% viability from-20 to 120 at 20 unit intervals. The x-axis is labeled as the test article concentration (nM) at a base 10 log interval from 10 -2 to 10 2.
FIG. 24A depicts PSMA cross-links for single and double paratope constructs (C3-101 FSY, square; C34-FSY, triangle; C36-FSY, circle). The y-axis is labeled as crosslinked PSMA (total%) from 0 to 100 at 20 unit intervals. The x-axis is labeled as the test article concentration (nM) at a base 10 log interval of from 0.01 to 1000. The figure corresponds to crosslinking at 1 hour.
FIG. 24B depicts PSMA cross-links of single and double paratope constructs (C3-101 FSY, square; C34-FSY, triangle; C36-FSY, circle). The y-axis is labeled as crosslinked PSMA (total%) from 0 to 100 at 20 unit intervals. The x-axis is labeled as the test article concentration (nM) at a base 10 log interval of from 0.01 to 1000. The figure corresponds to crosslinking at 6 hours.
FIG. 25A depicts Western blot analysis of FSY cross-linking kinetics at 1 hour and 6 hour time points for different concentrations of C3-101FSY and C34-FSY constructs. Lanes 1-6 represent C3-101FSY at 1 hour; lanes 7-12 represent C34-FSY at 1 hour; lanes 13-18 represent C3-101FSY at 6 hours, and lanes 19-24 represent C34-FSY at 6 hours. Six lanes per group depict the increase in construct concentration (left to right): UTC, 0.1, 1, 10, 100, 1000nM. The top panel depicts a-PSMA and the bottom panel depicts a-GAPDH. The band positions of GAPDH, PSMA, crosslinking monomers, and crosslinking dimers are labeled.
FIG. 25B depicts Western blot analysis of FSY cross-linking kinetics at 1 hour and 6 hour time points for different concentrations of C3-101FSY and C36-FSY constructs. Lanes 1-6 represent C3-101FSY at 1 hour; lanes 7-12 represent C36-FSY at 1 hour; lanes 13-18 represent C3-101FSY at 6 hours, and lanes 19-24 represent C36-FSY at 6 hours. Six lanes per group depict the increase in construct concentration (left to right): UTC, 0.1, 1, 10, 100, 1000nM. The top panel depicts a-PSMA and the bottom panel depicts a-GAPDH. The band positions of GAPDH, PSMA, crosslinking monomers, and crosslinking dimers are labeled.
FIG. 26A depicts SDS-PAGE analysis of cross-linking kinetics between EGFR and single paratope construct C4-109 FSY. The half maximum time is about 120 minutes. Bands corresponding to the cross-linked product, EGFR and sdAb monomers are labeled. The right panels show the inset of the receptor with the FSY containing sdAb monomer.
FIG. 26B depicts SDS-PAGE analysis of cross-linking kinetics between EGFR and the double paratope construct C37-FSY. Bands corresponding to the cross-linked product, EGFR and sdAb dimers are labeled. The right side shows an inset depicting the receptor engaged with the sdAb biparatopic construct containing FSY.
FIG. 27 depicts EGFR cross-linking kinetics for single paratope construct C4-109FSY (square) or double paratope construct C37-FSY (circular). The y-axis is labeled as% EGFR crosslinking from 0 to 100%. The x-axis is marked as time (minutes) from 0 to 400 at 100 minute intervals.
Fig. 28A-C depict exemplary conjugates described herein comprising a first targeting domain (e.g., constructs C1-C4) and a second targeting domain (e.g., constructs C1-C4), wherein the conjugates comprise unnatural amino acids (e.g., FSY). One exemplary conjugate also comprises a payload. Exemplary conjugates can include unnatural amino acids on the first targeting domain, the second targeting domain, or both targeting domains.
Detailed Description
Antibodies, antibody fragments, and antibody-related constructs, such as Antibody Drug Conjugates (ADCs), can be useful tools for research and clinical applications. However, in some cases, the use of these molecules is limited by the on/off rate and stability of the target. Provided herein are compositions and methods for specific and covalent binding of conjugates to targets. In some cases, the conjugate comprises at least one targeting domain having at least one Unnatural Amino Acid (UAA) residue near the interface between the target and the targeting domain, such that when the targeting domain binds to the target, a covalent bond is formed between the target and the UAA residue. In some cases, the conjugate comprises at least one targeting domain and at least one payload. In some cases, the targeting domain comprises at least one Unnatural Amino Acid (UAA) residue near the interface between the target and the targeting domain, such that when the targeting domain binds to the target, a covalent bond is formed between the target and the UAA residue. In some cases, once the conjugate reaches its target cell/tumor, the payload is now covalently bound to the target via the targeting domain. In some cases, covalent interactions eliminate or reduce the rate of binding of the conjugate to the target, increasing contact with the attached payload.
In some cases, the conjugate comprises (i) a first targeting domain and (ii) a second targeting domain, wherein the first targeting domain or the second targeting domain comprises at least one Unnatural Amino Acid (UAA). In some cases, the first or second targeting domain comprises at least one UAA present near an interface between the target and the first or second targeting domain, and when the first or second targeting domain binds to the target, a covalent bond is formed between the target and the at least one UAA. In some cases, the conjugate further comprises a payload. In some cases, when the conjugate reaches its target cell, such as a tumor or a cell in the tumor microenvironment, the payload is now covalently bound to the target via one of the targeting domains (e.g., the first targeting domain or the second targeting domain).
In some cases, the covalent interactions eliminate or reduce the rate of binding of the conjugate to the target, or otherwise stabilize contact with the target. In further cases, the first targeting domain and the second targeting domain in the conjugates provided herein target the same or different targets. In some cases, only the second targeting domain, but not the first targeting domain, comprises UAA. In some cases, only the first targeting domain, but not the second targeting domain, comprises UAA. In some cases, both the first targeting domain and the second targeting domain comprise UAA. In some cases, the payload may be attached to the first targeting domain or the second targeting domain. In some cases, the payload is attached to the first targeting domain. In some cases, the payload is attached to a second targeting domain.
Conjugate(s)
The conjugate may comprise a targeting domain and a payload. In some embodiments, the payload is attached to the conjugate with a linker. In some cases, the conjugate comprises at least one unnatural amino acid. In some cases, the targeting domain is configured to bind to a target, and one unnatural amino acid within the targeting domain forms a covalent bond with the target.
Conjugates provided herein can comprise (i) a first targeting domain and (ii) a second targeting domain. Conjugates provided herein may comprise (i) a first targeting domain, (ii) a second targeting domain, and (iii) a payload. The conjugates herein may include a first targeting domain and a second targeting domain, wherein the first targeting domain and the second targeting domain are configured to bind to the same cell. In some cases, the conjugate comprises at least one Unnatural Amino Acid (UAA) that is comprised in the first targeting domain, the second targeting domain, or each of the first targeting domain and the second targeting domain comprises at least one UAA. In some cases, the targeting domain is configured to bind to a target, and one of the UAAs within the targeting domain (e.g., the first and/or second targeting domain) forms a covalent bond with the target. In some cases, the first targeting domain comprises UAA and is configured to form a covalent bond with the first target such that at least one UAA is present in the first targeting domain near an interface between the first target and the first targeting region. In some cases, the first target binds to the first targeting domain and forms a covalent bond between the first target and UAA in the conjugate first targeting domain. In some cases, the second targeting domain comprises UAA and is configured to form a covalent bond with the second target such that at least one UAA is present in the second targeting domain near an interface between the second target and the second targeting domain. In some cases, the second target binds to the second targeting domain and forms a covalent bond between the second target and UAA in the second targeting domain of the conjugate. In some cases, the first target is the same as the second target. In some cases, the first target and the second target are located on the surface of the same cell.
In some embodiments, the first targeting domain and the second targeting domain bind to the same target, such as on the surface of a cell, and engagement of one targeting domain brings the other targeting domain into proximity to its respective target. In some cases, the first targeting domain binds to a first target and the second targeting domain binds to a second target, and at least one of the first and second targeting domains comprises UAA, and the UAA forms a covalent bond with the corresponding target. In some cases, one targeting domain forms a covalent bond with its respective target and the other targeting domain is non-covalently bound to its respective target. In some cases, the first targeting domain and the second targeting domain are both covalently bound to their respective targets.
Conjugates provided herein can be configured to bind to more than one target. In some cases, the second targeting domain can bind a different target than the first targeting domain. In some cases, the conjugate comprises at least 2, 3, 4, 5, 6, or more than 7 targeting domains. In some cases, the targeting domains (e.g., the first targeting domain and the second targeting domain) are attached to each other via a linker (e.g., a chemical linker, a fusion protein, or other linker provided herein). In some cases, multiple targeting domains (e.g., a first targeting domain and a second targeting domain) in the conjugates provided herein can provide a single-paratope or a double-paratope construct. In some cases, the first targeting domain and the second targeting domain bind to the same target. In some cases, the first targeting domain and the second targeting domain bind different epitopes of the same target. In some cases, the first targeting domain and the second targeting domain bind different targets. In some cases, the first targeting domain and the second targeting domain are linked to form a fusion protein.
The targeting domain (e.g., the first targeting domain or the second targeting domain) can direct a payload attached to the conjugate to the target. In some cases, the targeting domain comprises an antibody or fragment thereof. In some cases, the targeting domain includes monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, diabody, triabody, scFv-Fc, nanobody (i.e., single domain antibody, sdAb), minibody, igNAR, V-NAR, hcIgG, vhH, or peptibody (peptibody), darpin, monomer/FN 3, VNAR, repebody, darpin. In some cases, the targeting domain comprises a nanobody. In some embodiments, the targeting domain comprises a single domain antibody (sdAb). In some cases, the targeting domain includes one or more CDR regions. In some embodiments, the targeting domain binds to a cell surface molecule. In some cases, the conjugate is biparatopic. In some embodiments, the first targeting domain and the second targeting domain each comprise an antibody or antigen binding fragment, and the structure of such an antibody or antigen binding fragment may be the same or different. In some cases, the first targeting domain can be a single chain (e.g., fv) antibody fragment and the second targeting domain can be an sdAb, or in other cases, the first targeting domain can be an sdAb and the second targeting domain can be an sdAb.
In some cases, the targeting domain (e.g., the first targeting domain and/or the second targeting domain) is an antibody mimetic, such as affibody, DARPin or a mini-binding agent.
In some cases, the target comprises a cell surface protein. In some cases, the target comprises PSMA. In some cases, the targeting domain (e.g., the first targeting domain and/or the second targeting domain) comprises any one of SEQ ID's 1-4 or 16-64. In some cases, the targeting domain comprises a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70% or at least 65% identity to any of SEQ ID's 1-4 or 16-64. In some cases, the targeting domain comprises a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70% or at least 65% identity to any one of SEQ ID's 1-4 or 16-64 and at least one unnatural amino acid.
The unnatural amino acid can be located at any position in the conjugate. In some cases, one or both of the first targeting domain or the second targeting domain (e.g., the first targeting domain or the second targeting domain) comprises an unnatural amino acid. In some cases, the targeting domain is an antibody or antigen binding fragment comprising one or more Complementarity Determining Regions (CDRs). In some cases, one or more unnatural amino acids are contained within or near the CDRs in only one or two targeting domains. In some cases, the targeting domain comprises an unnatural amino acid. In some cases, the targeting domain comprises C1, C2, or C3. In some cases, the conjugate comprises C1, which has an unnatural amino acid in CDR1, CDR2, or CDR 3. In some cases, the conjugate comprises C1, which has an unnatural amino acid at any one of positions 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115. In some cases, the conjugate comprises C1, which has an unnatural amino acid at any one of positions 28, 102, 112, and 113. In some cases, the conjugate comprises C2, which has an unnatural amino acid in CDR1, CDR2, or CDR 3. In some cases, the conjugate comprises C2, which has an unnatural amino acid at any of positions 50, 52, 53, 54, 56, 58, or 100. In some cases, the conjugate comprises C4, which has an unnatural amino acid in CDR1, CDR2, or CDR 3. In some cases, the conjugate comprises C4 with an unnatural amino acid at position 109. In some cases, the conjugate comprises a C3 having an unnatural amino acid in CDR1, CDR2, or CDR 3. In some cases, the conjugate comprises C3 with an unnatural amino acid at any of positions 58, 62, 101, 103, or 107. In some cases, the conjugate comprises C8 with an unnatural amino acid at any of positions 52, 53, 54, 55, 56, 58, 60, 62, 64. In some cases, the conjugate comprises C9 with an unnatural amino acid at any of positions 53, 55, 56, 57, 58, 60, 64, 67. In some cases, the conjugate comprises a single domain antibody (sdAb) comprising an sdAb having a CDR region and at least one Unnatural Amino Acid (UAA) residue within or near the CDR region, where the sdAb comprises any one of SEQ ID nos 1-4 or 16-64. In some cases, the conjugate comprises a single domain antibody (sdAb) comprising an sdAb having a CDR region and at least one Unnatural Amino Acid (UAA) residue within or near the CDR region, where the sdAb comprises a sequence that is 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% identical to any of SEQ ID nos. 1-4 or 16-64. In some embodiments, the CDR regions comprise one or more of SEQ ID Nos. 5-13. In some embodiments, the CDR regions comprise sequences having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70% or at least 65% identity to one or more of SEQ ID nos. 5-13.
In some cases, the conjugate comprises a tag, such as a tag for purification (e.g., his6 tag). In some cases, the conjugate does not contain a label, or the label is removed prior to application. In some cases, the conjugate comprises a leader sequence, such as for expression or secretion. In some cases, the conjugate does not comprise a leader sequence, or the leader sequence is removed from the first targeting domain or the second targeting domain prior to formation of the conjugate, prior to attachment of the payload, or prior to administration of the conjugate.
In some cases, the conjugate comprises a signal sequence. The signal sequence may allow expression, folding or oxidation of the conjugate in a bacterial cell. In some cases, the signal sequence may allow the conjugate expressed in the bacterial cell to be transported to another location or environment to facilitate folding or function of the conjugate. In some cases, the signal sequence may be a PelB sequence. In some cases, the signal sequence may allow the conjugate to be transported to the periplasm of the bacterial cell. In some cases, the environment may be oxidizing or reducing to allow for disulfide formation or disulfide reduction.
Target(s)
Conjugates provided herein can be configured to bind to one or more targets. In some cases, the conjugate is configured to have a single targeting domain comprising UAA, and the conjugate binds to the target. In some cases, the conjugate is configured to have two (or at least two) targeting domains, and the first targeting domain and the second targeting domain bind to one or more targets. In some cases, the first targeting domain binds to a first target and the second targeting domain binds to a second target. In some cases, UAA contained in a targeting domain (e.g., a single targeting domain or a first targeting domain or a second targeting domain) forms a covalent bond between the targeting domain (e.g., a single targeting domain, or a first targeting domain or a second targeting domain) and a target (the respective target of the targeting domain, e.g., a first or second target). In some cases, the conjugate comprises a first targeting domain comprising a first UAA and a second targeting domain comprising a second UAA. These UAAs are the same in some cases, or different in other cases. In some cases, the target is a cell surface molecule (i.e., is present in whole or in part on the outer surface of the cell). In some cases, the first targeting domain and the second targeting domain bind to the same target, such as the same cell surface molecule. In some cases, the first targeting domain and the second targeting domain each bind to a different target, such as to a different cell surface molecule. Different cell surface molecules may be on the same cell.
In some cases, the target is a cell surface molecule present on a tumor cell. In some cases, the target is a monomer. In some cases, the target is contained in a multimeric structure of a homo-or hetero-unit. In some cases, the target is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.
In some cases, the conjugate comprises a first targeting domain and a second targeting domain, and both targeting domains bind to the same target, wherein the target is a cell surface molecule on a tumor cell. For example, both targeting domains bind to a target selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some cases, the conjugate comprises a first targeting domain and a second targeting domain that bind to different targets, wherein one or more of the different targets is a cell surface molecule on a tumor cell. For example, the first targeting domain and the second targeting domain bind to different targets, each target selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some cases, the conjugate carries the payload to the cell surface when the first or second target (or both) binds. In some cases, the conjugate has no payload and, when covalently bound to the first target, the second target, or both the first and second targets, the conjugate acts as a blocker or antagonist.
In some cases, the conjugate comprises a single targeting domain, and the targeting domain binds to a target, wherein the target is a cell surface molecule on a tumor cell. For example, the targeting domain binds to a target selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some cases, a single targeting domain carries the payload of the target. In some cases, the single targeting domain has no payload and the conjugate acts as a blocker or antagonist when covalently bound to the target.
Targets engaged by the targeting domain may include various structures. In some cases, the target comprises one or more epitopes that can be joined by a targeting domain. In some cases, the target comprises multiple epitopes and can be joined by multiple targeting domains, e.g., such as binding to a first epitope through a first targeting domain and binding to a second epitope through a second targeting domain. In some cases, the target comprises a monomer. In some cases, the target comprises a single-chain peptide. In some cases, the target comprises a multimeric molecule. In some cases, the multimeric molecule comprises two or more subunits. In some cases, the subunits have the same structure. In some cases, the subunits are of different construction, or a combination of identical and different constructions. In some cases, the multimeric molecule comprises two or more molecules in a complex. In some cases, the multimeric molecule comprises two protein compositions that complex or interact with each other.
Exemplary targeting domains and exemplary conjugates
The conjugates provided herein comprise a targeting domain that can be assembled from molecules that are linked to a target. In some cases, the targeting domain (single targeting domain or first targeting domain and/or second targeting domain) comprises an antigen binding region, wherein the antigen binding region is linked to a specific target. Such antigen binding regions may comprise CDRs, such as 3 CDRs typically found in the heavy or light chain of an antibody. In some cases, the targeting domain comprises an antigen binding fragment comprising CDRs, such as a VHH (also known as a nanobody or a single domain antibody). In some cases, the single domain antibody comprises one or more UAAs. In some cases, a single targeting domain or for a conjugate having multiple targeting domains, wherein one targeting domain (e.g., the first targeting domain or the second targeting domain) is selected from the single domain antibodies of table 1, such as C1, C2, C3, C4, or C5. In some cases, if conjugated to more than one targeting domain, each targeting domain (e.g., first targeting domain and second targeting domain) is independently selected from a single domain antibody of table 1, such as any of C1, C2, C3, C4, or C5, and one or more UAAs are present in or near CDRs of the single domain antibody. In some cases, the first targeting domain is selected from the single domain antibodies of table 1, such as any one of C1, C2, C3, C4, or C5. In some cases, the second targeting domain is selected from the single domain antibodies of table 1, such as any one of C1, C2, C3, C4, or C5.
In some cases, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or two targeting domains comprising construct C1. In some cases, the conjugate comprises construct C1 having an unnatural amino acid in CDR1, CDR2, or CDR3. In some cases, the conjugate comprises construct C1 having an unnatural amino acid at any of positions 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115 in SEQ ID nos. 1, 4, 19, or 73. In some cases, the conjugate comprises construct C1 having an unnatural amino acid at any one of positions 28, 102, 112, and 113 in SEQ ID NO. 1, 4, 19, or 73. In some cases, the conjugate comprises a single domain antibody (sdAb) and at least one Unnatural Amino Acid (UAA) within or near a CDR region within the sdAb. In some cases, such sdabs comprise SEQ ID No. 1. In some cases, the conjugate comprises a single domain antibody (sdAb) and at least one Unnatural Amino Acid (UAA) within or near a CDR region within the sdAb, where the sdAb comprises a sequence that has at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID No. 1. In some cases, the conjugate comprises a single targeting domain, a first targeting domain, or a second targeting domain, or both, that has at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% identity to SEQ ID No. 1.
In some cases, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or two targeting domains comprising construct C2. In some cases, the conjugate comprises construct C2 having an unnatural amino acid in CDR1, CDR2, or CDR 3. In some cases, the conjugate comprises construct C2 having an unnatural amino acid at any of positions 50, 52, 53, 54, 56, 58 or 100 of SEQ ID NO. 2 or 22. In some cases, the conjugate comprises a single domain antibody (sdAb) comprising the sdAb and at least one Unnatural Amino Acid (UAA) within or near the CDR regions within the sdAb, where the sdAb comprises SEQ ID NO:2. In some cases, the conjugate comprises a single domain antibody (sdAb) and at least one Unnatural Amino Acid (UAA) within or near a CDR region within the sdAb, where the sdAb comprises a sequence that has at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID No. 2. In some cases, the conjugate comprises a single targeting domain, a first targeting domain, or a second targeting domain, or both, having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID No. 2.
In some cases, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or two targeting domains comprising construct C3. In some cases, the conjugate comprises construct C3 having an unnatural amino acid in CDR1, CDR2, or CDR 3. In some cases, the conjugate comprises construct C3 having an unnatural amino acid at any of positions 58, 62, 101, 103, or 107 of SEQ ID NO. 3. In some cases, the conjugate comprises an unnatural amino acid in CDR1, CDR2, or CDR3 of C3. In some cases, the conjugate comprises an unnatural amino acid at position 109. In some cases, the conjugate comprises a single domain antibody (sdAb) and at least one Unnatural Amino Acid (UAA) within or near a CDR region within the sdAb, where the sdAb comprises SEQ ID NO:3. In some cases, the conjugate comprises a single domain antibody (sdAb) and at least one Unnatural Amino Acid (UAA) within or near a CDR region within the sdAb, where the sdAb comprises a sequence that has at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID No. 3. In some cases, the conjugate comprises a single targeting domain, a first targeting domain, or a second targeting domain, or both, having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID No. 3.
In some cases, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or two targeting domains comprising construct C4. In some cases, the conjugate comprises construct C4 having an unnatural amino acid in CDR1, CDR2, or CDR 3. In some cases, the conjugate comprises a single domain antibody (sdAb) and at least one Unnatural Amino Acid (UAA) within or near a CDR region within the sdAb, where the sdAb comprises SEQ ID NO:16. In some cases, the conjugate comprises a single domain antibody (sdAb) and at least one Unnatural Amino Acid (UAA) within or near a CDR region within the sdAb, where the sdAb comprises a sequence that has at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID No. 16. In some cases, the conjugate comprises a single targeting domain, a first targeting domain, or a second targeting domain, or both, having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID No. 16.
In some cases, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or two targeting domains comprising construct C5. In some cases, the conjugate comprises construct C5 having an unnatural amino acid in CDR1, CDR2, or CDR 3. In some cases, the conjugate comprises a single domain antibody (sdAb) and at least one Unnatural Amino Acid (UAA) within or near a CDR region within the sdAb, where the sdAb comprises SEQ ID NO:18. In some cases, the conjugate comprises a single domain antibody (sdAb) and at least one Unnatural Amino Acid (UAA) within or near a CDR region within the sdAb, where the sdAb comprises a sequence that has at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID NO:18. In some cases, the conjugate comprises a single targeting domain, a first targeting domain, or a second targeting domain, or both, having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID No. 18.
Conjugates described herein may comprise two or more targeting domains. In some cases, two or more targeting domains (e.g., a first targeting or a second targeting) are attached to each other via a linker. In some cases, the conjugate is a fusion protein. In some cases, the linker includes linker L1 (SEQ ID NO: 14). In some cases, the conjugate comprises any one of SEQ ID NOs 65-72. In some cases, the conjugate has at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70% or at least 65% sequence identity to any of SEQ ID NOs 65-72.
In some cases, the first targeting domain comprises any one of SEQ ID NOs 1-4 or 16-64. In some cases, the first targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs 1-4 or 16-64. In some cases, the second targeting domain comprises any one of SEQ ID NOs 1-4 or 16-64. In some cases, the second targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs 1-4 or 16-64. In some cases, the conjugate is configured with a single targeting domain, and the single targeting domain comprises any one of SEQ ID NOs 1-4 or 16-64. In some cases, a single targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs 1-4 or 16-64.
In some cases, the conjugate comprises SEQ ID NO 65-72. In some cases, the conjugate comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs 65-72. In some cases, the conjugate comprises any one of SEQ ID NOs 1-4 or 16-64. In some cases, the conjugate comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs 1-4 or 16-64. In some cases, the conjugates comprise a sequence having at least 70% sequence identity to SEQ ID NOS 65-72.
In some cases, the amino acid sequences associated with the conjugates described herein are in table 1A.
Table 1A: amino acid sequence
X represents the position of the FSY incorporation site. Sequences in the table with His6 purification tags and/or PelB leader sequences are also presented herein.
In some cases, DNA sequences associated with the conjugates described herein are in table 2.
Table 2: DNA sequence
Payload
The conjugate may comprise a payload. In some cases, the targeting domain (e.g., a single targeting domain or a first targeting domain or a second targeting domain) included in the conjugate can direct the payload to the target. In some cases, the targeting domain (e.g., a single targeting domain or a first targeting domain or a second targeting domain) comprises an antibody or fragment thereof that directs the payload to a cell (such as a tumor cell). In some embodiments, the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent. In some embodiments, the cytotoxic moiety comprises a small molecule drug, peptide, protein, or chemotherapeutic drug. In some embodiments, the payload comprises a radioligand reagent. In some embodiments, the radioligand reagent is selected from 35S、3H、111In、112In、14C、186Re、188Re、32P、153Sm、177Lu、86Y、88Y、90Y、131I、123I、124I、125I、149Tb、211At、212Pb/212Bi、213Bi、223Ra、225Ac、64Cu、67Cu and 227 Th. In some embodiments, the radioligand reagent further comprises a chelator. Examples of chelating agents include DOTA, DOTAGA, NOTA, MACROPA, THP and TRAP. In some embodiments, the radioligand reagent is selected from 99mTc、131I、201Tl、111 In and 67 Ga. In some cases, the imaging agent includes a dye. In some cases, the conjugate comprises two or more payloads. For example, the conjugate comprises a payload dye for visualizing tissue penetration and a payload chemotherapeutic agent for killing the tumor.
The payload may be attached to the conjugate. The payload may be covalently attached to the conjugate. In some cases, the payload is not attached to the conjugate via an Unnatural Amino Acid (UAA) residue. In some embodiments, the payload is attached to amino acid n+x (toward the C-terminus) relative to the UAA residue. In some embodiments, the site of attachment is defined as a position that is x amino acids from the unnatural amino acid position n. In some embodiments, the payload is attached to amino acid n-x (toward the n-terminus) relative to the UAA residue. In some embodiments, the UAA residues are contained within or near the region of the targeting domain (e.g., a single targeting domain or a first targeting domain or a second targeting domain) that interfaces with the target. In some cases, the payload is at least 2,5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 125 amino acids from the unnatural amino acid in the conjugate.
Unnatural Amino Acid (UAA)
Unnatural Amino Acids (UAA) can be incorporated into the conjugates described herein. In some cases, the UAA residue is present in the targeting domain of the conjugate. In some cases, the UAA is configured to covalently bind to the target. In some cases, the UAA is configured to covalently bind an amino acid present in the target. In some cases, the conjugate comprises one, two, three, four, or more UAAs. In some cases, the conjugate comprises a first targeting domain comprising a first UAA and a second targeting domain comprising a second UAA. In some cases, the UAA is configured within a targeting domain (e.g., when the targeting domain is first or second) to covalently bind an amino acid present in a target when the targeting domain is engaged with the target. In some cases, such amino acids include nucleophilic amino acids. In some cases, the UAA residue forms a covalent bond with lysine, histidine or tyrosine. In some cases, the UAA residue is located in a target binding domain. In some cases, once incorporated into the conjugate, the UAA is located in a CDR of a targeting domain, such as a targeting domain comprising an antibody or antigen binding fragment, e.g., a single domain antibody. In some embodiments, the UAA residue comprises an aryl fluorosulfate moiety. In some cases, the UAA residues are genetically encoded into conjugates described herein. In some cases, UAA residues include variants of tyrosine or lysine. In some embodiments, the unnatural amino acid residue comprises formula I: is a structure of (a). In some embodiments, the unnatural amino acid residue comprises formula II: is a structure of (a). In some embodiments, the UAA residues result from the incorporation of UAA as described herein. In some embodiments, the unnatural amino acid is 2-amino-3- (4- ((fluorosulfonyl) oxy) phenyl) propanoic acid In some embodiments, the unnatural amino acid is fluorosulfonyl tyrosine (FSY): in some embodiments, the unnatural amino acid is N6- (4- ((fluorosulfonyl) oxy) benzoyl) lysine: In some embodiments, the unnatural amino acid is fluorosulfonyloxy benzoyl-L-lysine (FSK): in some embodiments, the unnatural amino acid residue comprises formula III: is a structure of (a). In some embodiments, the unnatural amino acid is In some embodiments, the unnatural amino acid is
In some embodiments, the Unnatural Amino Acid (UAA) of the UAA residue has the structure of formula (IA):
(IA)，
wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is-O-or-NR-, m is 1; when Y is-n=m is 2.
In some embodiments, the UAA of formula (IA) has formula (IA-a): Is a structure of (a). In some embodiments, the UAA of formula (IA) has formula (IA-b): Is a structure of (a).
In some embodiments, the UAA of formula (IA) has formula (IB): Is a structure of (a).
In some embodiments, the UAA of formula (IB) has formula (IB-a): is a structure of (a). In some embodiments, the UAA of formula (IB) has formula (IB-b): Is a structure of (a).
In some embodiments, the UAA of formula (IA) has formula (IC): Is characterized by comprising the following structure:
in some embodiments, the UAA of formula (IC) has formula (IC-a): is a structure of (a). In some embodiments, the UAA of formula (IB) has formula (IB-b): is a structure of (a). In some preferred embodiments, R is hydrogen.
In some embodiments, the UAA of formula (IA) has formula (ID): Is characterized in that the structure of the (c) is that,
Wherein each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
In some embodiments, the UAA of formula (ID) has formula (ID-a): is a structure of (a). In some embodiments, the UAA of formula (ID) has formula (ID-b): Is a structure of (a).
In some embodiments, the UAA of formula (IA) has formula (IE): Is characterized in that the structure of the (c) is that,
Wherein each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
In some embodiments, the UAA of formula (IE) has formula (IE-a): Is a structure of (a). In some embodiments, the UAA of formula (IE) has formula (IE-b): Is a structure of (a).
In some embodiments, Y is a bond, -O-or-NR-, and m is 1. In other embodiments, Y is-n=, m is 2. In certain preferred embodiments, Y is-O-and m is 1. In other embodiments, Y is-NR-and m is 1. In other embodiments, Y is a bond and m is 1. In other embodiments, Y is O-or-NR-, and m is 1.
In some embodiments, the UAA of formula (IA) has formula (IIA): Is characterized in that the structure of the (c) is that,
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
In some embodiments, the UAA of formula (IIA) has formula (IIA-a): Is a structure of (a). In some embodiments, the UAA of formula (IIA) has formula (IIA-b): Is a structure of (a).
In some embodiments, the UAA of formula (IA) has formula (IIB): Is characterized in that the structure of the (c) is that,
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
In some embodiments, UAA of formula (IIB) has formula (IIB-a): is a structure of (a). In some embodiments, UAA of formula (IIB) has formula (IIB-b): Is a structure of (a).
In some embodiments, the Unnatural Amino Acid (UAA) has formula (IV)Is characterized in that the structure of the (c) is that,
Wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
Ring a is a 5 to 6 membered aryl or heteroaryl group;
Each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; each R X is optionally substituted alkyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
In some embodiments, a is a bond. In other embodiments, a is- (CH 2)n -. In some embodiments, n is 1,2, 3, or 4. In one embodiment, n is 1, in another embodiment, n is 2, in another embodiment, n is 3, in another embodiment, n is 4.
In some embodiments, ring a is a 5 membered ring. In some embodiments, ring a is a 6 membered ring. In some embodiments, ring a is aryl. In some embodiments, ring a is heteroaryl. In some embodiments, ring a is a 6 membered aryl. In some embodiments, ring a is a 6 membered heteroaryl. In a preferred embodiment, ring a is phenyl.
In some embodiments, p is 0. In some embodiments, p is an integer from 1 to 4. In some embodiments, p is an integer from 1 to 3. In one embodiment, p is 1. In another embodiment, p is 2. In another embodiment, p is 3. In another embodiment, p is 4.
In some embodiments, each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; each R X is optionally substituted alkyl. In some embodiments, each R A is independently-OH, halogen, or optionally substituted alkyl. In some embodiments, each R A is independently halogen or optionally substituted alkyl. In some embodiments, each R A is independently-OH or optionally substituted alkyl. In some embodiments, each R A is independently optionally substituted alkyl. In some embodiments, each R A is independently substituted alkyl. In some embodiments, each R A is independently unsubstituted alkyl. In one embodiment, each R A is iodine. In another embodiment, each R A is methyl. In a specific embodiment, wherein p is 1 and R A is iodine. In another specific embodiment, wherein p is 2, each R A is methyl.
In some embodiments, Y is a bond, -O-, -NR-, or-n=. In some embodiments, Y is-O-, -NR-, or-n=. In some embodiments, Y is a bond, -O-, or-NR-. In some embodiments, Y is a bond, -O-, or-n=. In some embodiments, Y is a bond, -NR-, or-n=. In some embodiments, Y is-O-or-NR-. In some embodiments, Y is-O-or-n=. In some embodiments, Y is-NR-or-n=. In one embodiment, Y is a bond. In another embodiment, Y is-O-. In another embodiment, Y is-NR-. In another embodiment, Y is-n=.
In some embodiments, L is- (CH 2)p -. In other embodiments, L is-C (O) NH- (CH 2)p -. In some embodiments, p is an integer from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2. In one embodiment, p is 1 to 4. In another embodiment, p is 1 or 2. In another embodiment, p is 1 or 4. In a preferred embodiment, L is-CH 2 -. In another preferred embodiment, L is-C (O) NH- (CH 2)4 -.
In some embodiments, R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, R is hydrogen or substituted or unsubstituted alkyl. In a preferred embodiment, R is hydrogen. In another embodiment, R is a substituted or unsubstituted C 1-6 alkyl. In another embodiment, R is unsubstituted C 1-6 alkyl. In one embodiment, R is methyl. In another preferred embodiment, R is hydrogen or methyl.
In some embodiments, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl. In embodiments, R' is hydrogen. In another embodiment, R' is substituted or unsubstituted. In another embodiment, R' is a substituted or unsubstituted aryl.
In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is fluoro. In some embodiments, R 1 is iodine. In some embodiments, R 2 is hydrogen. In other embodiments, R 2 is methyl. In some embodiments, R 1 is hydrogen and R 2 is hydrogen. In some embodiments, R 1 is fluoro and R 2 is hydrogen. In some embodiments, R 1 is hydrogen and R 2 is methyl. In some embodiments, R 1 is iodine and R 2 is hydrogen.
Connector body
In some embodiments, the conjugates provided herein comprise more than one linker. In some embodiments, the targeting domains (e.g., the first targeting domain and the second targeting domain) are linked via a first linker. The first linker and the second linker may be any of the linkers described herein. In some embodiments, the payload is linked to one of the targeting domains (the first or second targeting domain) via a second linker. In a preferred embodiment, the first linker is a peptide linker. In another preferred embodiment, the second linker is formed by chemical conjugation. In some embodiments, the second linker is a bifunctional linker for chemical conjugation. In another preferred embodiment, the second linker is a non-peptide linker.
In some embodiments, useful functional reactive groups for conjugating or binding a targeting domain to a payload described herein include, for example, zero or higher order linkers. In some cases, the conjugate moiety comprises a functional reactive group that reacts with a linker described herein (optionally pre-attached to the payload, targeting domain, or other moiety of the conjugate). In some embodiments, the linker comprises a reactive group that reacts with a natural amino acid in a payload or targeting domain described herein. The targeting domain (e.g., first targeting domain and second targeting domain) or one of the targeting domains (e.g., first targeting domain or second targeting domain) and the payload can be conjugated together by reacting a nucleophilic reactive moiety on the first targeting domain with an electrophilic reactive moiety on the second targeting domain, or by reacting a nucleophilic reactive moiety on the targeting domain with an electrophilic reactive moiety on the payload. In alternative embodiments, the first targeting domain and the second targeting domain, or the targeting domain (e.g., the first targeting domain or the second targeting domain) and the payload are conjugated together by reacting an electrophilic reactive moiety on the first targeting domain with a nucleophilic moiety on the second targeting domain, or by reacting an electrophilic reactive moiety on the targeting domain with a nucleophilic moiety on the payload. In some embodiments, an amide bond may be formed when an amine on a targeting domain (e.g., epsilon-amine of a lysine residue) reacts with a carboxyl group on another targeting domain, or an amine on a targeting domain reacts with a carboxyl group on a payload. In alternative embodiments, the targeting domain and or payload is derivatized with a derivatizing agent prior to conjugation.
In some cases, the higher order linker includes a bifunctional linker, such as a homobifunctional linker or a heterobifunctional linker. Exemplary homobifunctional linkers include, but are not limited to, lomant reagent dithiobis (succinimidyl propionate) DSP, 3 '-dithiobis (sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfoDST), ethylene glycol bis (succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N' -disuccinimidyl carbonate (DSC), dimethyl hexadiimidate (DMA), dimethyl pimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3, 3 '-Dithiodipropimidate (DTBP), 1, 4-di-3' - (2 '-pyridyldithio) propanamide) butane (DPDPB), bismaleimide hexane (BMH), aryl halide containing compounds (DFDNB), such as 1, 5-difluoro- [ 2, 5-bis- [ 2, 4' -bis-fluoro-4-nitro-4, 34 ] butanediyl-bis- [ 1, 4-bis-fluoro-4, 34-bis- [ bis-nitro-4, 4-fluoro-4-bis- [ bis-4, 4-fluoro-4-bis-nitro-4, 34 ] butanediyl-bis-4-fluoro-1, 4-bis-4-fluoro-nitro-1, bis-4-bis-fluoro-2-bis-fluoro-butanediyl-2 Carbohydrazide, o-toluidine, 3 '-dimethylaniline, benzidine, α' -p-diaminobiphenyl, diiodo-p-xylene sulfonic acid, N '-ethylenebis (iodoacetamide), or N, N' -hexamethylenebis (iodoacetamide).
In some embodiments, the bifunctional linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine reactive and thiol crosslinkers such as N-succinimidyl 3- (2-pyridyldithio) propionate (sPDP), N-succinimidyl long chain 3- (2-pyridyldithio) propionate (LC-sPDP), N-succinimidyl water soluble long chain 3- (2-pyridyldithio) propionate (thio-LC-sPDP), succinimidyloxycarbonyl-alpha-methyl-alpha- (2-pyridyldithio) toluene (sMPT), sulfosuccinimidyl-6- [ alpha-methyl-alpha- (2-pyridyldithio) toluylamino ] hexanoate (sulfo-LC-sMPT), succinimide-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide esters (MBs), m-maleimidobenzoyl-N-hydroxysuccinimide esters (sulfo-MBs), N-succinimidyl (4-iodoacetyl) aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-sIAB), succinimidyl-4- (p-maleimidophenyl) butyrate (sMPB), Sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate (sulfo-sMPB), N- (gamma-maleimidobutyryloxy) succinimidyl ester (GMBs), N- (gamma-maleimidobutyryloxy) sulfosuccinimidyl ester (sulfo-GMBs), 6- ((iodoacetyl) amino) caproic acid succinimidyl ester (sIAX), 6- [6- (((iodoacetyl) amino) caproyl) amino ] caproic acid succinimidyl ester (sIAXX), 4- (((iodoacetyl) amino) methyl) cyclohexane-1-carboxylic acid succinimidyl ester (sIAC), Succinimidyl 6- ((((4-iodoacetyl) amino) methyl) cyclohexane-1-carbonyl) amino) hexanoate (sIACX), p-nitrophenylaspartic acid esters (NPIA), carbonyl-reactive and mercapto-reactive cross-linking agents, such as 4- (4-N-maleimidophenyl) butanoic acid hydrazide (MPBH), 4- (N-maleimidomethyl) cyclohexane-1-carboxy-hydrazide-8 (M 2C2 H), 3- (2-pyridyldithio) propionyl hydrazine (PDPH), Amine reactive and photoreactive crosslinking agents such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl- (4-azidosalicylamino) hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2- (ρ -azidosalicylamino) ethyl-1, 3' -dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6- (4 ' -azido-2 ' -nitrophenylamino) hexanoate (sANPAH), sulfosuccinimidyl-6- (4-azido-2 ' -nitrophenylamino) hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxy succinimide (ANB-NOs), sulfosuccinimidyl-2- (m-azido-o-nitrobenzoylamino) -ethyl-1, 3' -dithiopropionate (sAND), N-succinimidyl-4 (4-azidophenyl) -1,3' -dithiopropionate (sADP), N-Succinimidyl (4-azidophenyl) -1,3 '-dithiopropionate (sulfo-sADP), 4- (ρ -azidophenyl) butanoic acid sulfosuccinimidyl ester (sulfo-sAPP), 2- (7-azido-4-methylcoumarin-3-acetamide) ethyl-1, 3' -dithiopropionic acid sulfosuccinimidyl ester (sAED), 7-azido-4-methylcoumarin-3-acetic acid sulfosuccinimidyl ester (sulfo-sAMCA), p-nitrophenyl diazopyruvate (ρNPDP), p-nitrophenyl-2-diazo-3, 3-trifluoropropionate (PNP-DTP), Thiol-reactive and photoreactive crosslinkers such as 1- (ρ -azidosalicylamino) -4- (iodoacetamido) butane (AsIB), N- [4- (ρ -azidosalicylamino) butyl ] -3'- (2' -pyridyldithio) propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl-reactive and photoreactive crosslinkers such as ρ -azidobenzoyl hydrazine (ABH), carboxylate-reactive and photoreactive crosslinkers such as 4- (ρ -azidosalicylamino) butylamine (AsBA) and arginine-reactive and photoreactive crosslinkers such as ρ -azidophenyl glyoxal (APG).
In some cases, the reactive functional group includes a nucleophilic group that reacts with an electrophilic group present on the binding moiety (e.g., on the payload moiety or on the targeting domain). Exemplary electrophilic groups include carbonyl groups such as aldehydes, ketones, carboxylic acids, esters, amides, ketenes, acid halides, or anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, amino groups, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aromatic hydrazides. In some embodiments, the unnatural amino acid incorporated into the conjugates described herein comprises an electrophilic group.
In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a dipeptide linker. In some embodiments, the dipeptide linker is valine-citrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), and valine-lysine (Val-Lys). In some embodiments, the dipeptide linker is valine-citrulline. The linker may comprise a cleavable sequence or a sequence recognized by a protease.
In various embodiments, the targeting domain (e.g., the first targeting domain and the second targeting domain) comprises a polypeptide linker sequence. The linker may be at the C-terminus of the targeting domain. The linker may be expressed via recombinant techniques and may be encoded using the expression of the nucleic acid sequence along with the targeting domain. The linker can join the targeting domain to form a fusion protein. The presence of the linker in the conjugate allows the targeting domain to function properly without being spatially disturbed by other targeting domains. The linker may link the targeting domain to a tag, such as an expression tag or a purification tag. The connector may be a flexible connector. The flexibility of the linker may allow the targeting domain to adopt an independent conformation with minimal interference from other targeting domains. In some embodiments, the linker is a peptide linker comprising, for example, at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some cases, the peptide linker comprises up to 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50 or fewer amino acids. In other cases, the peptide linker comprises about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In other cases, the polypeptide linker comprises about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some cases, the polypeptide linker comprises (GGGGSGGGS) x (SEQ ID NO: 14), wherein x is 1-10. In some embodiments, the linker is a polypeptide linker of length 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 amino acids and longer. The linker may comprise SPSTPPTPSPSTPP and the polypeptide linker may be a repeat of (GGGGS) x, (GGGS) x. The linker may include glycine, serine, threonine or proline. In some embodiments, the N-terminus of one targeting domain is fused to the C-terminus of the linker polypeptide, and the N-terminus of the linker polypeptide is fused to the N-terminus of another targeting domain.
In various embodiments, the targeting domains (e.g., the first targeting domain and the second targeting domain) are linked or separated by a linker. The linker may be a polypeptide linker. The linker may be expressed via recombinant techniques and may be encoded using the expression of the nucleic acid sequence along with the targeting domain. The linker can join the targeting domain to form a fusion protein. The presence of the linker in the conjugate allows the targeting domain to function properly without being spatially disturbed by other targeting domains. The connector may be a flexible connector. The flexibility of the linker may allow the targeting domain to adopt an independent conformation with minimal interference from other targeting domains. In some embodiments, the linker is a peptide linker comprising, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some cases, the peptide linker comprises up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50 or fewer amino acids. In other cases, the peptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In other cases, the polypeptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some cases, the polypeptide linker comprises (GGGGSGGGS) x (SEQ ID NO: 14), wherein x is 1-10. In some embodiments, the linker is a polypeptide linker of length 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 amino acids and longer. The linker may comprise SPSTPPTPSPSTPP and the polypeptide linker may be a repeat of (GGGGS) x, (GGGS) x. The linker may include glycine, serine, threonine or proline. In some embodiments, the N-terminus of one targeting domain is fused to the C-terminus of the linker polypeptide, and the N-terminus of the linker polypeptide is fused to the N-terminus of another targeting domain.
In some embodiments, the linker comprises a self-cleaving (self-immolative) linker moiety. In some embodiments, the self-cleaving linker moiety comprises para-aminobenzyl alcohol (PAB), para-aminophenoxycarbonyl (PABC), or a derivative or analog thereof. In some embodiments, the linker comprises a dipeptide linker moiety and an self-cleaving linker moiety. In some embodiments, the self-cleaving linker moiety is as described in U.S. patent No. 9089614 and WIPO application No. WO 2015038426.
In some embodiments, the cleavable linker is a glucuronide. In some embodiments, the cleavable linker is an acid cleavable linker. In some embodiments, the acid cleavable linker is hydrazine. In some embodiments, the cleavable linker is a reducible linker.
In some embodiments, the linker comprises a maleimide group. In some cases, maleimide groups are also referred to as maleimide spacers. In some cases, the maleimide group also includes caproic acid forming a maleimidocaproyl (mc). In some cases, the linker includes a maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other cases, the maleimide groups include maleimide methyl groups such as succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-sMCC) described above.
In some embodiments, the maleimide group is a self-stabilizing maleimide. In some cases, self-stabilizing maleimides utilize diaminopropionic acid (DPR) to incorporate basic amino groups adjacent to the maleimide to provide intramolecular catalysis for thiosuccinimide ring hydrolysis, thereby eliminating the maleimide elimination reaction by reverse Michael reaction. In some cases, the self-stabilizing maleimide is a maleimide group as described by Lyon et al ,"Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,"Nat.Biotechnol.32(10):1059-1062(2014). In some cases, the linker comprises a self-stabilizing maleimide. In some cases, the linker is a self-stabilizing maleimide.
In some cases, the linker comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more of carbonyl or dicarbonyl groups, oxime groups, hydroxylamine groups, or protected forms thereof. The TLR agonist connector derivatives or targeting domains may be the same or different, e.g., there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different sites in the derivative comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different reactive groups.
As described herein, the present disclosure provides a targeting domain coupled to another molecule having the formula "targeting domain-L-payload", wherein L is a linking group or a chemical bond. In some embodiments, L is stable in vivo. In some embodiments, L is hydrolyzable in vivo. In some embodiments, L is metastable in vivo.
The targeting domain and the payload may be linked together by L using standard linkers and procedures known to those skilled in the art. In some aspects, the targeting domain is fused directly to the payload, and L is a bond. In other aspects, the targeting domain and the payload are fused via a linking group L. For example, in some embodiments, the targeting domain and the payload are linked together via a peptide bond, optionally linked together by a peptide or amino acid spacer. In some embodiments, the targeting domain and the payload are linked together by chemical conjugation, optionally by a linking group (L). In some embodiments, L is conjugated directly to each of the targeting domain and the payload.
Chemical conjugation may occur by reacting a nucleophilic reactive group of one compound with an electrophilic reactive group of another compound. In some embodiments, when L is a bond, the targeting domain is conjugated to the payload by reacting the nucleophilic reactive moiety on the targeting domain with the electrophilic reactive moiety on the linker, or by reacting the electrophilic reactive moiety on the targeting domain with the nucleophilic reactive moiety on the payload. In embodiments, when L is a group linking the targeting domain and the payload together, the targeting domain and/or the payload may be conjugated to L by reacting a nucleophilic reactive moiety on the targeting domain and/or the payload with an electrophilic reactive moiety on L, or by reacting an electrophilic reactive moiety on the targeting domain and/or the payload with a nucleophilic reactive moiety on L. Non-limiting examples of nucleophilic reactive groups include amino groups, thiol groups, and hydroxyl groups. Non-limiting examples of electrophilic reactive groups include carboxyl groups, acid chlorides, anhydrides, esters, succinimidyl esters, alkyl halides, sulfonates, maleimidyl groups, haloacetyl groups, and isocyanates. In embodiments where the targeting domain and the payload are conjugated together by reaction of the carboxylic acid with an amine, an activating agent may be used to form an activated ester of the carboxylic acid.
The activated ester of a carboxylic acid may be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), mesylate, triflate, carbodiimide or hexafluorophosphate. In some embodiments, the carbodiimide is 1, 3-Dicyclohexylcarbodiimide (DCC), 1' -Carbonyldiimidazole (CDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), or 1, 3-diisopropylcarbodiimide (dic). In some embodiments, the hexafluorophosphate is selected from the group consisting of hexafluorophosphate benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy-tripyrrolidine phosphonium hexafluorophosphate (PyBOP), 2- (1H-7-azabenzotriazol-1-yl) -1, 3-tetramethylurea Hexafluorophosphate (HATU) and o-benzotriazol-N, N' -tetramethylurea-Hexafluorophosphate (HBTU).
In some embodiments, the targeting domain (e.g., first targeting domain and second targeting domain) comprises a nucleophilic reactive group (e.g., amino, thiol, or hydroxyl of a side chain of lysine, cysteine, or serine) that is capable of conjugation to an electrophilic reactive group on the payload or L. In some embodiments, the targeting domain comprises an electrophilic reactive group (e.g., carboxylate group of the side chain of Asp or Glu) that is capable of conjugation to a nucleophilic reactive group on the payload or L. In some embodiments, the targeting domain is chemically modified to comprise a reactive group capable of direct conjugation to a payload or L. In some embodiments, the targeting domain is modified at the N-terminus or C-terminus to comprise a natural amino acid having a nucleophilic side chain. In exemplary embodiments, the N-terminal or C-terminal amino acid of the targeting domain is selected from lysine, ornithine, serine, cysteine and homocysteine. For example, the N-terminal or C-terminal amino acids of the targeting domain can be modified to include lysine residues. In some embodiments, the targeting domain is modified at the N-terminal or C-terminal amino acid to comprise a natural amino acid having an electrophilic side chain, such as, for example, asp and Glu. In some embodiments, the internal amino acids of the targeting domain are replaced with natural amino acids having nucleophilic side chains, as previously described herein. In exemplary embodiments, the internal amino acid of the targeting domain that is replaced is selected from lysine, ornithine, serine, cysteine, and homocysteine. For example, an internal amino acid of the targeting domain may be replaced with a lysine residue. In some embodiments, the internal amino acids of the targeting domain are replaced with natural amino acids having electrophilic side chains, such as, for example, asp and Glu.
In some embodiments, the payload comprises a reactive group capable of being conjugated directly to a targeting domain or L. In some embodiments, the payload comprises a nucleophilic reactive group (e.g., amine, thiol, hydroxyl) capable of conjugation to an electrophilic reactive group on the targeting domain or L. In some embodiments, the payload comprises an electrophilic reactive group (e.g., carboxyl, activated form of carboxyl, compound with leaving group) capable of conjugation to a nucleophilic reactive group on the targeting domain or L. In some embodiments, the payload is chemically modified to comprise a nucleophilic reactive group capable of conjugation to the electrophilic reactive group on the targeting domain or L. In some embodiments, the payload is chemically modified to comprise an electrophilic reactive group capable of conjugation to a nucleophilic reactive group on the targeting domain or L.
In some embodiments, conjugation can be performed by organosilanes, for example, aminosilanes treated with glutaraldehyde, carbonyl Diimidazole (CDI) activation of silanol groups, or with dendrimers. A variety of dendrimers are known in the art and include poly (amidoamine) (PAMAM) dendrimers, which are synthesized by divergent methods starting from ammonia or ethylenediamine initiator core reagents; a subset of PAMAM dendrimers based on a triaminoethyleneimine core; radially layered poly (amidoamine-silicone) dendrimers (PAMAMOS) that are reverse single-molecule micelles consisting of hydrophilic, nucleophilic Polyamidoamine (PAMAM) interior and hydrophobic silicone (OS) exterior; poly (propylene imine) (PPI) dendrimers, which are typically polyalkylamines with primary amine as terminal groups, with the dendrimer interior consisting of many tertiary tripropyleneamines; poly (acrylamide) (POPAM) dendrimers; diaminobutane (DAB) dendrimers; amphiphilic dendrimers; micelle dendritic macromolecules which are single molecule micelles of water-soluble hyperbranched polyphenylenes; polylysine dendrimers; and dendrimers based on a hyperbranched backbone of a dibenzyl ether.
In some embodiments, conjugation may be by olefin metathesis. In some embodiments, the payload and targeting domain, the payload and L, or both the targeting domain and L comprise an alkene or alkyne moiety capable of undergoing metathesis. In some embodiments, a suitable catalyst (e.g., copper, ruthenium) is used to accelerate the metathesis reaction. Suitable methods for conducting olefin metathesis reactions are described in the art. See, for example, SCHAFMEISTER et al, J.am.chem.Soc.122:5891-5892 (2000), walensky et al, science 305:1466-1470 (2004) and Blackwell et al, angew, chem., int.ed.37:3281-3284 (1998).
In some embodiments, conjugation may be performed using click chemistry. The "click reaction" is widely available, easy to implement, uses only readily available reagents, and is insensitive to oxygen and water. In some embodiments, the click reaction is a cycloaddition reaction between an alkynyl group and an azido group to form a triazolyl group. In some embodiments, the click reaction uses a copper or ruthenium catalyst. Suitable methods for performing click reactions are described in the art. See, e.g., kolb et al, drug Discovery Today 8:1128 (2003); kolb et al Angew.chem.int.ed.40:2004 (2001); rostovtsev et al, angew.chem.int.ed.41:2596 (2002); tornoe et al, J.org.chem.67:3057 (2002); manetsch et al, J.am.chem.Soc.126:12809 (2004); lewis et al, angew.chem.int.ed.41:1053 (2002); speers, J.am.chem.Soc.125:4686 (2003); chan et al org. Lett.6:2853 (2004); zhang et al, J.am.chem.Soc.127:15998 (2005); and Waser et al, J.am.chem.Soc.127:8294 (2005).
Indirect conjugation via high affinity specific binding partners (e.g. streptavidin/biotin or avidin/biotin or lectin/carbohydrate) is also contemplated.
Reactive residues for conjugation
In some embodiments, the targeting domain and/or payload is functionalized to include a nucleophilic reactive group or electrophilic reactive group with an organic derivatizing agent. Such derivatizing agents are capable of reacting with selected side chains or N-or C-terminal residues of the targeting amino acids on the targeting domain and the functional groups on the payload. Reactive groups on the targeting domain and/or the payload include, for example, aldehydes, amino, esters, thiols, a-haloacetyl, maleimide, or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimidyl ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, or other agents known in the art. Alternatively, the targeting domains and/or payloads may be indirectly interconnected by an intermediate carrier (such as a polysaccharide or polypeptide carrier). Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and other amino acids (e.g., serine) to impart the desired solubility on the resulting loaded carrier.
Cysteine residues are most commonly reacted with a-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cysteine residues are also derivatized by reaction with bromotrifluoroacetone, α -bromo- β - (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkyl maleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuric-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1, 3-diazole.
Histidine residues were derivatized by reaction with diethyl pyrocarbonate at a pH of 5.5-7.0, as this reagent was relatively specific for histidyl side chains. P-bromophenylacetyl bromide is also useful; the reaction is preferably carried out in 0.1M sodium dimethylarsinate at pH 6.0.
Lysyl and amino terminal residues are reacted with succinic anhydride or other carboxylic anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysyl residues. Other suitable reagents for derivatizing the α -amino group containing residue include imine esters such as methyl picolinate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methyliso urea, 2, 4-pentanedione, and transaminase catalyzed reactions with glyoxylate.
The arginyl residues are modified by reaction with one or more conventional reagents, including phenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires reaction under basic conditions due to the high pKa of the guanidine functionality. In addition, these reagents can react with lysine groups and arginine epsilon amino groups.
Specific modifications may be made to tyrosyl residues, in particular by introducing spectroscopic tags into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.
The pendant carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with a carbodiimide (R-n=c=n-R '), wherein R and R' are different alkyl groups such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4, 4-dimethylpentyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation (T.E.Creighton,Proteins:Structure and Molecular Properties,W.H.Freeman&Co.,San Francisco,pp.79-86(1983))、 of alpha-amino groups of lysine, arginine and histidine side chains, deamidation of asparagine or glutamine, acetylation of the N-terminal amine and/or amidation or esterification of the C-terminal carboxylic acid group.
Another type of covalent modification involves chemically or enzymatically coupling glycosides to the peptide. The saccharide may be attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free thiol group such as cysteine, (d) a free hydroxyl group such as serine, threonine or hydroxyproline hydroxyl group, (e) an aromatic residue such as tyrosine or tryptophan residue, or (f) an amide group of glutamine. These methods are described in WO1987/05330 and Aplin and Wriston CRC Crit. Rev. Biochem., pages 259-306 (1981).
In some embodiments, L is a bond. In these embodiments, the targeting domain and the payload are conjugated together by reacting a nucleophilic reactive moiety on the targeting domain with an electrophilic reactive moiety on the payload. In alternative embodiments, the targeting domain and the payload are conjugated together by reacting an electrophilic reactive moiety on the targeting domain with a nucleophilic moiety on the payload. In an exemplary embodiment, L is an amide bond formed when an amine on the targeting domain (e.g., epsilon-amine of a lysine residue) reacts with a carboxyl group on M. In alternative embodiments, the targeting domain and or payload is derivatized with a derivatizing agent prior to conjugation.
In some embodiments, L is a linking group. In some embodiments, L is a bifunctional linker and comprises only two reactive groups prior to conjugation to the targeting domain and the payload. In embodiments where both the targeting domain and the payload have electrophilic reactive groups, L comprises two identical or two different nucleophilic groups (e.g., amine, hydroxyl, thiol) prior to conjugation to the targeting domain and the payload. In embodiments where both the targeting domain and the payload have nucleophilic reactive groups, L comprises two identical or two different electrophilic groups (e.g., carboxyl, activated form of carboxyl, compound with leaving group) prior to conjugation to the targeting domain and the payload. In embodiments where one of the targeting domain or payload has a nucleophilic reactive group and the other of the targeting domain or payload has an electrophilic reactive group, L comprises one nucleophilic reactive group and one electrophilic group prior to conjugation to the targeting domain and payload.
L may be any molecule (prior to conjugation to the targeting domain and payload) having at least two reactive groups capable of reacting with each of the targeting domain and payload. In some embodiments, L has only two reactive groups and is difunctional. L (prior to conjugation to the peptide) may be represented by formula VI: A-L-B, wherein A and B are independently nucleophilic or electrophilic reactive groups. In some embodiments a and B are both nucleophilic groups or are both electrophilic groups. In some embodiments one of a or B is a nucleophilic group and the other of a or B is an electrophilic group.
In some embodiments, a and B may include alkene and/or alkyne functional groups suitable for alkene metathesis reactions. In some embodiments, a and B comprise moieties suitable for click chemistry (e.g., alkene, alkyne, nitrile, azide). Other non-limiting examples of reactive groups (a and B) include pyridyl dimercapto, aryl azide, diazide, carbodiimide and hydrazide.
In some embodiments, L is hydrophobic. Hydrophobic linkers are known in the art. See, e.g., bioconjugate Techniques, g.t. hermanson (ACADEMIC PRESS, san Diego, CA, 1996), which is incorporated by reference in its entirety. Suitable hydrophobic linking groups known in the art include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid. The hydrophobic linking group comprises at least two reactive groups (a and B) prior to conjugation to the peptide of the composition, as described herein and as follows: a- (hydrophobic linker) -B.
In some embodiments, the hydrophobic linking group includes a maleimide group or iodoacetyl group, and a carboxylic acid or activated carboxylic acid (e.g., NHS ester) as reactive groups. In these embodiments, the maleimide group or iodoacetyl group can be coupled to a thiol moiety on the targeting domain or payload, and the carboxylic acid or activated carboxylic acid can be coupled to an amine on the targeting domain or payload with or without the use of a coupling reagent. Any coupling agent known to those skilled in the art may be used to couple the carboxylic acid with the free amine, such as, for example DCC, DIC, HATU, HBTU, TBTU and other activators described herein. In particular embodiments, the hydrophilic linking group comprises an aliphatic chain of 2 to 100 methylene groups, wherein a and B are carboxyl groups or derivatives thereof (e.g., succinic acid). In other embodiments, L is iodoacetic acid.
In some cases, the hydrophilic linking group comprises at least two reactive groups (a and B) prior to conjugation to the peptide of the composition, as described herein and as follows: a- (hydrophilic linker) -B. In a specific embodiment, the linking group is polyethylene glycol (PEG). In certain embodiments, the molecular weight of the PEG is from about 100 daltons to about 10,000 daltons, such as from about 500 daltons to about 5000 daltons. In some embodiments, the molecular weight of the PEG is from about 10,000 daltons to about 40,000 daltons.
In some embodiments, the hydrophilic linking group includes a maleimide group or iodoacetyl group, and a carboxylic acid or activated carboxylic acid (e.g., NHS ester) as the reactive group. In these embodiments, the maleimide group or iodoacetyl group can be coupled to a sulfhydryl moiety on a targeting domain or payload, and the carboxylic acid or activated carboxylic acid can be coupled to an amine on the targeting domain or payload with or without the use of a coupling reagent. Any suitable coupling agent known to those skilled in the art may be used to couple the carboxylic acid with the amine, such as, for example, DCC, DIC, HATU, HBTU, TBTU and other activators described herein. In some embodiments, the linking group is maleimide-polymer (0.1-2.5 kDa) -COOH, iodoacetyl-polymer (0.1-2.5 kDa) -COOH, maleimide-polymer (0.1-2.5 kDa) -NHS, or iodoacetyl-polymer (0.1-2.50 kDa) -NHS.
In some embodiments, the linking group consists of an amino acid, dipeptide, tripeptide, or polypeptide, wherein the amino acid, dipeptide, tripeptide, or polypeptide comprises at least two activating groups as described herein. In some embodiments, the linking group (L) comprises a moiety selected from the group consisting of amino, ether, thioether, maleimide, disulfide, amide, ester, thioester, alkene, cyclic olefin, alkyne, triazole, carbamate, carbonate, cathepsin B cleavable, and hydrazone.
In some embodiments, L comprises an atomic chain of 1 to about 60, or 1 to 30 or more, 2 to 5, 2 to 10, 5 to 10, or 10 to 20 atoms long. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from C, O, N and S. In some cases, the chain atoms and linkers are selected according to their intended solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, L provides a functional group that can be cleaved by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell. In some embodiments, the length of L is sufficient to reduce the likelihood of steric hindrance.
In some embodiments, L is stable in biological fluids such as blood or blood components. In some embodiments, L is stable in serum for at least 5 minutes, e.g., less than 25%, 20%, 15%, 10% or 5% of the conjugate is cleaved when incubated in serum for a period of 5 minutes. In other embodiments, L is stable in serum for at least 10, or 20, or 25, or 30, or 60, or 90, or 120 minutes, or 3, 4,5, 6, 7, 8, 9, 10, 12, 15, 18, or 24 hours. In these embodiments, L does not contain a functional group capable of undergoing hydrolysis in vivo. In some exemplary embodiments, L is stable in serum for at least about 72 hours. Non-limiting examples of functional groups that are not capable of significant hydrolysis in vivo include amides, ethers, and thioethers.
In some embodiments, L is hydrolyzable in vivo. In these embodiments, L comprises a functional group capable of undergoing hydrolysis in vivo. Non-limiting examples of functional groups that are capable of undergoing hydrolysis in vivo include esters, anhydrides, and thioesters.
In some exemplary embodiments, L is unstable and undergoes substantial hydrolysis in plasma at 37 ℃ within 3 hours, and complete hydrolysis within 6 hours. In some exemplary embodiments, L is not unstable.
In some embodiments, L is metastable in vivo. In these embodiments, L comprises a functional group (e.g., an acid labile, reduction labile, or enzyme labile functional group) that is capable of being cleaved, either chemically or enzymatically, in vivo, optionally over a period of time. In these embodiments, L may include, for example, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety. While L is metastable, and not intending to be bound by any particular theory, the targeting domain-L-M conjugate is stable in the extracellular environment, e.g., stable in serum for the period of time described above, but unstable in the intracellular environment or conditions mimicking the intracellular environment, such that it will lyse upon entry into a cell. In some embodiments, when L is in a metastable state, L is stable in serum for at least about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or 48 hours, such as at least about 48, 54, 60, 66, or 72 hours, or about 24-48, 48-72, 24-60, 36-48, 36-72, or 48-72 hours.
In some cases, a suitable polymer backbone has the formula X-polymer-LY, wherein the polymer is polyethylene glycol, and X is a functional group that does not react with an azide group, and Y is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminooxy, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine, and vinylpyridine, and ketone. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, triflate (tresylate) and tosylate.
The linker may have a wide range of molecular weights or molecular lengths. A larger or smaller molecular weight linker may be used to provide the desired spatial relationship or conformation between the targeting domain and the attached entity or between the attached entity and its binding partner, if any. Linkers having longer or shorter molecular lengths may also be used to provide the desired space or flexibility between the targeting domain and the attached entity, or between the attached entity and its binding partner.
In some embodiments, the linker comprises a water-soluble bifunctional linker having a dumbbell structure comprising: a) An azide, alkyne, hydrazine, hydrazide, hydroxylamine, or carbonyl-containing moiety on at least a first end of the polymer backbone; and b) at least a second functional group on a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In some embodiments, the second functional group is not reactive with the first functional group. In some embodiments, the water-soluble compound comprises at least one arm of a branched molecular structure. For example, the branched molecular structure may be dendritic.
In exemplary embodiments, the polymer is linked to the targeting domain or modified targeting domain by a linker. For example, a linker may comprise one or two amino acids, one end of which is bound to a polymer, such as an albumin binding moiety, and the other end of which is bound to any available position on the polypeptide backbone. Additional exemplary linkers include hydrophilic linkers, such as chemical moieties containing at least 5 non-hydrogen atoms, where 30-50% of these are N or O.
Optionally, multiple targeting domains or modified targeting domain molecules can be linked by a linker polypeptide, wherein the linker polypeptide is optionally 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 amino acids in length, and longer, wherein optionally the N-terminus of one targeting domain is fused to the C-terminus of the linker polypeptide, and the N-terminus of the linker polypeptide is fused to the N-terminus of another targeting domain.
The terms "electrophile", and the like, as used herein, refer to an atom or group of atoms that can accept electron pairs to form a covalent bond. "electrophilic groups" as used herein include, but are not limited to, halides, carbonyls, and epoxide containing compounds. Common electrophiles include halides such as thiophosgene, dichlorophenone, phthaloyl chloride, succinyl chloride, chloroacetyl chloride, chlorosuccinyl chloride, and the like; ketones such as chloroacetone, bromoacetone, and the like; aldehydes such as glyoxal and the like; isocyanates such as hexamethylene diisocyanate, toluene diisocyanate, m-xylylene isocyanate, cyclohexylmethane-4, 4-diisocyanate, and derivatives of these compounds.
The terms "nucleophilic group", "nucleophile", and the like, as used herein, refer to an atom or group of atoms having an electron pair capable of forming a covalent bond. Groups of this type may be ionizable groups that react as anionic groups. "nucleophilic groups" as used herein include, but are not limited to, hydroxyl, primary amine, secondary amine, tertiary amine, and thiol.
In general, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, wherein the attacking nucleophile brings the carbon electrophile with electron pairs to form new bonds between the nucleophile and the carbon electrophile.
Non-limiting examples of carbon nucleophiles include, but are not limited to, alkyl, alkenyl, aryl, and alkynyl grignard reagents, organolithium, organozinc, alkyltin reagents, alkenyltin reagents, aryltin reagents, and alkynyl tin reagents (organotin), alkylborane reagents, alkenylborane reagents, arylborane reagents, and alkynyl borane reagents (organoboranes and organoborates); these carbon nucleophiles have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphoylide, enolic, and enolate reagents; these carbon nucleophiles have the advantage of being relatively easy to generate from precursors well known to those skilled in the art of synthetic organic chemistry. When a carbon nucleophile is used in combination with a carbon electrophile, a new carbon-carbon bond is created between the carbon nucleophile and the electrophile.
Non-limiting examples of non-carbon nucleophiles suitable for coupling to the carbon electrophile include, but are not limited to, primary and secondary amines, thiols, thiolates and sulfides, alcohols, alkoxides, azides, semicarbazides, and the like. When used in combination with a carbon electrophile, these non-carbon nucleophiles typically produce a heteroatom bond (C-X-C), where X is a heteroatom, including but not limited to oxygen, sulfur, or nitrogen.
In some cases, the polymers used herein terminate at one end with a hydroxyl or methoxy group, i.e., X is H or CH 3 ("methoxy PEG"). Alternatively, the polymer may terminate with reactive groups, thereby forming a difunctional polymer. Typical reactive groups may include those reactive groups commonly used to react with functional groups found in 20 common amino acids (including but not limited to maleimide groups, activated carbonates (including but not limited to p-nitrophenyl esters), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters) and aldehydes), and functional groups inert to 20 common amino acids but specifically reactive with complementary functional groups (including but not limited to azide groups, alkyne groups). Notably, the other end of the polymer (represented by Y in the formula above) will be attached to the targeting domain via an amino acid, either directly or indirectly. For example, Y is an amide, carbamate, or urea attached to an amine group of a polypeptide (including but not limited to the epsilon amine or N-terminus of lysine). Alternatively, Y is a maleimide linked to a thiol group (including but not limited to a thiol group of cysteine). Alternatively, the alkynyl groups on the polymer may react with azide groups present in the targeting domain to form a similar product. In some embodiments, strong nucleophiles (including but not limited to hydrazines, hydrazides, hydroxylamines, semicarbazides) can react with aldehydes or ketones present in the targeting domain to form hydrazones, oximes, or semicarbazides (as applicable), which in some cases can be further reduced by treatment with an appropriate reducing agent. Alternatively, the strong nucleophile may be incorporated into the targeting domain via an amino acid and used to preferentially react with ketone or aldehyde groups present in the water soluble polymer.
Any molecular mass of the polymer may be used according to practical needs, including but not limited to about 0.1 daltons (Da) to 2,500Da or higher. The molecular weight of the polymer may be a wide range including, but not limited to, between about 100Da and about 5000Da or greater. In some cases, the polymer is 50-5000Da, 50-3000Da, 50-2500Da, 100-2500Da, 250-5000Da, or 500-5000Da. Branched polymers, including but not limited to polymer molecules having a molecular weight of each strand ranging from 0.1 to 5kDa, 0.1 to 4kDa, 0.1 to 3kDa, 0.1 to 2.5kDa, 0.1 to 1.5 kDa.
The polymer may comprise azide-and acetylene-containing polymer derivatives comprising a water-soluble polymer backbone having an average molecular weight of about 800Da to about 100,000 Da. The polymer backbone of the water-soluble polymer may be poly (ethylene glycol). However, it should be understood that a wide variety of water-soluble polymers, including but not limited to poly (ethylene glycol) and other related polymers, including poly (dextran) and poly (propylene glycol), are also water-soluble, and the use of the term PEG or poly (ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, poly (ethylene glycol) in any form thereof, including difunctional PEG, multi-arm PEG, derivatized PEG, bifurcated PEG, branched PEG, pendent PEG (i.e., PEG or related polymer having one or more functional groups pendent to the polymer backbone), or PEG having a degradable linkage therein.
In addition to these forms of polymers, the polymers may also be prepared with weak or degradable linkages in the backbone. For example, the polymer may have ester linkages prepared in the polymer backbone that undergo hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into lower molecular weight fragments: -polymer-CO 2 -polymer- +h 2 O-polymer-CO 2 h+ho-polymer-.
The linkers may include polymers such as those comprising a water-soluble backbone. In some embodiments, the water-soluble polymer backbone comprises from 2 to about 300 termini. Examples of suitable polymers include, but are not limited to, other poly (alkylene glycols) such as poly (propylene glycol) ("PPG"), copolymers thereof (including, but not limited to, copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone may vary, it is typically in the range of about 800Da to about 100,000Da, typically in the range of about 6,000Da to about 80,000 Da. The molecular weight of each strand of the polymer backbone may be between about 100Da and about 100,000Da, including but not limited to 100,000Da、95,000Da、90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60,000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20,000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3,000Da、2,000Da、1,000Da、900Da、800Da、700Da、600Da、500Da、400Da、300Da、200Da and 100Da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 100Da and about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100Da and about 40,000 Da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 1,000da and about 40,000 da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 5,000da and about 40,000 da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 10,000da and about 40,000 da.
Suitable physiologically cleavable linkages include, but are not limited to, esters, carbonates, carbamates, sulfates, phosphates, acyloxyalkyl ethers, acetals and ketals. Such conjugates should have a physiologically cleavable bond that is stable upon storage and administration. For example, the polymer-linked or modified targeting domain should maintain its integrity when the final pharmaceutical composition is manufactured, when dissolved in an appropriate delivery vehicle (if used), and when administered by whatever route.
The invention also includes phosphate-based linkers with tunable stability as disclosed in US2017/0182181, incorporated herein by reference, for intracellular delivery of drug conjugates. The phosphate-based linker comprises a mono-, di-, tri-or tetraphosphoric acid group (phosphate group) covalently linked to the distal end of the linker arm, which comprises, in the distal to proximal direction, an adjustable element, an optional spacer element and a reactive functional group. The phosphate group of the phosphate-based linker can be conjugated to a payload, and the reactive functional group can be conjugated to a cell-specific targeting ligand (such as an antibody). The general structure of the phosphate-based linker is: phosphate group-adjustable element-optional spacer element-functional reactive group. The phosphate-based linker conjugated to the payload has the general structure: payload-phosphate group-adjustable element-optional spacer element-functional reactive group, and has the general structure when conjugated to a targeting ligand: payload-phosphate group-adjustable element-optional spacer element-targeting ligand. These phosphate-based linkers have differentiated and adjustable stability in blood and in the intracellular environment (e.g., lysosomal compartment). The rate at which phosphate groups cleave in the intracellular environment to release the payload in its natural or active form may be influenced by the structure of the adjustable element, further influences being mediated by the substitution of the phosphate groups and whether the phosphate groups are mono-, di-, tri-or tetraphosphoric acid. Furthermore, these phosphate-based linkers provide the ability to construct conjugates, such as antibody-drug conjugates, wherein the conjugates have a reduced propensity to form aggregates as compared to conjugates using non-phosphate-based linkers disclosed herein to conjugate the same payload to an antibody or targeting ligand.
In some cases, the targeting domain is linked to the payload via a water-soluble polymer via methods described herein. In some embodiments, the method comprises contacting an isolated targeting domain comprising a reactive amino acid side chain with a linker. In some cases, the conjugate is synthesized by reacting a functional group present on the targeting domain with a reactive group present on the linker. In some cases, the conjugate is synthesized by reacting a functional group present on the linker with a reactive group present on the payload. In some cases, the payload-linker moiety is conjugated to the targeting domain. In some cases, the targeting domain-linker moiety is conjugated to the payload. In some embodiments, the targeting domain is linked to a linker comprising a water soluble polymer.
In other embodiments, the targeting domain is conjugated to the payload via a linker. In some cases, the linker comprises a polymer. In some embodiments, the targeting domain is conjugated directly or indirectly to a linker, polymer, or biologically active molecule. In some embodiments, the linker is a cleavable or non-cleavable linker.
In some embodiments, the linker is 0.1kDa to 5kDa. In other embodiments, the linker is 0.1kDa to 2.5kDa. In other embodiments, the linker or polymer is a linear, branched, multimeric, or dendrimer. In another embodiment, the linker or polymer is a difunctional or multifunctional linker or difunctional or multifunctional polymer.
In other embodiments, the polymer is a water-soluble polymer. In other embodiments, the water-soluble polymer is polyethylene glycol (PEG). In some embodiments, the molecular weight of PEG is between 0.1kDa and 10kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 5kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 4 kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 3 kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 2 kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 2.5kDa. In some embodiments, the poly (ethylene glycol) molecule has a molecular weight of about 0.1kDa to about 10kDa. In some embodiments, the molecular weight of the poly (ethylene glycol) molecule is from 0.1kDa to 50kDa. In some embodiments, the molecular weight of the poly (ethylene glycol) is 0.1kDa to 2.5kDa, or 0.2 to 2.2kDa, or between 0.5kDa and 2 kDa. For example, in some cases, the molecular weight of the poly (ethylene glycol) polymer is about 0.5kDa, or about 1kDa, or about 2kDa, or about 2.5kDa. For example, in some cases, the molecular weight of the poly (ethylene glycol) polymer is 0.1kDa or 0.5kDa or 1kDa or 2.5kDa. In some embodiments, the poly (ethylene glycol) molecule is branched PEG. In some embodiments, the poly (ethylene glycol) molecule is branched 1K PEG. In some embodiments, the poly (ethylene glycol) molecule is branched 2.5K PEG. In some embodiments, the poly (ethylene glycol) molecule is branched 5K PEG. In some embodiments, the poly (ethylene glycol) molecule is linear PEG. In some embodiments, the poly (ethylene glycol) molecule is linear 2.5K PEG. In some embodiments, the poly (ethylene glycol) molecule is linear 10K PEG. In some embodiments, the poly (ethylene glycol) molecule is linear 2K PEG. In some embodiments, the poly (ethylene glycol) molecule is linear 0.5K PEG. In some embodiments, the molecular weight of the poly (ethylene glycol) polymer is the average molecular weight. In certain embodiments, the average molecular weight is a number average molecular weight (Mn). The average molecular weight may be determined or measured using GPC or SEC, SDS-PAGE analysis, RP-HPLC, mass spectrometry or capillary electrophoresis.
Therapeutic method
The conjugates described herein are useful for treating conditions and/or diseases. In some cases, the disease includes a proliferative disease. In some cases, the proliferative disease includes cancer. In some cases, the cancer comprises one or more tumors. In some cases, the cancer comprises a solid or liquid tumor. In some cases, the conjugate is administered to kill or inhibit the growth of rapidly dividing cells (such as tumor cells). In some cases, a method of treating a proliferative disease or condition in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a conjugate described herein. In some embodiments, the proliferative disease or condition is cancer. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the disease includes PCa (prostate cancer), CRPCa (castration-resistant prostate cancer), solid tumors (neovasculature), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal cancer), GC (gastric cancer), CRC (colorectal cancer), SCLC (small cell lung cancer), MPM (mesothelioma), PDAC (ductal pancreatic adenocarcinoma), ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), MSI-high tumors, melanoma, DLBCL (diffuse large B-cell lymphoma), endometrial cancer, cervical cancer, bladder cancer, brCa (breast cancer), TNBC (triple negative breast cancer), NE-PCa (neuroendocrine prostate cancer), GBM (glioblastoma), and RCC (renal cell carcinoma).
In some cases, the tumor cells targeted herein overexpress one or more targets. In some cases, the target comprises a surface marker or receptor. In some cases, the target is selected from one or more of PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some cases, the target is selected from two or more of PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some cases, when the conjugate binds to one or more targets on a tumor cell and the payload kills the tumor cell, inhibits or slows down the growth of the tumor cell, the conjugate will carry the payload to the tumor cell.
In some cases, the conjugates herein are administered for imaging a particular cell or cell population. In some cases, the cell or population of cells is a tumor cell, or is present in a tumor microenvironment. In some cases, when the conjugate binds to one or more targets on the tumor cell and the payload is an imaging agent (such as a visual dye or radiolabel), the conjugate will bring the payload to the tumor cell and the tumor cell is imaged due to the binding of the conjugate to the tumor cell.
The pharmaceutical composition is administered in a manner suitable for the disease to be treated (or prevented). The appropriate dosage, appropriate duration and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. Generally, the appropriate dosages and treatment regimens provide the compositions in amounts sufficient to provide a therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome), or to reduce the severity of the symptoms. The optimal dose is typically determined using experimental models and/or clinical trials. The optimal dose depends on the body mass, weight or blood volume of the patient.
In one embodiment, the injectable pharmaceutical compositions described herein are used to prepare a medicament for treating a disease or condition in a mammal that would benefit from administration of any one of the injectable pharmaceutical combinations of the disclosed conjugates. A method of treating any disease or condition described herein in a mammal in need of such treatment involves administering to the mammal a pharmaceutical composition comprising at least one compound described herein, or a pharmaceutically acceptable salt, active metabolite, prodrug, or pharmaceutically acceptable solvate thereof, in a therapeutically effective amount.
In certain embodiments, compositions containing the compounds described herein are administered for prophylactic and/or therapeutic treatment. In certain therapeutic applications, the composition is administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially alleviate at least one symptom of the disease or condition. The effective amount of such an administration depends on the severity and course of the disease or condition, previous treatments, the health condition of the patient, the weight and response to the drug, and the discretion of the attendant physician. The therapeutically effective amount is optionally determined by methods including, but not limited to, up-dosing and/or dose-range clinical trials.
In prophylactic applications, compositions containing the compounds described herein are administered to patients susceptible to or at risk of a particular disease, disorder or condition. Such an amount is defined as a "prophylactically effective amount or dose". In such use, the precise amount will also depend on the health, weight, etc. of the patient. When used in a patient, such an effective amount for use will depend on the severity and course of the disease, disorder or condition, previous treatments, the health of the patient and the response to the drug, and the discretion of the attending physician. In one aspect, prophylactic treatment comprises administering to a mammal a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, to prevent recurrence of symptoms of a disease or condition, wherein the mammal previously experienced at least one symptom of the disease being treated, and is currently in remission.
In certain embodiments, administration of the compound is for a prolonged period of time, i.e., for an extended period of time, including throughout the patient's life, to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.
Method of manufacture
Conjugates described herein can be synthesized using in vivo or in vitro methods or combinations of methods. In some cases, the method is an in vivo method. In some cases, the method is an in vitro method. In some cases, targeting domains comprising unnatural amino acids are synthesized in vivo and payloads are attached using in vitro chemistry.
In some cases, the method is an ex vivo method. In some cases, conjugates described herein comprising natural amino acid mutations or unnatural amino acid mutations are recombinantly produced or chemically synthesized. In some cases, the targeting domains described herein are recombinantly produced, e.g., by a host cell system or a cell-free system.
In general, methods for preparing target polypeptides comprising non-standard amino acids are known. In some cases, the aminoacyl tRNA synthetase/tRNA pair that is homologous to the unnatural amino acid is orthogonal to the cellular component of the cell in which it is used. The orthogonality (and thus suitability) of the exogenous aminoacyl tRNA synthetase/tRNA pair depends on the type of host organism. Four major orthogonal aminoacyl tRNA synthetases have been developed for genetic code expansion: methanococcus jannaschii (Methanococcus janaschii) tyrosyl-tRNA synthetase (MjTyrRS)/tRAN CUA pair, escherichia coli (ESCHERICHIA COLI) tyrosyl-tRNA synthetase (EcTyrRS)/tRAN CUA pair, escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNA CUA pair, and pyrrolysiyl-tRNA synthetase (PyleRS)/tRAN CUA pair from certain Methanocamphora (Methanosarcina). The PyleS/tRAN CUA pair is orthogonal in bacteria, eukaryotic cells and animals (see, e.g ,Chin,Jason W."Expanding and reprogramming the genetic code of cells and animals."Annual review of biochemistry 83(2014):379-408).
In some cases, an Unnatural Amino Acid (UAA) provided herein is incorporated using a pyrrolysinyl-tRNA synthetase (tRNA pyl). Unnatural Amino Acids (UAA) are introduced into transfer RNA molecules (tRNA) to make them available for translation. However, the attachment of the unnatural amino acid to the tRNA is not necessarily accomplished by a naturally occurring aminoacyl-tRNA synthetase. Thus, an engineered aminoacyl tRNA synthetase, such as engineered tRNA pyl prepared and selected from a library of generated tRNA pyl mutants, can be used to attach a desired UAA to a tRNA, so that the desired UAA can be incorporated by mutagenesis.
In some embodiments, an engineered mutant tRNA pyl variant capable of attaching such UAA can be used to attach a UAA provided herein (e.g., a UAA of formula (IA)) to a tRNA. In some embodiments, the mutant tRNA pyl variant introduces an amino acid mutation (i.e., incorporates UAA). In other embodiments, the mutant tRNA pyl variant introduces multiple amino acid mutations. In some embodiments, one of skill in the art prepares and screens libraries of tRNA pyl variants to select the appropriate tRNA pyl variants for attachment to the desired UAA. In some embodiments, the variant of tRNA pyl for introducing UAA in the conjugates provided herein is a variant of tRNA pyl described in US 8,735,093, US 9,133,449, WO2020206341, each of which is incorporated herein by reference.
In various embodiments, a mutant pyrrolysiyl-tRNA synthetase can be used. The mutant pyrrolysiyl-tRNA synthetase is derived from Methanopyrrococcus pasteurii (Methanosarcina barkeri), methanopyrrococcus equi (Methanosarcina mazei), methanopyrrococcus enteromethane (Methanosarcina alvus), or Methanopyrrococcus jannaschii (Methanosarcina jannaschii), or a variant or chimeric thereof.
In some embodiments, a mutant pyrrollysyl-tRNA synthetase provided herein comprises at least 5 amino acid residue substitutions within the substrate binding site of the mutant pyrrollysyl-tRNA synthetase. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase comprises at least 5 amino acid residue substitutions in the amino acid sequence of SEQ ID NO. 90. In some embodiments, the substrate binding site comprises alanine at position 302, leucine at position 305, tyrosine at position 306, leucine at position 309, isoleucine at position 322, asparagine at position 346, cysteine at position 348, tyrosine at position 384, valine at position 401, and tryptophan residue 417, as set forth in the amino acid sequence of SEQ ID NO. 90. In some embodiments, the at least 5 amino acid residue substitutions are selected from the group consisting of an alanine substitution at position 302, an asparagine substitution at position 346, a cysteine substitution at position 348, a tyrosine substitution at position 384, and a tryptophan substitution at position 417 set forth in the amino acid sequence of SEQ ID NO. 90. In some embodiments, at least 5 amino acid residues are replaced with isoleucine at alanine 302, threonine at asparagine 346, isoleucine at cysteine 348, leucine at tyrosine 384 and lysine at tryptophan 417 set forth in the amino acid sequence of SEQ ID NO. 90.
In some embodiments, a mutant pyrrolysiyl-tRNA synthetase provided herein has the amino acid sequence of SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase comprises the amino acid sequence of SEQ ID NO. 84. In some embodiments, the mutant pyrrollysyl-tRNA synthetase has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 80% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 85% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 90% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 91% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 92% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 93% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 94% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 95% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 96% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 97% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 98% identical to SEQ ID NO. 84. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 99% identical to SEQ ID NO. 84.
In some embodiments, a mutant pyrrolysiyl-tRNA synthetase provided herein has the amino acid sequence of SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase comprises the amino acid sequence of SEQ ID NO. 87. In some embodiments, the mutant pyrrollysyl-tRNA synthetase has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 80% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 85% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 90% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 91% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 92% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 93% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 94% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 95% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 96% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 97% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 98% identical to SEQ ID NO. 87. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 99% identical to SEQ ID NO. 87.
In some embodiments, a mutant pyrrolysiyl-tRNA synthetase provided herein is encoded by the nucleic acid sequence of SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that comprises the sequence of SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 80% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 85% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 90% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 91% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 92% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 93% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 94% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 95% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 96% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 97% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 98% identity to SEQ ID NO. 85. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 99% identity to SEQ ID NO. 85.
In some embodiments, a mutant pyrrolysiyl-tRNA synthetase provided herein is encoded by the nucleic acid sequence of SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that comprises the sequence of SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 80% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 85% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 90% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 91% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 92% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 93% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 94% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 95% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 96% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 97% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 98% identity to SEQ ID NO. 88. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 99% identity to SEQ ID NO. 88.
In some embodiments, a mutant pyrrolysiyl-tRNA synthetase provided herein has the amino acid sequence of SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase comprises the amino acid sequence of SEQ ID NO. 92. In some embodiments, the mutant pyrrollysyl-tRNA synthetase has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 80% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that has at least 85% identity to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 90% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 91% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 92% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 93% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 94% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 95% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that has at least 96% identity to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 97% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 98% identical to SEQ ID NO. 92. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase has an amino acid sequence that is at least 99% identical to SEQ ID NO. 87.
In some embodiments, a mutant pyrrolysiyl-tRNA synthetase provided herein is encoded by the nucleic acid sequence of SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that comprises the sequence of SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 80% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 85% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 90% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 91% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 92% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 93% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 94% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 95% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 96% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 97% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 98% identity to SEQ ID NO. 91. In some embodiments, the mutant pyrrolysiyl-tRNA synthetase is encoded by a nucleic acid sequence that has at least 99% identity to SEQ ID NO. 91.
In some cases, the sequences associated with the mutant pyrrolysiyl-tRNA synthetases herein are in table 1B.
TABLE 1B
In some cases, the targeting domain is recombinantly produced by the host cell system. In some cases, the host cell is a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell, or a plant cell) or a prokaryotic cell (e.g., a gram-positive or gram-negative bacterium). In some cases, the eukaryotic host cell is a mammalian host cell. In some cases, the mammalian host cell is a stable cell line, or a cell line that incorporates the genetic material of interest into its own genome and is capable of expressing the product of that genetic material after multiple cell divisions. In other cases, the mammalian host cell is a transient cell line, or a cell line that incorporates the genetic material of interest into its own genome and has no ability to express the genetic material product after multiple cell divisions.
Exemplary mammalian host cells include 293T cell lines, 293A cell lines, 293FT cell lines, 293F cells, 293H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, expi293F cell TM cells, flp-In TMT-RExTM cell lines, Flp-In TM -293 cell line, flp-In TM -3T3 cell line, flp-In TM BHK cell line, flp-In TM -CHO cell line, Flp-In TM -CV-1 cell line, flp-In TM The Jurkat cell line, freeStyle TM 293-F cell, freeStyle TM CHO-S cell, GripTite TM 293MSR cell line, GS-CHO cell line, heparg TM cell, T-REx TM Jurkat cell line, per.C6 cell, T-REx TM -293 cell line, T-REx TM -CHO cell line and T-REx TM HeLa cell line.
In some embodiments, the eukaryotic host cell is an insect host cell. Exemplary insect host cells include Drosophila (Drosophila) S2 cells, sf9 cells, sf21 cells, and Cellular High Five TM cells.
In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris strains such as GS115, KM71H, SMD1168H and X-33, and saccharomyces cerevisiae (Saccharomyces cerevisiae) strains such as INVSCl.
In some embodiments, the eukaryotic host cell is a plant host cell. In some cases, the plant cells include cells from algae. Exemplary plant cell lines include strains from Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) 137c or Synechococcus elongatus (Synechococcus elongatus) PPC 7942.
In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21、BL21(DE3)、Mach1TM、DH10BTM、TOP10、DH5α、DH10BacTM、OmniMaxTM、MegaXTM、DH12STM、INV110、TOP10F'、INVαF、TOP10/P3、ccdB survival, PIR1, PIR2, stbl2 TM、Stbl3TM, or Stbl4 TM.
In some cases, suitable nucleic acid molecules or vectors for producing the targeting domains described herein include any suitable vector derived from eukaryotic or prokaryotic sources. Exemplary nucleic acid molecules or vectors include vectors from bacterial (e.g., E.coli), insect, yeast (e.g., pichia), algal, or mammalian sources. Bacterial vectors include, for example, pACYC177, pASK75, pBAD series vectors, pBADM series vectors, pET series vectors, pETM series vectors, pGEX series vectors, pHAT2, pMal-C2, pMal-p2, pQE series vectors, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, PFLAG SHIFT-12C, pTAC-MAT-1, pFLAG CTC or pTAC-MAT-2.
Insect vectors include, for example, ,pFastBac1、pFastBac DUAL、pFastBac ET、pFastBac HTa、pFastBac HTb、pFastBac HTc、pFastBac M30a、pFastBac M30b、pFastBac、M30c、pVL1392、pVL1393M 10、pVL1393M11、pVL1393M 12、FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2 or MAT vectors such as pPolh-MAT1 or pPolh-MAT 2.
Yeast vectors include, for example, a pDOST TM vector, a pDOST TM vector, a pDOST TM vector, a pDOST TM vector, a pYES-DEST52 vector, a pBAD-DEST49Target vector, a pAO815 Pichia vector, a pFLD Pichia vector, a pGAPZA, a Pichia C vector, a Pichia pPICC 3.5K vector, a pPIC 6A, B and Pichia C vector, a pPIC9K vector, a pTEF1/Zeo, a pYES2 yeast vector, a pYES2/CT yeast vector, pYES2/NT A, B and C yeast parent or a pYES3/CT yeast vector.
Algal vectors include, for example, pChlamy-4 vectors or MCS vectors.
Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8、pFLAG-Myc-CMV19、pFLAG-Myc-CMV 23、pFLAG-CMV 2、pFLAG-CMV 6a、b、c、pFLAG-CMV 5.1、pFLAG-CMV 5a、b、c、p3xFLAG-CMV 7.1、pF-CMV 20、p3xFLAG-Myc-CMV 24、pCMV-FLAG-MAT1、pCMV-FLAG-MAT2、pBICEP-CMV 3 or pBICCMV-4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3、p3xFLAG-CMV 9、p3xFLAG-CMV 13、pFLAG-Myc-CMV 21、p3xFLAG-Myc-CMV 25、pFLAG-CMV 4、p3xFLAG-CMV 10、p3xFLAG-CMV14、pFLAG-Myc-CMV 22、p3xFLAG-Myc-CMV 26、pBICEP-CMV 1 or pBICEP-CMV 2. In some cases, the targeting domains described herein are generated using a cell-free system. In some cases, the cell-free system includes a mixture of cytoplasmic and/or nuclear components from the cell and is suitable for in vitro nucleic acid synthesis. In some cases, the cell-free system utilizes a prokaryotic cell component. In other cases, the cell-free system utilizes eukaryotic cell components. Nucleic acid synthesis is achieved in cell-free systems based on, for example, drosophila cells, xenopus (Xenopus) eggs, archaea or HeLa cells. Exemplary cell-free systems include E.coli S30 extract system, E.coli T7S 30 system, or XpressCF and XpressCF +.
Cell-free translation systems variously include components such as plasmids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, molecular chaperones, translation initiation and elongation factors, natural and/or unnatural amino acids, and/or other components for protein expression. These components are optionally modified to increase yield, increase synthesis rate, increase fidelity of the protein product, or incorporate unnatural amino acids. In some embodiments, the targeting domains described herein that contain unnatural amino acids are synthesized using the cell-free translation systems described in US 8,778,631, US2017/0283469, US2018/0051065, US 2014/0315245, or US 8,778,631. In some embodiments, the cell-free translation system includes modified release factors, or even one or more release factors are removed from the system. In some embodiments, the cell-free translation system comprises a reduced protease concentration. In some embodiments, the cell-free translation system includes a modified tRNA with a reassigned codon that encodes an unnatural amino acid. In some embodiments, the synthetases described herein for incorporating unnatural amino acids are used in cell-free translation systems. In some embodiments, the tRNA is preloaded with the unnatural amino acid using an enzymatic or chemical process prior to adding the tRNA to the cell-free translation system. In some embodiments, the components for the cell-free translation system are obtained from a modified organism, such as a modified bacterium, yeast, or other organism.
In some embodiments, the targeting domain is produced in a circular arrangement via an expression host system or by a cell-free system.
The orthogonal or amplified genetic code can be used to generate a targeting domain described herein, wherein one or more specific codons present in a nucleic acid sequence of the targeting domain are assigned to encode an unnatural amino acid, such that it can be genetically incorporated into a conjugate (e.g., a targeting domain) by using an orthogonal tRNA synthetase/tRNA pair. The orthogonal tRNA synthetase/tRNA pair is capable of charging the tRNA with an unnatural amino acid and is capable of incorporating the unnatural amino acid into a polypeptide chain in response to a codon.
In some cases, the codon is an amber codon, an ocher codon, an opal codon, or a quadruple codon. In some cases, the codon corresponds to an orthogonal tRNA that will be used to carry an unnatural amino acid. In some cases, the codon is an amber codon. In other cases, the codons are orthogonal codons.
In some cases, the codon is a four-way codon, which can be decoded by an orthogonal ribosomal ribose-Q1. In some cases, the quadruple codons are as described in Neumann et al ,"Encoding multiple unnatural amino acid analysis of a quadruplet-decoding ribosome,"Nature,464(7287):441-444(2010).
In some cases, codons used in the disclosure are recoded codons, e.g., synonymous codons or rare codons replaced by replacement codons. In some cases, the recoded codons are as described in Napolitano et al ,"Emergent rules for codon choice isolated by editing of a ray array code in Escherichia coli,"PNAS,113(38):E5588-5597(2016). In some cases, the recoded codons are as described in Ostrov et al, "Design, synthesis, AND TESTING TRANSLATED A-code gene," Science 353 (6301): 819.sub.822 (2016).
Definition of the definition
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that have been modified later, for example, hydroxyproline, g-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a natural amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as the natural amino acid. Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a natural amino acid.
The terms "non-naturally occurring amino acids" and "non-natural amino acids" refer to amino acid analogs, synthetic amino acids, and amino acid mimics that are not found in nature, e.g., amino acid residues comprising aryl amides, vinyl sulfonamides, sulfonyl fluorides, aryl sulfonyl fluorides, and 4-Sulfotetrafluorophenyl (STP) esters.
Amino acids may be referred to herein by their common three letter symbols or by the single letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee. Likewise, nucleotides may also be referred to by their commonly accepted single-letter codes.
The term "amino acid side chain" refers to a functional substituent contained on an amino acid. For example, the amino acid side chain may be a side chain of a naturally occurring amino acid. Naturally occurring amino acids are those encoded by the genetic code (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine), and those that are later modified, such as hydroxyproline, g-carboxyglutamic acid, and O-phosphoserine. In some aspects, the amino acid side chain may be an unnatural amino acid side chain. In some aspects, the amino acid side chain is H, In some embodiments, the unnatural amino acid side chain isIn some embodiments, the unnatural amino acid side chain is
The term "unnatural amino acid side chain" or "xaa" refers to a functional replacement for a compound having the same basic chemical structure as the natural amino acid, i.e., a carbon bound to hydrogen, carboxyl, amino, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium, allylalanine, 2-amino isobutyric acid. Unnatural amino acids are naturally occurring or chemically synthesized non-protein amino acids. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as the natural amino acid. Non-limiting examples include exo-cis-3-aminobicyclo [2.2.1] hept-5-ene-2-hydrochloride, cis-2-aminocycloheptane hydrochloride, cis-6-amino-3-cyclohexene-1-hydrochloride, cis-2-amino-2-methylcyclohexane carboxylate, cis-2-amino-2-methylcyclopentane hydrochloride, 2- (Boc aminomethyl), benzoic acid, 2- (Boc amino) suberic acid, boc-4, 5-dehydro-Leu-OH (dicyclohexylammonium salt), boc-4- (Fmoc-amino) -L-phenylalanine, boc-P-Homopyr-OH, Boc- (2-indenyl) -Gly-OH, 4-Boc-3-morpholinoacetic acid, boc pentafluoro-D-phenylalanine, boc-pentafluoro-L-phenylalanine 、Boc-Phe(2-Br)-OH、Boc-Phe(4-Br)-OH、Boc-D-Phe(4-Br)-OH、Boc-D-Phe(3-Cl)-OH、Boc-Phe(4-NH2)-OH、Boc-Phe(3-NH2)-OH、Boc-Phe(3,5-F2)-OH、2-(4-Boc- -piperazinyl) -2- (3, 4-dimethoxyphenyl) acetic acid purum, 2- (4-Boc-piperazinyl) -2- (2-fluorophenyl) acetic acid purum, 2- (4-Boc-piperazinyl) -2- (3-fluorophenyl) acetic acid purum, 2- (4-Boc-piperazinyl) -2- (4-fluorophenyl) acetic acid purum, 2- (4-Boc-piperazinyl) -2- (4-methoxyphenyl) acetic acid purum, 2- (4-Boc-piperazinyl) -2-phenylacetic acid purum, 2- (4-Boc-piperazinyl) -2- (3-pyridinyl) acetic acid purum, 2- (4-Boc-piperazinyl-2- [4- (trifluoromethyl) phenyl ] acetic acid purum, 2-P- (2-quinolinyl) -Ala-OH, NBoc-1,2,3, 6-tetrahydro-2-pyridinecarboxylic acid, boc-P- (4-thiazolyl) -Ala-OH, bo-b (2-thienyl) -D-Ala-OH, fmoc-N- (4-Boc-aminobutyl) -Gly-OH, fmoc-N- (2-Boc-aminoethyl) -Gly-OH, fmoc-N- (2, 4-dimethoxybenzyl) -Gly-OH, fmoc- (2-diindenyl) -Gly-OH, fmoc-pentafluoro-L-phenylalanine, fmoc-Pen (Trt) -OH, fmoc-Phe (2-Br) -OH, fmoc-Phe (4-Br) -OH, fmoc-Phe (3, 5-F2) -OH, Fmoc-P- (4-thiazolyl) -Ala-OH, fmoc-P- (2-thienyl) -Ala-OH, 4- (hydroxymethyl) -D-phenylalanine. In some embodiments, the unnatural amino acid comprises formula I: is a structure of (a). In some embodiments, the unnatural amino acid comprises formula II: is a structure of (a). In some embodiments, the unnatural amino acid is 2-amino-3- (4- (fluorosulfonyl) oxy) phenyl) propanoic acid: in some embodiments, the unnatural amino acid is fluorosulfonyl tyrosine (FSY): In some embodiments, the unnatural amino acid is N6- (4- ((fluorosulfonyl) oxy) benzoyl) lysine: In some embodiments, the unnatural amino acid is fluorosulfonyloxy benzoyl-L-lysine (FSK):
"conservatively modified variants" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, "conservatively modified variants" refers to those nucleic acids which encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, many nucleic acid sequences will encode any given protein. For example, both codons GCA, GCC, GCG and GCU encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each said sequence.
As regards amino acid sequences, one of skill in the art will recognize that a single substitution, deletion, or addition of a nucleic acid, peptide, polypeptide, or protein sequence that alters, adds, or deletes a single amino acid or a small percentage of amino acids in the coding sequence is a "conservatively modified variant" where the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are complements of the polymorphic variants, interspecies homologs, and alleles of the disclosure, and do not exclude them.
The following eight groups each contain amino acids that are conservative substitutions for one another: (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N) and glutamine (Q); (4) arginine (R), lysine (K); (5) Isoleucine (I), leucine (L), methionine (M), valine (V); (6) Phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); and (8) cysteine (C), methionine (M). (see, e.g., cright on, proteins (1984)).
In some embodiments, the pyrrolysin-tRNA synthetase (tRNA pyl) described herein is an aminoacyl-tRNA synthetase that catalyzes the reaction required to attach the α -amino acid pyrrolysine or similar unnatural amino acid to a cognate tRNA, thereby allowing incorporation of pyrrolysine or similar unnatural amino acid during the proteinogenic process of the amber stop codon (i.e., UAG). Wild-type tRNA pyl from Methanocaulis species (which naturally incorporates pyrrolysine) is orthogonal to endogenous tRNA and aminoacyl tRNA synthetases in E.coli and eukaryotic cells. Using this pair and its synthetically evolved derivatives or variants, we and others directed the efficient incorporation of unnatural amino acids, including post-translationally modified amino acids, chemical handles, and optically controlled (photocaged) amino acids, at specific sites in E.coli, yeast, and mammalian cells for the desired protein.
In some embodiments, a tRNA pyl described herein includes any recombinant or naturally occurring form of a pyrrolysiyl-tRNA synthetase, or variant, homologue, or isoform thereof, that retains tRNA pyl activity (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the activity range compared to wild-type tRNA pyl). In some embodiments, the variant, homologue, or isoform has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150, or 200 consecutive amino acid portions) as compared to the naturally occurring pyrrolysiyl-tRNA synthetase. In some embodiments, the mutant tRNA pyl catalyzes the attachment of an Unnatural Amino Acid (UAA) (e.g., UAA of formula (IA), such as fluorosulfate L-tyrosine (FSY)) to tRNA pyl, in order to incorporate an unnatural amino acid.
In some embodiments, the tRNA pyl provided herein is a tRNA pyl derivative or variant that can be engineered by one of skill in the art. In some embodiments, tRNA's pyl provided herein are single stranded RNA molecules containing about 70 to 90 nucleotides that fold via intra-strand base pairing to form a characteristic clover structure that carries a particular amino acid (e.g., UAA of formula (IA), such as FSY) and matches it to a corresponding codon on the mRNA during protein synthesis.
An "imaging ligand" or "detectable agent" is a composition that is detectable by suitable means, such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging or other physical means. For example, useful detectable reagents include 3H、14C、18F、33P、35S、45Ti、47Sc、52Fe、59Fe、62Cu、64Cu、67Cu、67Ga、68Ga、77As、86Y、90Y、89Sr、89Zr、94Tc、94Tc、99mTc、99Mo、105Pd、105Rh、111Ag、111In、112In、123I、124I、125I、131I、142Pr、143Pr、149Pm、153Sm、154-158Gd、161Tb、166Dy、166HO、169Er、175Lu、177Lu、186Re、188Re、189Re、194Ir、198Au、199Au、211At、211Pb、212Bi、212Pb、213Bi、223Ra、225Ac、153Sm、177Lu、86Y、88Y、90Y、123I、124I、125I、131I、149Tb、212Pb/212Bi and 227Th、Cr、V、Mn、Fe、Co、Ni、Cu、La、Ce、Pr、Nd、Pm、Sm、Eu、Gd、Tb、Dy、Ho、Er、Tm、Yb、Lu、32 P, fluorophores (e.g., fluorescent dyes), electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxin, paramagnetic molecules, paramagnetic nanoparticles, ultra-small superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, monocrystalline iron oxide nanoparticles, monocrystalline iron oxide, nanoparticle contrast agents, liposomes, or other delivery vehicles containing gadolinium chelate ("Gd chelate") molecules, gadolinium, radioisotopes, radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15, fluoro-18, rubidium-82), fluorodeoxyglucose (e.g., fluorine-18 labeled), any gamma-emitting radionuclide, positron emitting radionuclide, radiolabelled glucose, radiolabelled water, radiolabelled ammonia, biocolloids, microbubbles (e.g., including microbubbles containing albumin, galactose, lipids, and/or polymers); microbubble gas cores comprising air, heavy gas, perfluorocarbon, nitrogen, octafluoropropane, perfluorohexane lipid microspheres, perfluoropropane, etc.), iodinated contrast agents (e.g., iohexol, iodixanol, ioversol, ioisophthalol, ioxilan, iopromide, diatrizoate, mediatrizoic acid, and iodic acid), barium sulfate, thorium dioxide, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins or other detectable entities, they are prepared, for example, by incorporating a radiolabel into a peptide or antibody that specifically reacts with the target peptide. The detectable moiety is a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.
According to embodiments of the present disclosure, radioactive materials (e.g., radioisotopes) that may be used as imaging and/or labeling agents include, but are not limited to 3H、14C、18F、33P、35S、45Ti、47Sc、52Fe、59Fe、62Cu、64Cu、67Cu、67Ga、68Ga、77As、86Y、90Y、89Sr、89Zr、94Tc、94Tc、99mTc、99Mo、105Pd、105Rh、111Ag、111In、112In、123I、124I、125I、131I、142Pr、143Pr、149Pm、153Sm、154-158Gd、161Tb、166Dy、166HO、169Er、175Lu、177Lu、186Re、188Re、189Re、194Ir、198Au、199Au、211At、211Pb、212Bi、212Pb、213Bi、223Ra、225Ac、153Sm、177Lu、86Y、88Y、90Y、123I、124I、125I、131I、149Tb、212Pb/212Bi and 227 Th. Paramagnetic ions that may be used as additional imaging agents according to embodiments of the present disclosure include, but are not limited to, transition metal and lanthanide metal ions (e.g., metals having atomic numbers 21-29, 42, 43, 44, or 57-71). These metals include Cr, V, mn, fe, co, ni, cu, la, ce, pr, nd, pm, sm, eu, gd, tb, dy, ho, er, tm, yb and Lu ions.
The term "bond" or "linker" as used herein refers to a bond or chemical moiety formed by a chemical reaction between a functional group of a linker and another molecule. Such linkages may include, but are not limited to, covalent and non-covalent linkages, and such chemical moieties may include, but are not limited to, ester, carbonate, phosphoramidate, hydrazone, acetal, orthoester, peptide linkage, and oligonucleotide linkage. Hydrolytically stable bonds means that these bonds are substantially stable in water and do not react with water at useful pH values, including but not limited to extended periods of time under physiological conditions, possibly even indefinitely. Hydrolytically unstable or degradable linkages means that these linkages are degradable in water or in aqueous solutions (including, for example, blood). An enzymatically labile or degradable bond means that the bond can be degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in the linking group between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed from the reaction of PEG carboxylic acid or activated PEG carboxylic acid with alcohol groups on the bioactive agent, wherein these ester groups are typically hydrolyzed under physiological conditions to release the bioactive agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine bonds resulting from the reaction of an amine and an aldehyde; a phosphate bond formed by reacting an alcohol with a phosphate group; hydrazone bond, which is the reaction product of hydrazide and aldehyde; an acetal bond, which is the reaction product of an aldehyde and an alcohol; orthoester linkages, which are the reaction product of a formate and an alcohol; peptide bonds formed from amine groups, including but not limited to at the end of a polymer such as PEG and the carboxyl group of a peptide; and oligonucleotide linkages formed from phosphoramidite groups, including, but not limited to, phosphoramidite groups at the polymer terminus and 5' hydroxyl groups of the oligonucleotide. Linkers include, but are not limited to, short linear, branched, multi-arm, or dendrimers, such as polymers. In some embodiments, the linker may be branched. In other embodiments, the linker may be a bifunctional linker. In some embodiments, the linker may be a trifunctional linker. Mechanisms by which reagents are released from these linker groups include, for example, irradiation of photolabile bonds and acid-catalyzed hydrolysis. The length of the linker may be predetermined or selected according to the desired spatial relationship between the polypeptide and the molecule to which it is attached.
The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein in embodiments the polymer may be conjugated to a moiety that is not composed of amino acids. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. "fusion protein" refers to a chimeric protein that encodes two or more separate protein sequences that are recombinantly expressed as a single portion.
The amino acid or nucleotide base "position" is represented by a number that identifies each amino acid (or nucleotide base) in the reference sequence based on its positional order relative to the N-terminus (or 5' -terminus). Since deletions, insertions, truncations, fusions, etc. must be considered in determining the optimal alignment, in general the number of amino acid residues in a test sequence determined by simple counting from the N-terminus is not necessarily the same as the number at its corresponding position in the reference sequence. For example, where the variant has a deletion relative to the aligned reference sequence, there will be no amino acids in the variant that correspond to the location of the deletion in the reference sequence. If there is an insertion in the aligned reference sequences, the insertion will not correspond to the numbered amino acid position in the reference sequence. In the case of truncation or fusion, the stretch of amino acids in the reference or alignment sequence may not correspond to any amino acid in the corresponding sequence.
The term "reference … … to a numbered" or "corresponding to" when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to numbering of residues of a given reference sequence when comparing the given amino acid or polynucleotide sequence to the reference sequence.
Amino acid residues in a protein "correspond" to a given residue when they occupy the same essential structural position in the protein as the given residue. For example, a selected residue in a selected protein corresponds to Ala302 of a PyleS protein when the selected residue has the same requisite spatial or other structural relationship with Ala302 in the PyleS protein. In embodiments, when the selected protein is aligned for maximum homology with the PylRS protein, the position in the aligned selected protein aligned with Ala302 is considered to correspond to Ala302. In addition to primary sequence alignment, three-dimensional structural alignment may be used, for example, to align the structure of the selected protein for maximum correspondence with the PylRS protein and the overall structure of the comparison. In this case, the amino acid occupying the same essential position as Ala302 in the structural model is considered to correspond to the Ala302 residue.
"Percent sequence identity" is determined by comparing two optimally aligned sequences within a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in the two sequences where the same nucleobase or amino acid residue occurs to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.
In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "percent identity" refers to two or more sequences or subsequences that have the same or a specified percentage of the same amino acid residues or nucleotides (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, over a specified region when compared and aligned for maximum correspondence over a comparison window or specified region, using a BLAST or BLAST 2.0 sequence comparison algorithm having default parameters described below, or as measured by manual alignment and visual inspection (see, e.g., NCBI website ncbi.nih.gov/BLAST/etc.). Such a sequence is referred to as "substantially identical". This definition also refers to or may be applied to the complement of the test sequence. The definition also includes sequences with deletions and/or additions, as well as sequences with substitutions. As described below, the preferred algorithm may take into account gaps, etc. Preferably, identity exists over a region of at least about 25 amino acids or nucleotides in length, or more preferably over a region of 50-100 amino acids or nucleotides in length.
An "antibody" is a large complex protein with a complex internal structure. The natural antibody molecule comprises two pairs of identical polypeptide chains, each pair having a light chain and a heavy chain. Each light and heavy chain in turn consists of two regions: a variable ("V") region involved in binding to a target antigen, and a constant ("C") region that interacts with other components of the immune system. The light and heavy chain variable regions are clustered together in three dimensions to form variable regions that bind antigen (e.g., receptors on the surface of a cell). Within each light or heavy chain variable region, there are three short fragments (10 amino acids in average length) called complementarity determining regions ("CDRs"). Six CDRs in the antibody variable domain (three from the light chain and three from the heavy chain) are folded together in three dimensions to form the actual antibody binding site that interfaces with the target antigen. Kabat, E. ,Sequences of Proteins of Immunological Interest,U.S.Department of Health and Human Services,1983,1987, et al, precisely define the location and length of CDRs. The portion of the variable region that is not included in the CDR is referred to as the framework ("FR") which forms the environment of the CDR.
The term "antibody" is used in accordance with its known meaning in the art. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bond in the hinge region to produce dimers of F (ab)' 2-Fab, which are themselves light chains linked to VH-CH1 via disulfide bonds. The F (ab) '2 may be reduced under mild conditions to break disulfide bonds in the hinge region, thereby converting the F (ab) '2 dimer into Fab ' monomers. The Fab' monomer is essentially a Fab with a partial hinge region (see Fundamental Immunology (Pailed., 3 d. 1993)). While various antibody fragments are defined in terms of digestion of intact antibodies, those skilled in the art will appreciate that these fragments may be synthesized de novo, either chemically or by using recombinant DNA methods. Thus, the term antibody as used herein also includes antibody fragments produced by modification of whole antibodies, or antibody fragments synthesized de novo using recombinant DNA methods (e.g., single chain Fv), or antibody fragments identified using phage display libraries (e.g., mcCafferty et al, nature 348:552-554 (1990)).
Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" (about 50-70 kD) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. Fc (i.e., fragment crystallizable region) is the "base" or "tail" of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of antibody. By binding to a specific protein, the Fc region ensures that each antibody produces an appropriate immune response to a given antigen. The Fc region also binds to various cellular receptors (such as Fc receptors) and other immune molecules (such as complement proteins).
An antibody "variant" as provided herein refers to a polypeptide that is capable of binding to an antigen and that includes one or more domains of an antibody or fragment thereof. Non-limiting examples of antibody variants include single domain antibodies (nanobodies), affibody (polypeptides that are smaller than monoclonal antibodies and are capable of binding antigen with high affinity and mimic monoclonal antibodies (e.g., about 6 kDa)), monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, diabodies, trispecific diabodies, scFv-Fc, minibodies, igNAR, V-NAR, hcIgG, vhH, or peptibodies. "peptibody" as provided herein refers to a peptide moiety attached (via a covalent or non-covalent linker) to the Fc domain of an antibody. Other non-limiting examples of antibody variants known in the art include antibodies raised by cartilaginous fish or camelids. General descriptions of camelid antibodies and their variable regions, and methods of making, isolating and using them, can be found in references WO 97/49805 and WO 97/49005, which are incorporated herein by reference in their entirety for all purposes. Also, antibodies and variable regions thereof from cartilaginous fish and methods of producing, isolating and using the same are found in WO2005/118629, which is incorporated herein by reference in its entirety for all purposes.
"Single domain antibody" or "nanobody" refers to an antibody fragment having a single monomeric variable antibody domain. Like an intact antibody, it is capable of selectively binding to a specific antigen. In some embodiments, the single domain antibody is a human or humanized single domain antibody. In some embodiments, the single domain antibody is a camelidae single domain antibody. The single domain antibody may be an engineered single domain antibody and may comprise unnatural amino acids.
The term "antigen" as provided herein refers to a molecule capable of binding to an antibody binding domain as provided herein. An "antigen binding domain" as provided herein is a region of an antibody that binds an antigen (epitope). As described above, the antigen binding domain may comprise one constant domain and one variable domain of each of the heavy and light chains (VL, VH, CL and CH1, respectively). In embodiments, the antigen binding domain comprises a light chain variable domain and a heavy chain variable domain. In embodiments, the antigen binding domain comprises a light chain variable domain and does not comprise a heavy chain variable domain and/or a heavy chain constant domain. Paratope or antigen binding sites are formed at the N-terminus of the antigen binding domain. The two variable domains of the antigen binding domain may bind to an epitope of an antigen. For example, antibodies exist in the form of intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bond in the hinge region to produce F (ab)' 2, which is a dimer of Fab, which itself is a light chain linked to VH-CH1 by a disulfide bond. The F (ab) '2 may be reduced under mild conditions to break disulfide bonds in the hinge region, thereby converting the F (ab) '2 dimer into Fab ' monomers. Fab' monomers are essentially antigen binding moieties with a partial hinge region (see Fundamental Immunology (Pailed., 3 d. Ed. 1993)). While various antibody fragments are defined in terms of digestion of intact antibodies, those skilled in the art will appreciate that these fragments may be synthesized de novo, either chemically or by using recombinant DNA methods. Thus, the term antibody as used herein also includes antibody fragments produced by modification of whole antibodies, or antibody fragments synthesized de novo using recombinant DNA methods (e.g., single chain Fv), or antibody fragments identified using phage display libraries (see, e.g., mcCafferty et al, nature 348:552-554 (1990)).
Examples
Example 1 identification of PSMA sdAb coupling sites
To assess the compatibility of PSMA as a target coupled to a targeting molecule containing FSY using a single domain antibody (sdAb) C1 (SEQ ID NO: 1), FSY sites were screened by replacing each selector codon within the CDR1, CDR2, and CDR3 regions with a TAG stop codon and generating the encoded protein to incorporate FSY in place. Approximately 19 potential sites were identified, most of which were clustered in the CDR1 and CDR3 regions. These TAG sites are located in CDR1 (positions 26-35:GYTDSNYYMS,SEQ ID NO:5); CDR2 (50-66:VNTGRGSTSYADSVKG,SEQ ID NO:6), CDR3 (99-116, excluding Cys101, cys104 AACHFCDSLPKTQDEYIL, SEQ ID NO: 7). A similar evaluation was made for the second sdAb C2 (SEQ ID NO: 2) for the FSY modification site. About 8 sites were found in the C2 CDR as potential sites for FSY, most of which were clustered in CDR2 and CDR 3. These TAG sites are located in CDR1 (site 26-35RFMISEYSMH,SEQ ID NO:8);CDR2(50-65:TINPAGTTDYAESVKG,SEQ ID NO:9),CDR3(96-100DGYGY,SEQ ID NO:10)).
Example 2 expression of FSY-modified sdabs
The vector pBAD sequence (Invitrogen # 43001) lacking ORF insert was PCR amplified with forward primer (SEQ ID NO: 80) and reverse primer (SEQ ID NO: 81), respectively.
The resulting PCR amplified vector was gel extracted and purified using Zymo PCR purification kit (Zymoclean gel DNA recovery kit Cat#D4002). The dsDNA sequences of C2 WT His and C1 WT His were ligated to the PCR-amplified pBAD backbone and transformed into E.coli DH10B chemocompetent cells (Fisher Thermo Scientific TM DH10B competent cells; high efficiency; FEREC 0113). Clones were miniprep and sequence verified using pBAD forward primers. The expression cassette also incorporates a PelB leader sequence and His6 tag. The DNA sequences of C1 and C2 wt are SEQ ID NO:73 and SEQ ID NO:74, respectively. For constructs incorporating FSY residues, the TAG codon is engineered to the desired position of the amino acid substitution in the open reading frame of the sdAb or biparatopic construct in the DNA sequence.
C1 Construction of TAG library. A polynucleotide (dsDNA sequence) containing TAG codons was synthesized by IDT in place of each codon in the site identified in the CDR region of C1 (see example 1). These TAG sites are located in CDR1 (positions 26-35); CDR2 (50-66), CDR3 (99-116, excluding Cys101, cys 104). These sites are underlined in the following sequences:
QVQLQESGGGSVQAGGSLRLSCTAPGYTDSNYYMSWFRQAPGKERE
WVAGVNTGRGSTSYADSVKGRFTISQDNAKNTMFLQMNSLKPEDT
AIYYCAVAACHFCDSLPKTQDEYILWGQGTQVTVSSAAAYPYDVPDYG(SEQ ID NO:1)
polynucleotides containing each of these TAG mutants were cloned into the pBAD backbone and the sequences verified as described above.
C2 Construction of TAG library. A polynucleotide sequence containing a single TAG codon was synthesized by IDT, replacing each residue in the CDR region of C2 identified in example 1. C2 These regions in the amino acid sequence of the WT His protein sequence are underlined in the following sequence:
EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVRQAPGKGLE
WVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVTVSS(SEQ ID NO:2)
polynucleotides containing each of these TAG mutants were cloned into the pBAD backbone and the sequences verified as described above.
Library strain generation. The pEVOL-FSYRS plasmid expressing the engineered Mb FSYRS aminoacyl-tRNA synthetase was synthesized by Genscript (order number U168 NGE; U168 NGE; cat#SC1010) (ref: J.am. Chem. Soc.2018,140,15, 4995-4999) and transformed into chemically competent DH10b competent cells using standard methods to generate the parental strain FSYRS-DH10b. A single plasmid encoding each C1 CDR TAG or C2 CDR TAG variant was transformed into chemwell FSYRS-DH10b using standard methods. Single colonies were inoculated into 2XYT medium (Teknova #Y0166) containing 100ug/ml ampicillin and 34ug/ml chloramphenicol and grown overnight at 37℃with shaking at about 220 rpm. The overnight cultures were mixed with sterile 50% glycerol 1:1 and stored at-80 ℃.
Expression and purification. Strains harboring plasmids encoding sdabs with a single CDR residue mutated to TAG in the FSYRS-DH10b parental background were grown in 2xYT medium (Teknova #y0166) in the presence of 100ug/ml ampicillin and 34ug/ml chloramphenicol. Prior to induction, cells were grown at a temperature of 37 ℃. At OD 600 of 0.6, cultures were supplemented with 1mM FSY and induced by the addition of 0.2% arabinose. At induction, the cultures were moved to 25 ℃ while shaking overnight at 220RPM for a total of 16 hours.
Expression was collected by precipitating cells at 2200Xg for 30 minutes at 4 ℃. The supernatant was removed, and the cell pellet was weighed and stored at-80 ℃. The cell pellet was resuspended for lysis by adding B-PER protein extraction reagent (ThermoFisher # 78243) at 4mL/g pellet. The resuspended pellet was lysed by placing the sample at room temperature on an orbital shaker for 15 minutes at medium speed. The lysed cells were then spun at 2200xg for 30 minutes. The soluble fraction of the supernatant was removed for further purification.
To purify the soluble fraction, hisPur-Ni-NTA resin (ThermoFisher # 88222) was used to capture soluble material from the lysate. The resin storage buffer was removed and equilibrated by batch washing in wash buffer (40 mM sodium phosphate, pH 7.2, 300mM NaCl,20mM imidazole). Batch washes were repeated for a total of 50x resin volumes. The clarified lysate was added to the washed Ni-NTA resin and combined with constant rotation for 1 hour at room temperature. After binding, the protein-bound resin was washed batchwise in a 50x resin volume in wash buffer to remove unbound contaminants. The protein-bound resin was then transferred to a spin column and briefly spun at 700Xg to remove residual wash buffer. The target protein was then eluted using a buffer with elution buffer (40 mM sodium phosphate, 7.2, 300mM NaCl,500mM imidazole). Elution buffer was added at 2x resin volume and incubated for 5 minutes at room temperature. The sample was briefly spun and the eluted protein was collected in a new tube. The elution procedure was repeated twice using the same conditions. The eluted fractions were pooled and quantified by a 280 on a Nanodrop 2000 and a blank was run with elution buffer. The Ni-NTA purified protein was then concentrated using a 0.5mL, 3kDa MWCO PES spin filter (ThermoFisher # 88512) and buffer exchanged to 1xPBS via repeated dilutions at 8-10 Xvolumes of sample and concentration.
Example 3 covalent interactions of FSY-modified sdAb and PSMA
The semi-purified sdAb product from example 2 was incubated overnight in 1xPBS with commercial PSMA (Sino Bio, cat# 15877-H07H) at a molar ratio of about 7:1 (sdAb: PSMA, PSMA final concentration of 0.125mg/mL,1.25 uM) at 37 ℃. Crosslinking was analyzed by reducing SDS-PAGE (FIGS. 1A-1E).
The sites assessed in CDR1 and CDR3 of the C1 sdAb demonstrate the cross-linking ability profile for PSMA, as shown in table 3 below.
The position of the site is listed with respect to SEQ ID NO. 1.
The crosslinking efficiency of the second PSMA-specific sdAb C2 was evaluated using a method similar to that of example 1, except that the sdAb to PSMA molar ratio was about 8:1. The crosslinking results are shown in FIGS. 2A-2C. C2 The estimated sites in CDR2 and CDR3 of sdAb indicate the cross-linking ability profile for PSMA, as shown in table 4 below.
The position of the site is listed relative to SEQ ID NO. 2.
EXAMPLE 4 kinetics of crosslinking
A subset of FSY modified sdabs were selected to assess the kinetics of PSMA crosslinking. The sdAb was incubated with PSMA at a 5:1 molar ratio (PSMA final concentration of 0.125mg/mL,1.25 uM). Samples were taken at time points of 0-180 minutes and the percentage of crosslinked PSMA was assessed by SDS-PAGE. As shown in fig. 3A and 3B, the modified sdAb exhibits a range of coupling kinetics. Percent crosslinked bands were calculated by quantifying sdAb PSMA crosslinked bands, and PSMA band intensities were quantified using ImageJ.
Example 5 PSMA targeting constructs with modified CDR3
The sdAb C1 construct targeting PSMA was generated by modifying the two cysteine residues in CDR3 (SEQ ID NO: 7) of C1 (SEQ ID NO: 1) to alanine (C101A and C104A) to generate C39, removing these residues. The construct was then further modified to install FSY (C1-C101A/C104A/H102 (FSY), SEQ ID NO:4, hereafter referred to as C39-102 FSY) at residue 102. A diagram of these sdAb constructs is shown in FIG. 4. The construct was synthesized with pelB leader sequence (SEQ ID NO: 15) cleaved from the mature protein and His-Tag purified using six C-terminal histidines. The cross-linking ability of these constructs was evaluated using the method of example 3. SDS-PAGE analysis showed that under these conditions the level of cross-linking of all constructs was comparable.
PSMA-targeted sdAb C39-FSY C1, containing FSY, was cloned and expressed as described in example 2. By ligating the C39-FSY C1 with an additional copy of C1-C101A/C104A/H102H (SEQ ID NO:71, free of FSY), a double paratope construct (C1-C101A/C104A/H102 (FSY) -L1-C1-C39, SEQ ID NO:21, hereinafter C40-FSY) was created. Constructs were designed by adding a GGGGSGGGGS linker between the two sdAb amino acid sequences. The biparatopic construct was then cloned into the pBAD vector and expressed as described in example 2. The construct was synthesized with pelB leader sequence (SEQ ID NO: 15) cleaved from the mature protein and His-Tag purified using six C-terminal histidines.
Crosslinking of single and double paratope constructs to PSMA was assessed by SDS-PAGE and time course. SDS-PAGE analysis showed that dimeric form C40-102FSY had twice the rate of crosslinking than monomeric C39-102FSY (dimer had a maximum time of about 30 minutes for t1/2, compared to about 60 minutes for t 1/2).
(FIGS. 5A-5B)
C39-102FSY amino acid sequence (102 FSY position, SEQ ID NO:4, alanine modification underlined):
QVQLQESGGGSVQAGGSLRLSCTAPGYTDSNYYMSWFRQAPGKEREWVAGVNTGRGSTSYADSVKGRFTISQDNAKNTMFLQMNSLKPEDTAIYYCAVAAA*FADSLPKTQDEYILWGQGTQVTVSSAAAYPYDVPDYGSCHHHHHH
EXAMPLE 6 Synthesis of constructs
Site-specific conjugation via engineered C-terminal cysteine residues. To achieve site-specific bioconjugation with sdAb compounds, additional Cys residues were designed near the C-terminus of the construct. The sdAb-Cys compound was prepared and formulated to 1mg/ml in 1xPBS,pH 7.4,5mM EDTA. TCEP was added to 10mM for light reduction and incubated for 15 min at room temperature. The samples were then buffer exchanged using a Zeba centrifuge column to remove TCEP and exchanged to 1xPBS,pH 7.4,1mM EDTA. Then the maleimide reactive payload compound (such as AZDye 647 maleimide cat# 1122-5) is added in a 10x molar excess and incubated for 2 hours at 37℃or 16 hours at 4 ℃. Unreacted maleimide compound is removed from the sample using size exclusion chromatography, desalting column, dialysis or TFF. Samples were subjected to SDS-PAGE under reducing and non-reducing conditions to observe labelling efficiency and analyzed using LC-MS to quantify the complete mass and relative proportions of labelled and unlabelled species.
EXAMPLE 7 determination of crosslinking Activity
The specific binding and cross-linking of the molecules to the target tumor antigen is determined. To assess the cell binding capacity of PSMA-targeted constructs, specific FSY-or Tyr-containing sdabs were used to test the effect of covalent cross-linking on a specific antigen of interest. These test preparations were serially diluted and associated with the in vitro cultured prostate cancer cell lines LnCAP (psma+) and PC3 (PSMA-). After incubation at 37 ℃ for various times, the cells were washed and the medium was changed to remove unbound material. Samples were analyzed by flow cytometry using anti-His tag antibodies or anti-VHH to measure the bound population as a function of input concentration. To measure crosslinking efficiency and specificity, cells were collected, lysed, and western blotted with anti-his or anti-VHH antibodies to measure covalently bound populations via gel shift measurement and compared to gel shift western blots with anti-PSMA antibodies to measure total target antigen crosslinked to sdAb assay preparations.
The functional activity of the conjugate molecule is additionally measured, in this case the ability to specifically deliver a toxic payload to tumor cells expressing the target antigen in vitro. Constructs containing engineered C-terminal or proximal Cys residues were coupled to a cytotoxic payload, such as MMAE or other classes of highly cytotoxic compounds, using the general method described in example 8. Dose response curves were generated and samples were incubated with LnCAP and PC3 cells in vitro. After incubation for various times, the cells are washed to remove free compounds and cell viability is measured using an assay (such as Promega CytoxGreen) or other assays that measure cell viability via reporter compound detection, live cells by prest Blue, CCK-8 or similar reagents, and/or apoptosis via annexin V-FITC, propidium iodide staining or similar reagents.
Example 8 in vivo delivery
To measure the ability of the construct to specifically deliver a payload to tumor tissue in vivo and to compare tumor exposure of the construct to the non-covalent sdAb conjugate, a fluorophore label was used as representative of the payload to enable tracking/biodistribution measurements over time. sdAb constructs (e.g., C1, C2, or C3) with single residues replaced with Tyr or FSY provide for conjugation of maleimide chemistry to chemical fluorophores (Alexa 647 or the like) via engineered cysteine residues and purification as described in example 6. In psma+ or-tumor-bearing male NSG mice, fluorophore-labeled sdADC conjugate molecules were administered via tail IV injection and the biodistribution of the test article was observed over time via whole animal imaging using a AmiX imager or similar device. Tumor-specific and peripheral exposure were quantified via image densitometry and a comparison was made between FSY and Tyr versions of sdAb.
Example 9: design of further FSY modified SdAb
C3 was constructed by cloning the C3 wt sequence (synthesized by IDT, SEQ ID NO: 3) into the digestion vector (synthesized by Genscript) by NdeI and HindIII restriction enzymes
Wt anti-PSMA sdAb. The expression cassette also incorporates a PelB leader sequence and His6 tag. The C3wt DNA sequence is shown in SEQ ID NO. 75.
At the site selected for FSY modification, the amino acid sequence of the CDR region was changed to the TAG codon in the plasmid. These TAG mutants in the ORF were prepared by Genscript via site directed mutagenesis.
C3 CDR1(26-35:GWPYSTYSMN,SEQ ID NO:11);CDR2(50-65:GISSTMSGIIFAESKAG,SEQ ID NO:12);CDR3(99-113:RRDYSLSSSSDDFDY,SEQ ID NO:13).
Example 10 selection of FSY-modified sdabs
The C3 construct from example 9 was co-transformed with pEVOL FSYRS into DH10b competent cells as described in example 2. Single colonies were picked from the transformation and inoculated into 1mL of 2XYT in a 24-deep well plate that was shaken at 220rpm, 37℃and that 24-deep well plate was supplemented with 100ug/mL Amp+34ug/mL Cm. When the OD reached 0.5, the temperature was reduced to 25 ℃. Expression was induced per well by the addition of 0.2% arabinose +1mM FSY. The product was expressed overnight at 25 ℃. After overnight, cells were transferred to 1.5mL Eppendorf tubes and centrifuged using a bench top centrifuge. The supernatant was removed. The pellet was treated with 50uL B-per (ThermoFisher # 78243) for 15 min for cell lysis. After that, the cell lysate was spun at maximum speed using a 4℃bench centrifuge.
The cross-linking reaction was initiated by incubating 3uL of supernatant with 3uL of PBS or 3uL of 0.25mg/mL PSMA. The reaction was incubated at 37℃for a short time window of 3 hours. Thereafter, the incubation mixture was treated with 2 XSDS-supported dye. Crosslinking was studied by running gel on SDS-PAGE stained with Coomassie blue. The results are shown in fig. 6A and 6B and summarized in table 5 below.
The position of the site is listed with respect to SEQ ID NO. 3.
Example 11 identification and kinetics of FSK modified FAP sdabs
Sequence analysis (abYsis) was used to define Complementarity Determining Region (CDR) loop positions in Fap antibody sequences. Libraries were constructed to replace each CDR residue with a TAG codon individually and the resulting protein expressed with incorporation of the unnatural amino acid FSK at each position.
C8 FAP library generation. To assess the different FSK incorporation sites in the sdAb, the vector pBAD-C8 WT (SEQ ID NO: 82) was constructed as follows: c8 The WT sequence (SEQ ID: 23) was codon optimized for E.coli expression and synthesized by IDT company. The pBAD-C8 WT vector was constructed by cloning the C8 WT gblock sequence (SEQ ID NO: 78) into the digested pBAD vector (Genscript) by NdeI and HindIII restriction enzymes.
Library strain generation. For expression of the corresponding synthetase, a pEVOL-FSKRS plasmid (Genscript) was synthesized, which codes for an engineered Ma FSKRS aminoacyl tRNA synthetase (SEQ ID NO: 87). pEVOL-FSKRS (SEQ ID NO: 89) was transformed into chemically competent DH10b competent cells to produce the parental strain FSKRS-DH10b. A single plasmid encoding each C8 CDR TAG variant was transformed into chemically competent FSKRS-DH10b cells. Single colonies were inoculated into Superbroth medium (Teknova #S1530) containing 100ug/ml ampicillin and 34ug/ml chloramphenicol and grown overnight at 37℃with shaking at about 220 rpm.
Expression and purification. Strains harboring plasmids encoding sdabs with a single CDR residue mutated to TAG in the FSKRS-DH10b parental background were grown in Superbroth in the presence of 100ug/ml ampicillin and 34ug/ml chloramphenicol. Individual colonies in each pool were inoculated into 5mL of Superbroth medium containing the relevant antibiotic and grown overnight at 37 ℃. The next day, the cells were diluted 1:10 to 30mL (3 mL to 30mL dilution) of Superbroth medium containing 100ug/mL ampicillin and 34ug/mL chloramphenicol, 1mM FSK, 0.2% arabinose. Cells were induced for FSK incorporation at 30℃and 220rpm for 6 hours. After expression, FSK modified sdabs (C8-FSK) were harvested and purified as described in example 2.
Crosslinking kinetics. FAP receptor (Acrobiosystem # AP-H5263-100 ug) and the pooled antibody mixture (Table 6) were incubated at 37℃for 1 hr, 2 hr or overnight at 1XPBS, pH 7.4 at a molar ratio of about (antibody: receptor) 8:1. After incubation, the cross-linking reaction was stopped by adding 1X Laemmli sample buffer containing 100mM DTT. The samples were heated at 95℃for 5 min and then subjected to Tris-GLCYINE SDS-PAGE (4-20%TGX TM). The relative band intensities were then quantified using ImageJ software. The results of FSK-combined translocation indicate that FAP receptors are translocated via crosslinking activity within libraries 2 and 3 (fig. 7A).
TABLE 6 CDR pool designed for expression of C8 FSK mixtures
Library numbering CDR regions
1 CDR1(26-35)
2 CDR2(50-57)
3 CDR2(58-65)
4 CDR3(98-106)
Identification of a single FSK site responsible for cross-linking with FAP. To identify the single FSK site responsible for cross-linking, each TAG mutant was co-transformed with pEVOL-FSKRS into DH10b cells, expressed and purified as described above. Each individual FSK mutant in the candidate pool was incubated with FAP receptor for studying gel shift (fig. 7B). The 9 FSK mutants in CDR2 were found to crosslink with FAP (52, 53, 54, 55, 56, 58, 60, 62, 64). One site (56) was found to have the highest crosslinking yield of all crosslinking sites within 1 hour.
Measurement of the kinetics of cross-linking of C8-FSK with FAP receptor. To evaluate FSK crosslinking kinetics, C8-54FSK and C8-56FSK were incubated with FAP receptor for 15, 30, 60 and 120 minutes and the crosslinking yields were checked. The 56FSK sites were found to have the highest crosslinking rate compared to the other sites, indicating that C8-56FSK had the fastest crosslinking rate (crosslinking >50% in 2 hours) (FIG. 7C).
Example 12 identification and kinetics of FSY-modified Her3-SdAb
C9 library generation. Sequence analysis (abYsis) was used to determine the Complementarity Determining Region (CDR) loop positions in Her-3 antibody sequences. Libraries were constructed to replace each CDR residue with a TAG codon individually and expressed under conditions where each position incorporates the unnatural amino acid FSY.
The vector pBAD-C9 WT was constructed. C9 The WT sequence (SEQ ID NO: 24) was codon optimized for E.coli expression. The pBAD-C9 WT vector (SEQ ID NO: 83) was constructed by cloning the C9-gblock sequence (SEQ ID NO: 79) into the digested pBAD vector (GENESCRIPT CAT #SC1010) by NdeI and HindIII restriction enzymes. A library of C9-FSY variants was created as described in example 2, except that the pEVOL-FSYRS plasmid and the single mutant pBAD-C9 mutants were transformed and expressed in BL21 cells instead of DH10b cells. Expression of FSY-modified C9-sdAb (C9-FSY) follows the method described in example 2, except that the FSY-modified C9-sdAb is expressed and purified in parent strain FSYRS-BL 21.
Identification of the pool responsible for crosslinking. To initially identify CDR regions containing cross-linked compatible FSY sites, strains containing plasmids with TAG sites in multiple CDR regions were initially pooled (table 7). The pooled strains were expressed and purified by Ni-NTA affinity, normalized to concentration and cross-linked via SDS-PAGE gel displacement assessment as described in example 2. The C9-FSY pool mix was incubated with Her3 (Acrobiosystem # ER3-H5223-100 ug) at a 3:1 ratio (3 ug C9 FSY to 1ug Her3 receptor) overnight at 37 ℃, 1XPBS, pH 7.4. The reaction is between 4 and 20 percentTGX TM followed by staining with coomassie blue. The results of the combined translocation show Her3 receptor crosslinking activity within pools 2, 3 and 4 (fig. 8A).
TABLE 7 CDR pool designed for expression of C9 FSY mixtures
Library numbering CDR regions
1 CDR1(28-37)
2 CDR2(52-58)
3 CDR2(59-63)
4 CDR2(64-68)
5 CDR3(101-106)
6 CDR3(107-112)
7 CDR3(113-118)
Identification of a single FSY site responsible for cross-linking with Her 3. To identify each individual FSY site responsible for cross-linking, each TAG mutant was co-transformed with pEVOL-FSYRS into BL21 cells, expressed and purified as described above. Each FSY mutant in the candidate pool was incubated with Her3 receptor overnight at 37 ℃. By a proportion of between 4 and 20% The cross-linking was assessed by reaction on TGX TM with Coomassie blue staining. (FIG. 8B). Eight C9-FSY mutants in CDR2 were found to support cross-linking with Her3 (53, 55, 56, 57, 58, 60, 64, 67). The two sites (55, 57) were found to have the highest crosslinking yield among all crosslinking sites.
The C9-FSY crosslinking site was identified at the fastest rate. To identify FSY crosslinking sites at the fastest rate, C9 with FSY sites identified above were incubated with Her3 receptor (Acrobiosystem) and the crosslinking yield was determined. The C9-FSY mutant was incubated with Her3 receptor in 1 XPBS at pH 7.4 at a molar ratio of 8:1. After 1 hour, incubation was stopped by addition to final 1X SDS-loaded dye containing 100mM DTT, and by 4-20% MiniTGX TM was stained with coomassie blue to analyze the reaction.
The unique 55FSY site was found to have the highest crosslinking yield (> 50% within 1 hour) compared to the other sites, indicating that 55FSY has the fastest crosslinking rate (fig. 8C). C9-55FS was further analyzed by incubating C9-55FSY with Her3 receptor and stopping the reaction during the time of 0, 15, 30, 60, 120 and 180 minutes. As shown in fig. 8D, crosslinking progressed rapidly and was detected within 15 minutes, with crosslinking exceeding 50% within 1 hour.
Example 13 binding affinity of FSY modified and unmodified C2 sdabs
The C2sdAb with FSY at position 54 (C2-54 FSY) was selected to compare binding affinities to the C2sdAb with tyrosine at the same position (C2-54 TYR). The protein was prepared as described in example 2 and then further purified via FPLC size exclusion chromatography (HiLoad 16/600Superdex 200pg size exclusion column Cytiva # 28989335). 1 XDPBS was used as running buffer and the protein was collected by isocratic elution. For each C2sdAb, monomeric peak fractions were analyzed by reducing SDS-PAGE, and pooled and dialyzed into anion exchange running buffer (20 mM Tris, 7.5, and 20mM NaCl) overnight at 4 ℃. To remove endotoxin, the dialyzed sample library was run in flow-through mode on a HiTrap Q XL 1ml column (Cytiva # 17515801) with endotoxin bound to the column. The flow-through was collected and checked for endotoxin levels. Finally, the fully purified samples were dialyzed into 1x DPBS as final formulation buffer. The samples were aliquoted and stored at-80 ℃.
Binding kinetics were measured using a biological layer interferometry with an AHC sensor (Sartorius project # 18-5060). PSMA (50 nM), with Fc tag (Acrobiosystem PSA-H5264-100 ug), with C2-54FSY or C2-54TYR sdAb (protein concentration 400nM, 200nM, 100nM, 50 nM). The step of making OCET measurements served as baseline: 60 seconds; loading the receptor: 300 seconds, eluting unbound receptor: 300 seconds; loading the associated sdAb:100 seconds; dissociation: 600 seconds. The KD for C2-54TYR is 9.1nM and the KD for C2-54FSY is 10.9nM, indicating that the incorporation of FSY instead of tyrosine at position 54 does not alter binding affinity.
Example 14 cell binding assay
Flow cytometry was used to assess the binding of C2-54FSY and C2-54TYR sdabs to human prostate tumor cell lines LNCaP (PSMA+) and PC3 (PSMA-). The protein from example 13 was formulated with FACS buffer (1xpbs+2% HI-FBS) to 3 μΜ (1000 μl) and then serially diluted 5x (200 μl sdAb to 800 μl FACS buffer) to generate 8 concentration spots for assay, lowest concentration of 0.0000384 μΜ. Control sdAb (human Alexa Fluor Alexa)647-Conjugated antibody) was formulated with FACS buffer (1xpbs+2% HI-FBS) to 1 μΜ (450 μl), and then serially diluted 3x (150 μl test article to 300 μl FACS buffer), at a minimum concentration of 0.00045 μΜ.
Binding assay. LNCaP and PC3 cell lines (ATCC) were maintained in RPMI-1640 and F12K medium supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher) in a humidified environment at 37℃with 5% CO 2. Cells were harvested at the exponential growth phase, counted and aliquoted into v-bottom 96-well plates, 100 μl cells per well. Cells were pelleted by centrifugation. C2 sdAb and control sdAb samples were added to the cells and incubated on ice for one hour. For cells treated with C2 sdAb, after incubation, cells were washed twice with FACS buffer and incubated on ice for 30min with 100 μl of ice-cold FACS buffer (Alexa Fluor 647AffiniPure goat anti-alpaca IgG, VHH domain) containing 5 μg/mL secondary antibody. The cells were then washed twice with FACS buffer and fixed with 4 ℃ fixing buffer (Thermofisher, cat.no. 0082249) for 30 minutes. For cells treated with control sdabs, after incubation, cells were washed twice with FACS buffer and fixed with 4 ℃ fixation buffer (Thermofisher, cat No. 0082249) for 30 minutes. After fixation, the cells were washed twice and analyzed by Attune flow cytometry (thermo fisher).
Raw data (FCS file) (Flowjo 10.7.2) is analyzed. FSC-A and SSC-A were used to isolate total cells, and intact cells were gated with FSC-A and SSC-A. The single peak is then gated with FSC-A for FSC-H. The single peak was separated with Alexa Fluror-647. The geometric mean intensity of Alexa Fluror-647 signal intensities of the samples were used for further analysis and mapping. The mean value of the geometric mean intensity and STDEV values (for Microsoft 365MSO Version 2202Build 16.0.14931.2012864-bit) of samples Alexa Fluror-647 were calculated). Curve fitting and mapping was performed using 'log (agonist) and response-variable slope (four parameters)' (GRAPHPAD PRISM 9.2.0).
C2-54TYR and C2-54FSY, which bound to LNCaP cells in a dose-dependent manner, did not show binding to PSMA-negative PC3 cells (FIG. 9). Binding affinities of C2-54TYR and C2-54FSY were measured according to EC50 values after LNCaP cell flow staining (Table 8).
TABLE 8 binding affinity of C2 sdabs in LNCaP cells
C2-54TYR C2-54FSY
EC50 14.88nM 12.25nM
EC50 range 8.83NM to 27.05nM 8.98NM to 16.88nM
Square R 0.978 0.99
Example 15 crosslinking of FSY-modified sdabs with target cells
The effect of crosslinking of C2-54TYR, C2-54FSY and C3-101FSY on cell binding was compared. LNCaP and PC3 cells were cultured as described in example 14. Cells were plated in 6-well plates in their growth medium at a density of 500,000 cells/well. 48 hours after inoculation, the medium was removed and 0.8mL of sdAb-containing medium was added at the indicated concentrations and time points (see tables 9 and 10). Cells were rinsed twice (1 mL/well 1 xPS) and 0.2 mL/well 0.25% EDTA-trypsin (Gibco, cat. No. 25200-056) was added. After five minutes incubation in the incubator, 1ml of complete medium was added to neutralize the trypsin. Cells were washed and pelleted.
Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Santa Cruz Biotechnology, cat. No. SC-24948A). Denatured samples were run by electrophoresis in 4-20% standard TGX gel (Bio-Rad, cat. No. 5671095) and then Western blotted with kits (Bio-Rad, cat. No. 1704271) and PIERCE FAST Western blotting kit (Thermo Scientific, cat. No.35060, 35061) and analyzed using primary anti-human PSMA (Invitrogen, cat. No. 37-3900) or anti-alpaca VHH (Jackson ImmunoResearch, cat.No.128-035-232), internal standard anti-GAPDH (CELL SIGNALING Technology, cat. No. CST-2118).
The image was acquired by Azure Biosystem C600,600. GIMP 2.10.28 is used to process images. The density of bands in the Western blot was determined using ImageJ 1.51j8 according to the instructions (Imagej. Nih. Gov/ij/docs/guide/146-30.Html # -info box: densitomery, section 30.13). The percentage of crosslinked PSMA to total PSMA was calculated as follows: density of crosslinked PSMA/(density of crosslinked psma+density of uncrosslinked PSMA) x 100%.
The major band detected with anti-PSMA antibody (about 100 kD) was observed in western blots of LNCaP cells (fig. 10A), but no band of similar molecular weight was detected in PC3 cells (fig. 10B), demonstrating the specificity of anti-PSMA antibody and confirming the specificity of PSMA expression in LNCaP cells. In LNCaP cells treated with C2-54FSY, additional bands above PSMA were detected. The strength of the tape increases in a time and concentration dependent manner, indicating specific crosslinking. However, in LNCaP cells treated with different concentrations of C2-54TYR for different times, similar intensities of non-crosslinked PSMA bands were observed, but no crosslinked bands, indicating that sdabs without FSY did not crosslink with PSMA in LNCaP cells (fig. 10B). Quantification of crosslinking showed that C2-54TYR crosslinked with PSMA in LNCaP cells in a time and dose dependent manner. (Table 9 and FIG. 10C)
TABLE 9 kinetics of crosslinking of C2-54FSY with PSMA in LNCaP cells
A similar pattern was observed in LNCaP cells treated with C3-101FSY, with the intensity of the crosslinked bands (PSMA-sdAb) increasing in a time and concentration dependent manner. Since the molecular weight of C3-101FSY is higher than that of C2-54FSY, the apparent molecular weight of the migrating crosslinked bands is higher than that of C2-54FSY (FIG. 11A). A comparison of the crosslinking kinetics of C2-54FSY and C3-101FSY is shown in Table 10 and FIG. 11B.
TABLE 10 crosslinking kinetics of C2-54FSY and C3-101FSY with PSMA in LNCaP cells
EXAMPLE 16 in vivo crosslinking assay
This study assessed in vivo cross-linking, systemic and intratumoral exposure of C2-54TYR and C2-54FSY with PSMA in two mouse xenograft tumor models LNCaP and PC 3. LNCaP and PC3 cell lines (ATCC) were maintained in RPMI-1640 and F12K medium supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher) in humidified environment at 37℃and 5% CO 2, respectively. Cells were harvested at the exponential growth phase and centrifuged at 335Xg in a cryocentrifuge and the medium aspirated. For cell seeding, the cell pellet was resuspended in 100. Mu.L of serum-free F-12K medium (for PC 3) or RPMI (for LNCaP) plus 100. Mu. L MATRIGEL. 200 uL containing 500 ten thousand PC3 cells or 300 ten thousand LNCaP cells was implanted into the flank of male NSG mice (Jackson Labs). When the tumor size reached about 200mm 3, C2-54TYR and C2-54FSY were injected by tail vein. Peripheral blood samples were collected by cheek bleeding. After 6 hours, mice were sacrificed and both peripheral blood samples and tumors were collected, weighed and frozen at-80 ℃.
To a tumor sample (about 30 to 40 mg) was added 1ml of RIPA buffer (Santa Cruz Biotechnology, cat. No. sc-24948 a) containing a protease inhibitor cocktail, which was then homogenized with a Qiagen Tissuelyser II sample pulverizer. After homogenization, the sample was centrifuged at 12,000rpm for 10 minutes at 4 ℃. The supernatant was retained and the precipitate was discarded. The centrifugation process was repeated once (twice in total).
Protein concentration of tumor lysates was quantified using BCA protein assay kit (Thermo Scientific, cat.no. 23225). Samples were formulated to the same concentration with RIPA buffer and then heated at 100 ℃ for 10 minutes after addition of 6x reducing loading buffer (ALFA AESAR, CAT.NO.J61337). Denatured samples were analyzed by electrophoresis in 4-20% standard TGX gel (Bio-Rad, cat.no. 5671095) followed by western blotting (Bio-Rad, cat.no.1704271 and Thermo Scientific, cat.no.35060, 35061) with anti-PSMA and anti-VHH to detect cross-linked and anti-GAPDH antibodies as loading controls. The image was acquired by Azure Biosystem C600,600. GIMP 2.10.28 is used to process images. Plasma was ELISA and data were processed and plotted (GRAPHPAD PRISM 9.2.0). Mean and STDEV were calculated and plasma concentrations were assessed for significance using the unpaired two-tailed student t-test.
Samples of LNCaP and PC3 tumors from animals administered C2-FSY or C2-TYR were prepared as described above and western blotted with anti-VHH antibodies to detect non-crosslinked C2 compounds (15 kD region of blot) and crosslinked C2 compounds (approximately 100kD region above PSMA). In LNCaP tumor samples from animals administered C2-54FSY, anti-VHH Western blotting detected bands above the PSMA region (approximately 100 kD) in all samples, whereas no crosslinked bands were observed in C2-54TYR or vehicle samples (FIG. 12A), indicating that C2-54FSY had specific and reproducible crosslinking. No anti-VHH signal was observed in PC3 tumor samples, while different band patterns were observed in LNCaP samples treated with C2-54TYR and C2-54 FSY. The uncrosslinked/free sdAb was shown in the approximately 15kD region of the blot, and both C2-54TYR and C2-54FSY treated LNCaP samples showed bands of similar intensity, indicating the presence of similar levels of uncrosslinked sdAb. In the LNCaP tumor tissue treated with C2-54FSY, an additional band was observed to migrate specifically at about 100kD, whereas in the PC3 or LNCaP samples from the C2-54TYR treated animals no band was observed in this region, indicating that C2-54FSY was crosslinked with PSMA.
To estimate and compare systemic exposure of sdabs, plasma concentrations of C2-54TYR and C2-54FSY were measured using ELISA 30 minutes and 6 hours post-dosing (table 11 and fig. 12B).
TABLE 11 plasma concentrations (ng/ml) of C2-54TYR and C2-54FSY
The results indicate that after a single IV dose, C2-54TYR and C2-54FSY entered the tumor and accumulated in a PSMA-dependent manner. However, in tumors, only C2-54FSY specifically crosslinked with PSMA.
EXAMPLE 17 construction of FSK-modified and FSY-modified sdabs for target-specific crosslinking
Libraries and library strains were constructed, expressed and purified according to the general methods of examples 2 and 11. To initially identify CDR regions containing sites compatible with FSY or FSK insertions, strains of plasmids containing TAG codons within the coding region of the CDR regions were constructed. Table 12 shows the sdAb and CDR sequences used for screening.
Table 12: sdAb with FSY and FSK insertions
And (5) screening. Strains of the plasmid having TAG codons in the coding region containing the CDR regions were selected in the library and cross-linked determined according to the general method of examples 11 and 12. Libraries exhibiting crosslinking activity towards targets were further analyzed by assessing crosslinking of individual FSY or FSK mutants. Individual mutants showing cross-linking are shown in table 13.
Table 13: identification of FSY and FSK insertions for cross-linked sdAbs
Reagents for cross-linking the targets of the assay. CEACAM5 is available from Acrobiosystem (#CE5-HF 255-25. Mu.g) and Sinobiological (#11077-H02H-100. Mu.g). FAP receptors are available from Acrobiosystem (#AP-H5263-100 ug). Human FolRa (folate receptor alpha) receptors were purchased from Acrobiosystem (#FO1-H5253-100 ug). MSLN (mesothelin) was purchased from Sinobiological (Cat: 13128-HNCH, cat: 13128-H01H-B) and Acrobiosystem (#MSN-H526 x-100 ug). Human MSLN ectodomain is purchased from Acrobiosystem (#MSN-H5253-100 ug, #MSN-HF223-25 ug). Her3 ectodomain was purchased from Acrobiosystem (#ER3-H5223-100 ug). The extracellular domain of human CD123 protein was purchased from SinoBiological (# 10518-H02H-50 ug). Human 5T4 extracellular domain was purchased from Acrobiosystem (#TPG-H5253-100 ug).
For the C8 FSY sdAb, the cross-linking kinetics were analyzed as in example 11. FIG. 13 shows crosslinking for 0-180 minutes. The C8-54 FSY sites were found to have the highest crosslinking rate (about 50% crosslinking in 2 hours) compared to the other sites. Similarly, the rates of cross-linking of C18-FSY and C18-FSK sdabs with the HER3 receptor were compared. Between positions 33, 101 and 103, it is observed that position 101FSY achieves the highest crosslinking efficiency and rate. For the C18 sdAb FSK variants at positions 30, 32, 35, it was observed that position 35FSK achieved the highest crosslinking efficiency and rate for Her 3. Evaluation of the 3 sites in the C20 FSY sdAb indicated that position 57 achieved the highest crosslinking efficiency and rate for CD 123.
Example 18 identification and kinetics of FSY-modified DARPin
Her2 was screened for its ability to bind DARPin (C15) by inserting FSY amino acids for cross-linking. Libraries and library strains were constructed, expressed and purified according to the general procedure of example 2. C15 The WT sequence (SEQ ID NO 29) is shown in Table 14, the region screened by FSY insertion is shown in bold, and the amino acid position of the FSY substitution is shown in the second row of the table.
TABLE 14 amino acid positions for replacement of FSY into HER2 DARPin
Each individual C15-FSY mutant was incubated with Her2 receptor for crosslinking efficiency following the general procedure of example 11. Sites 9, 12, 37, 66, 68 in C15 show cross-linking with Her2 receptor as shown in figure 14. To identify FSY crosslinking sites at the fastest rate, DARPin with the FSY sites identified above was incubated with Fc-labeled Her2 (Acrobiosystem: acrobiosystem: #her2-H5253-100 ug) and checked for crosslinking at 15 and 30 minutes. The C15-66 FSY site was found to have a higher crosslinking yield than the other sites.
Double paratope DARPin (C15-66 FSY-C16, hereinafter C38-FSY) was constructed by fusing a DARPin copy (SEQ ID NO 30) without FSY substitution C16 to the C-terminus of C15-66FSY with a 5 amino acid GGGGS linker to prepare fusion proteins. The C38-FSY protein was incubated with Fc labeled Her2 and checked for cross-linking following the general procedure of example 11. The double paratope C38-FSY crosslinked Her2 in a time dependent manner, 50% in 2.5 hours, as shown in FIG. 15.
Example 19 cell binding to Targeted imaging payload
C22-TYR and C22-FSY (SEQ ID Nos. 39 and 40, respectively) were expressed and purified according to the general procedure of example 2, except that after elution from the Ni-NTA resin, the purified protein buffer was exchanged into anion exchange running buffer (20 mM Tris, 7.5 and 20mM NaCl) using a dilution factor of 1:100 at 4C overnight. The dialyzed sample library was passed through a HiTrap Q XL 5ml column (Cytiva # 17515801) in flow-through mode, where endotoxin was bound to the column. The sdAb monomer-circulating fractions were analyzed by reducing SDS-PAGE and pooled. Purified C22-TYR and C22-FSY were then conjugated to AZ Dye 680 (CLICK CHEMISTRY Tools, catalyst # 1578-25).
AZ Dye680 (CLICK CHEMISTRY Tools, catalog # 1578-25) was conjugated to C22-FSY and C22-TYR using a sortase-mediated linkage to produce C23-TYR and C23-FSY. The sortase gblock sequence was codon optimized and sequenced by IDT, cloned into the pBAD vector, and prepared as described (PubMed: 21697512). To conjugate the sdAb construct with AZ Dye680, 20mg of the purified protein in PBS at pH 7.4 was incubated with 1mM CaCl 2, 0.5mM AZ Dye680, 2mg sortase in a shaker at 30 ℃ (final concentration of sdAb was about 0.7 mg/mL). After 2 hours, 2mM MTSET was added to quench the reaction. The quenched reaction was batch-bound to 2mL (PBS equilibrated) Ni resin to remove sortase and unconjugated material, and turned around for 30 minutes at room temperature. The beads were spun to facilitate separation of the resin from unbound fraction (700 g x 10min,20 ℃), and the flow through was collected by loading a glass column and washing the beads with PBS to collect the remaining protein. The flow-through and wash solutions were combined and concentrated (5K MWCO,4k rcf,10C,1.5h) and then loaded onto HiLoad 26/600Superdex 75pg (Cytiva, # 28-9893-34) to remove unconjugated dye. Fractions were collected and analyzed by SDS-PAGE and pooled and stored at-80 ℃.
The binding of the resulting conjugates C23-TYR and C23-FSY to human epidermoid squamous cell carcinoma line a431 (egfr+) and human colorectal cancer cell line COLO320DM (EGFR-) was assessed by flow cytometry. Proteins were formulated to 30 μm (10 x final concentration) with FACS buffer (1 xpbs+2% HI-fbs+5mM EDTA) and then serially diluted 5x (20 μl sdAb to 80 μl FACS buffer) to generate 8 concentration spots for assay, with a minimum concentration of 0.000384 μm (10 x final concentration). Both the A431 and COLO320DM cell lines (ATCC) were maintained in an humidified environment of 5% CO 2 at 37℃in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher). Cells were harvested at the exponential growth phase, counted, resuspended in ice-cold FACS buffer at 1.1X10 6/mL and aliquoted at 90. Mu.L/well into v-bottom 96-well plates (Corning 3897). The C23-TYR and C23-FSY proteins were added to A431 cells at 10 uL/well to give a final concentration of 1X. To control the specificity of binding to egfr+ cells, test preparations of the same dilution range were added to COLO320DM cells. After 2 hours incubation on ice, cells were pelleted at 2000rpm for two minutes, then washed twice with 200 μl FACS buffer, then resuspended in 100 μl ice-cold FACS buffer, and analyzed with NovoCyte flow cytometer (ACEA/Aglient).
Raw data (FCS file) was analyzed using FlowJo 10.7.2. Total intact cells were identified using FSC-A and SSC-A, and then FSC-A was used to screen FSC-H for single peaks. The geometric mean fluorescence intensity of AZ680 of the samples was used for further analysis and mapping. The geometric mean fluorescence intensity (GeoMean or GeoMFI) of the 'log (agonist) and response-variable slope (four parameters)', AZ680 channels was used to calculate EC50 in GRAPHPAD PRISM (V9.3.1).
As shown in fig. 16, C23-FSY and C23-TYR bound to a431 cells in a dose-dependent manner did not show binding to EGFR-negative COLO320DM cells. As shown in Table 15, the binding affinities of C23-FSY and C23-TYR were measured based on staining of A431 cells and EC50 values after flow cytometry.
TABLE 15 binding affinity of C23-FSY and C23-TYR AF680 conjugates for A431 cells
C23-TYR C23-FSY
EC50(nM) 95 18
EC50 range 415.2-613.1 444.7-618.2
Square R 0.99 1.00
Example 20 in vivo tumor locking Using Targeted imaging payloads
This study assessed in vivo cross-linking of fluorescently labeled sdabs. The C22-FSY and C22-TYR were modified as in example 19 to produce AZ680 conjugates C23-FSY and C23-TYR (SEQ ID No.42, 41, respectively). In vivo cross-linking of C23-FSY and C23-TYR with EGFR was evaluated in A431 epidermoid squamous cell carcinoma xenograft tumor models. Human colorectal cancer xenograft model COLO320DM served as EGFR control. Both cell lines were cultured according to the supplier's protocol (ATCC). On the day of injection, cells were harvested, washed in serum-free medium, counted and resuspended in cold serum-free medium. The A431 cell pellet was resuspended in 100mL serum-free DMEM and COLO320DM 100. Mu.L RPMI1640, and the cells were then mixed with 100mL Matrigel to a final concentration of 5X 10 6 viable cells/100. Mu.L. 200mL volumes containing 1000 ten thousand A431 or COLO320DM cells were implanted into the right upper flank (CHARLES RIVER) of 6-8 week old female Balb/c nude mice. When the tumor size reached about 200mm 3 mg/kg of C23-FSY, C23-TYR or vehicle control (PBS) was injected through the tail vein in a volume of 10 mL/kg. Peripheral blood samples were collected via cheek bleeding 0.5 hours post-dosing to confirm dosing accuracy.
As shown in table 16, the biodistribution of the test articles was studied at 2 different time points. Animals were euthanized, tumors were harvested, weighed and placed on ice for IVIS imaging ex vivo, and then flash frozen for Western blot analysis.
TABLE 16 time points of biodistribution analysis
And (5) tissue treatment. A piece of tumor (about 30 to 50 mg) was weighed and cut into small pieces with razor blades. Excised tumor tissue was placed in CK28-R tubes (Bertin Instruments, cat.P000916 LYSK0-A) and 0.5ml of T-PER buffer (Thermo Scientific Cat.78510) containing a mixture of Hall protease and phosphatase inhibitors (Thermo Scientific Cat.78446) was added. The tissue was then homogenized with PRECELLYS a 24 tissue homogenizer. After homogenization, the samples were centrifuged at 12,000x rpm for 10 minutes at 4 ℃. The supernatant was then retained and the precipitate discarded. The centrifugation process was repeated once more (twice in total).
Gel imaging. Protein concentration of tumor lysates was quantified using PIERCE RAPID Gold BCA protein assay kit (Thermo Scientific cat.a 53225) according to the manufacturer's instructions. 7-point 2-fold dilutions of BSA starting from 1mg/mL BSA in the kit were used as standard. Samples were formulated to the same concentration (3.6 mg/mL) with T-PER buffer, and then heated at 95℃for 10 minutes in 1 Xreduced loading buffer, resulting in a final concentration of 3 mg/mL. Next, 30ug of total protein per sample was loaded onto a standard TGX gel (Bio-Rad, cat.5671085) of 4-20% for electrophoresis. Images were acquired by Azure Biosystem C600,600 using the NIR-700 channel. GIMP 2.10.28 is used to process images.
Figure 17 shows the time-dependent manner of intratumoral release and EGFR cross-linked sdAb in a431 (egfr+) and COLO320DM (EGFR-) tumors. Free sdAb was detected in the about 15kD region (lower panel, labeled free VHH) and EGFR crosslinked sdAb band was detected in the about 175kD region (upper panel, labeled VHH-EGFR crosslink). In EGFR+A431 tumors, time-dependent crosslinking of EGFR with C23-FSY was observed, but no time-dependent crosslinking with C23-TYR was observed. Neither free sdAb retention nor cross-linking was observed in EGFR-COLO320DM tumors.
In vitro IVIS imaging data analysis: tumors were dissected and imaged with AMI HTX; imaging data was analyzed using Aura software (Spectral Instruments imaging, version 4.0.7). The average radiation efficiency value (Ellipse ROI tools in Aura) was obtained by creating an ROI around a single tumor. The total emission (photons/s) was measured and recorded. The total emission values were normalized to tissue weight (photons/s/g). Statistical significance was determined in Prism (v 9.1.0 (221)) using paired T-test (=p.ltoreq.0.05, =p.ltoreq.0.005). FIG. 18 shows ex vivo imaging of A431 and COLO320DM tumor tissue 8 and 24 hours after administration of C23-FSY and C23-TYR.
FIG. 18A shows photons/s/g tumor tissue in three replicates. Values from individual animals are shown, the center bar shows the mean intensity, and the error bars represent SEM (by paired t-test, × p=0.024, × p=0.002). At the time points of 8 hours and 24 hours, the level of C23-FSY in A431 tumors was significantly higher compared to the C23-TYR protein. After 24 hours, C23-TYR was observed at very low levels in A431 tumor tissues, whereas C23-FSY, in particular EGFR-VHH cross-linked species, were prominent (FIG. 17, 18A). Since the test preparations have similar affinities (fig. 16), these results together demonstrate that tumor AUC of sdAb containing FSY can be increased by covalency.
FIG. 18B shows the quantitative ex vivo fluorescence intensity of A431 and COLO320DM tumors in animals administered C23-FSY 8 hours and 24 hours after dosing. The figure shows photons/s/g tumor tissue in three replicates. Values from individual animals are shown, with the center bar showing average intensity and the error bars representing SEM. At the 8 hour and 24 hour time points, the level of C23-FSY in A431 tumors was significantly higher than in EGFR-COLO320DM tumors, indicating the tumor locking specificity of the C23-FSY protein.
These results indicate that a targeting domain with cross-linked compatible unnatural amino acids, such as an sdAb, can carry a payload (here an imaging dye) into a tumor with target specificity, and can also improve the retention of the payload at the target site as compared to a targeting domain without cross-linked compatible unnatural amino acids.
Example 21 in vivo PSMA tumor locking using targeted imaging payloads
This study assessed in vivo PSMA crosslinking, systemic and intratumoral exposure of fluorescent labeled sdabs in LNCaP mice prostate cancer xenograft tumor models.
PSMA-targeted sdabs conjugated to AZ Dye680 were prepared according to the method of example 19 using C29-101TYR and C29-101FSY (SEQ ID NOs: 53 or 54) (with TYR or FSY at position 101) to produce C30-TYR and C30-FSY (SEQ ID NOs: 55, 56, respectively). LNCaP cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum in a humidified environment at 37℃with 5% CO 2. On the day of implantation, cells were harvested, washed in serum-free medium, counted and resuspended in cold serum-free medium. The cell pellet was resuspended in 50 μl serum-free RPMI and mixed with 50 μl L MATRIGEL to a final concentration of 5x 10 6 viable cells per 100 μl. A volume of 100. Mu.L containing 500 tens of thousands of LNCaP cells was implanted into the right upper flank of male NU/J mice (Jackson Labs). When the tumor size reached about 200mm 3 mg/kg of C30-TYR or C30-FSY was injected through the tail vein in a volume of 10 mL/kg. Peripheral blood samples were collected via cheek bleeding 0.5 hours post-dosing to confirm dosing accuracy. As shown in table 17, the biodistribution of the test articles was studied at 8 different time points (15 minutes, 1h, 6h, 24h, 48h, 72h and 96h before administration, after administration). After euthanasia, pooled tumor tissues were harvested, weighed, IVIS imaged ex vivo, and then flash frozen for western blot analysis.
Table 17 time points of PK sampling
Tissue treatment, protein quantification and normalization were performed using the method of example 20. To quantify free and PSMA-crosslinked sdAb assay preparations, a 2-fold 16-point standard curve of C30-FSY was prepared in T-PER buffer starting from 500 ng/mL. The standard was heated in 1x reducing loading buffer at 95 ℃ for 10 minutes. Different ranges of standards were used for different gels based on an estimate of the concentration of the test article in the sample. A standard curve was generated using a linear fit function in Excel, using the band area of the standard versus its concentration. The density of bands in the image was determined using ImageJ 1.51j8 according to the instructions (https:// Imagej. Nih. Gov/ij/docs/guide/146-30.Html # info box: densitometry, section 30.13). After obtaining the area of the bands of the samples with ImageJ, the amount of TA in the lanes was calculated using the formula derived from the linear fit of the standard curve. When 30 μg of total protein was loaded, the amount of TA in the lane was also the amount of TA in 30 μg of total protein. Tumor sdAb concentration (pg sdAb/mg tumor tissue) =amount of sdAb in lane (pg) ((protein concentration (mg/ml) (0.5 ml×1000/30 μg)/tumor tissue weight (mg)) the resulting standard curve contains at least 5 points of R2>0.99 using a trapezoidal rule to calculate the area under the curve (AUC) using GRAPHPAD PRISM, where the area between two adjacent points is calculated as Δx ([ (y1+y2)/2 ] -baseline ] (https:// www.graphpad.com/guides/prism/last/station_area_un_t he_curve.htm) C30-TYR AUC is 1676, and C30-Y AUC is 4902 fsm.
FIG. 19 shows fluorescent images of SDS-PAGE gels showing the time-dependent manner of tumor-associated free sdAb and PSMA cross-linked sdAb following administration of C30-TYR and C30-FSY. At designated time points after treatment, tumors were collected and subjected to gel electrophoresis treatment to detect fluorophore conjugated sdAb assay preparations. Free (non-crosslinked) sdAb-AF680 was detected in the approximately 20kD region (lower panel), and PSMA-crosslinked sdAb-AF680 species of C30-FSY migrated in the 100kD region (upper panel). Lanes marked with x show vehicle samples.
Figure 20 shows quantitative analysis of tumor-associated free and PSMA cross-linked test preparations. Fluorescence band intensities from the free and PSMA crosslinked species bands of the above gels were quantified via densitometry and compared to a standard curve. Total intratumoral test article concentration (free and crosslinked PSMA, pg/mg tumor tissue) was plotted against time (h). Data points expressed as x represent samples below the detection and quantification limit. The tumor exposure of C30-FSY was increased to about 3X compared to non-covalent C30-TYR.
Example 22 delivery of cytotoxic payload
C25 and C27 sdabs were prepared with and without FSY substitutions to generate C25-54TYR, C25-54FSY, C27-101TYR, and C27-101FSY (SEQ ID NOS: 45, 46, 49, 50, respectively). Proteins were expressed and purified according to the general method of example 2, except that after elution from the Ni-NTA resin, the purified protein buffer was exchanged into anion exchange running buffer (20 mM Tris,7.5 and 20mM NaCl) using a dilution factor of 1:100 at 4C overnight. The dialyzed sample library was passed through a HiTrap Q XL 5ml column (Cytiva # 17515801) in flow-through mode, where endotoxin was bound to the column. The sdAb monomer-circulating fractions were analyzed by reducing SDS-PAGE and pooled.
The sdAb construct was conjugated to MC-PEG8-VC-PABC-MMAE (structure shown in the following figure), forming C26-54 TYR, C26-54 FSY, C28-101TYR and C28-101FSY. The samples were first reduced with 1mM EDTA and 1.5 equivalents of TCEP (10 mM in deoxygenated water) and incubated for 1 hour at room temperature. After reduction, any residual TCEP was removed using a Zeba Spin desalting column (Thermo P/N8989891, 7k MWCO) using 1mM EDTA in 1X PBS as equilibration buffer. Propylene Glycol (PG) and Dimethylacetamide (DMA) were added to the reaction mixture (cf=10%v/v) to increase the linker payload solubility and 1.5 equivalents of linker payload was added. The reaction was mixed by gentle vortexing and incubated overnight at 4 ℃. The conjugate mixture was added to a pre-hydrated dialysis cartridge (about 1mL sample: 3500mL buffer volume) and dialyzed for at least 2 hours. The dialysis buffer was changed and the sample was dialyzed overnight at 4 ℃ before being removed for analysis by a280 and analytical SEC. The unconjugated linker payload was removed using Cytiva PD-10 columns (4.3 mL Sephadex G-25 adsorbent per column) according to standard PD-10 spin protocols (equilibration buffer: 1xPBS, pH 7.4). After collecting the eluate, the column was washed with another 1mL of 1XPBS at pH 7.4. After purification on PD-10, the samples were subjected to a final dialysis step using a Thermo SCIENTIFIC SLIDE-A-Lyzer Mini dialysis apparatus (3.5 k MWCO) using 50mL of 1 xPBS. All samples were passed through a 0.2 μm sterile PES filter and stored at-20 ℃.
MC-PEG8-VC-PABC-MMAE：
To measure the ability of FSY-containing sdAb conjugates to specifically deliver cytotoxic payloads to tumor cell lines expressing immortalized PSMA in vitro as compared to non-covalent sdAb-conjugated agents, cell permeable payload monomethyl auristatin was conjugated to sdabs at a drug-to-antibody ratio (DAR) of 1. The PC3pip cell line and PC3 flu PSMA negative cell line engineered for PSMA expression (provided by CASE WESTERN RESERVE University, cleveland, OH, professor XinningWang and Warren D.Heston) were maintained in growth medium consisting of RPMI-1640 (Thermo Scientific, 11875-903) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher, FB-12) in a humidified environment at 37℃with 5% CO 2. Cells were harvested during the exponential growth phase using TrypLE Express (Thermo Scientific, 14175-095) and inoculated in 100 μl of growth medium at 800 cells/well in a black 96 well flat transparent bottom plate (Costar, 3603). Cells were then incubated overnight at 37℃in a humidified environment of 5% CO 2 to allow the cells to adhere to the plates. The sdAb-MMAE conjugates listed in the following table were diluted to 50x final concentration in growth medium and diluted in 3-fold increments to form a 10-point dilution series. Each 50x test article dilution series of 2 μl volumes of solution was added to the wells in triplicate to achieve 1x final concentration (the highest concentration for each series is indicated in table 18).
TABLE 18 test article conjugates
Name of the name Highest concentration in dose response (1X)
C26-54TYR 158nM
C26-54FSY 137nM
C28-101TYR 208nM
C28-101FSY 127nM
The plates were returned to the incubator for 4 days, after which cell viability was assessed. CELLTITER G1o (Promega, G5737) was added to all plates at 50. Mu.L/well, and the plates were shaken at 1000rpm for one minute at room temperature. Luminescence was then read on a Victor X5 plate reader (PerkinElmer) and reported as Relative Luminescence Units (RLU). Viability was calculated in Excel (applicable to Microsoft 365MSO,Version 2202Build 16.0.14931.20128, 64-bit) Viability% = 100 x (treated RLU/untreated RLU). Data are plotted in GRAPHPAD PRISM (V9.3.1) as% viability versus concentration of test articles and IC50 is calculated using 1og inhibitor versus reaction-variable slope (four parameters).
The sdAb-PEG8-VC-PABC-MMAE conjugate was observed to have a very low level of cytotoxic activity on the PC3flU cell line, consistent with the lack of PSMA target expression on this cell line. The estimated affinity of C26-54TYR for PMSA was 14.9nM (as shown in example 14 for unconjugated compounds on LNCaP cells), showing a dose-dependent increase in cytotoxicity to PC3pip cells, consistent with targeted delivery of the payload via PSMA binding and internalization. As shown in Table 19, the potency of C26-54FSY in delivering the payload was 3.57x for C26-54TYR, indicating that the covalency enhanced the cellular potency of the single domain antibody drug conjugate. In a second example, C28-101TYR with an affinity for PSMA >500nM had lower cytotoxic activity against PC3pip cells, while the crosslinking activity of C28-101FSY shifted the potency by more than 400x, confirming the effect of covalency on cell potency. Figure 21A (C26 construct) and figure 21B (C28 construct) show cytotoxicity comparisons of test preparations showing concentration of sdAb conjugated to MMAE versus cell viability curve. Viability of psma+pc3pip and PSMA-PC3flu cells was assessed by CellTiter-Glo assay after 4 days incubation with sdAb-PEG8-VC-PABC-MMAE conjugate. Dose response analysis was performed in triplicate, symbols represent averages, and error bars represent STDEV.
TABLE 19 IC50 values for cytotoxicity of sdab-PEG8-VC-PABC-MMAE conjugates
Example 23 delivery of cytotoxic payload to HER2 target
HER 2-targeted sdabs (C17; SEQ ID NO: 31) were evaluated to identify independent positions of FSY insertion that provided cross-linking with HER2 targets. Expression and purification of sdabs is typically cloned according to the method of example 2, except that specific individual sites are selected for modification and testing, rather than using pooled screens. Selected sites for FSY insertion are shown in table 20.
TABLE 20 FSY insert sdAb library of interest to target HER2
Positions 52 and 54 are identified as cross-linked to HER2 target. To evaluate FSY crosslinking kinetics, the 52FSY and 54FSY variants of C17 were incubated with Her2 receptor and the crosslinking efficiency was checked. As shown in fig. 22, the 54FSY site was found to have the highest crosslinking rate, crosslinking >90% in one hour, while 52FSY crosslinked to 90% in 2 hours. Both constructs were selected, and their non-FSY (TYR) counterparts were conjugated to the cytotoxic payload.
Table 21 conjugates based on C17 variants
Name of the name
C33-54FSY
C33-52FSY
C33-52TYR
C33-54TYR
To conjugate the sdAb construct with GGG-PEG8-vc-PABC-MMAE, C32-54TYR, C32-54 FSY, C32-52TYR, and C32-52FSY compounds were purified as described above. The compounds were incubated in PBS at pH 7.4, with 1mM CaCl 2, 10 equivalents of linker payload (10 mM stock solution in 10% DMA) and sortase (2 mg sortase/10 mg sdAb), and incubated overnight at 4 ℃. The reaction was batch bound to 2mL (PBS equilibrated) Ni resin to remove sortase and unconjugated material, and turned around for 30 minutes at room temperature. The beads were spun to facilitate separation of the resin from unbound fraction (700 g x 10min,20 c) and the flow through was collected by loading a glass column and washing the beads with PBS to collect the remaining protein. The flow-through and wash solutions were combined and concentrated (5K MWCO,4k rcf,10C,1.5h) and loaded onto HiLoad 26/600Superdex 75pg (Cytiva, # 28-9893-34) to remove unconjugated dye. Fractions were collected and analyzed by SDS-PAGE, pooled and stored at-80 ℃.
GGG-PEG8-VC-PABC-MMAE：
The binding of C33 conjugates containing TYR or FSY at positions 52 and 54 (table 21) to human breast cancer lines BT-474 (her2+) was assessed. Proteins were diluted to 3 μm (1 x final) in FACS buffer (1 xpbs+2% HI-fbs+5mM EDTA) and then serially diluted 3x to generate 8 concentration points for assay, with a minimum concentration of 0.00384 μm (1 x final).
The BT-474 cell line (ATCC HTB-20) was maintained in a growth medium consisting of RPMI-1640 (Thermo Scientific, 11875-903) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher, FB-12) in a humidified environment at 37℃with 5% CO 2. Cells were harvested at the exponential growth phase and resuspended at 1.0x10 6/mL in ice-cold FACS buffer (PBS pH6.0/2% FBS/5mM EDTA) at pH6.0 and aliquoted at 100 μl/well into v-bottom 96-well plates (Corning 3897). Cells were pelleted at 2000rpm for 2 minutes and then resuspended in titration of test preparation in pH6.0 FACS buffer, 100. Mu.L/well in duplicate. After 2 hours incubation on ice, cells were pelleted at 2000rpm for 2 minutes and then washed twice with 200 μl FACS buffer. The cells were then resuspended in 100. Mu.L of ice-cold FACS buffer (pH 7.4) containing 1:100 dilutions of AF488 goat anti-alpaca VHH antibody (Jackson ImmunoResearch-547-232). After incubation on ice for 30 minutes, the cells were pelleted at 2000rpm for two minutes, washed twice with 200 μl FACS buffer, then resuspended in 100 μl FACS buffer pH 7.4, and analyzed with NovoCyte flow cytometer (ACEA/Aglient).
Raw data (FCS file) was analyzed using FlowJo 10.7.2. FSC-A and SSC-A were used to identify total intact cells, and then FSC-A was used to gate A single peak for FSC-H. The geometric mean fluorescence intensity of sample AF488 was used for further analysis and mapping. The geometric mean fluorescence intensity of the AF488 channel (GeoMean or GeoMFI) was used for calculation of EC50 in GRAPHPAD PRISM (V9.3.1) using 'log (agonist) and response-variable slope (four parameters)'. The test article binds to BT-474 cells in a dose dependent manner. As shown in table 22, the binding affinities of the test preparations were measured according to staining of BT-474 cells and EC50 values after flow cytometry.
TABLE 22 binding affinity of BT-474 cells
Test article (conjugate) EC50(nM)pH6.0
C33-54FSY4 .6
C33-54TYR2 .5
C33-52FSY 49
C33-52TYR 160
The conjugates were then used in a cell-based assay to measure the ability of the covalent sdAb conjugate to specifically deliver a cytotoxic payload to an immortalized HER2 expressing tumor cell line in vitro with a non-covalent sdAb conjugate. The BT-474 cell line was maintained as described above. Cells were harvested at the exponential growth phase using TrypLE Express (Thermo Scientific, 14175-095) and inoculated at 5000 cells/well in 100 μl of growth medium in a black 96 well flat transparent bottom plate (Costar, 3603). The cells were then incubated overnight at 37 ℃ in a humidified environment of 5% CO 2 to allow the cells to adhere to the plates. The next day, one plate was analyzed by CTG to determine background. The remaining plates were treated with C33-TYR and C33-FSY conjugates as follows: the conjugates listed in table 23 were diluted in growth medium to 11x final concentration (1.1 μm) and then diluted in 5-fold increments to form a 10-point dilution series. Each of a 10uL volume dilution series of 11x test article was added to the wells in triplicate to achieve a final concentration of 1 x. A set of plates was returned to the incubator for 5 hours, after which each well was gently rinsed once with 100 μl of growth medium, then the growth medium was replaced (100 μl/well), and the cells were cultured in a humidified environment of 5% CO 2 at 37 ℃ for 6 days, thereby washing out the test article. Another set of plates was incubated with the test articles in a humidified environment of 5% CO 2 at 37 ℃ for an entire 6 days. On day 6, to assess cell viability, the growth medium of all the cultured plates was removed, CELLTITER GLO (Promega, G5737) was mixed with an equal volume of PBS and added to all plates at 100 μl/well, and the plates were then shaken at 1000rpm for one minute at room temperature. Luminescence was then read on a Victor X5 plate reader (PerkinElmer) and reported as Relative Luminescence Units (RLU). Viability was calculated in Excel (applicable to Microsoft 365MSO,Version 2202Build 16.0.14931.20128,64 bits) Viability% = 100 x (treated RLU-background RLU/untreated RLU-background RLU). Data are plotted as% viability versus concentration of test articles in GRAPHPAD PRISM (V9.3.1) and IC50 is calculated using log [ inhibitor ] and response-variable slope (four parameters).
The results are shown in FIG. 23 and Table 23. Fig. 23A shows washout for 5h, and fig. 23B shows exposure for 6 consecutive days. All dose response assays were performed in triplicate, symbols represent averages, and error bars represent STDEV. FSY variants showed increased cytotoxicity relative to their TYR counterparts in 5 hour wash out studies and 6 day continuous exposure experiments, demonstrating superior cellular potency in covalent form.
Table 23.5 hour wash out and 6 day exposure IC50 values for cytotoxicity.
Name of the name IC50(nM)-5h IC50 (nM) -6 days
C33-52TYR ＞100 8.417
C33-52FSY ＞100 1.599
C33-54TYR 21.56 0.3279
C33-54FSY 1.00 0.106
EXAMPLE 24 evaluation of double paratope constructs-binding
As shown in table 24, a series of single and double paratope sdabs with and without FSY were prepared and compared for their ability to cross-link PSMA targets. The protein was expressed according to the general procedure of example 2 and modified in example 19.
Table 24 single and double paratope constructs
The test article was evaluated for binding to psma+lncap human prostate tumor cells using flow cytometry following the general procedure of example 14, and the following modifications were made.
C34-TYR, C35-TYR and C2-54TYR were formulated with FACS buffer to 3uM (1 Xfinal), and then serially diluted 5 Xto generate 12 concentration points for measurement at a minimum concentration of 0.06pM (1 Xfinal). In a separate experiment, C24-101TYR was formulated with FACS buffer and then serially diluted 5X to generate 8 concentration spots for measurement, the lowest concentration was 38.4pM (1X final). The LNCaP cells in this assay were 120,000 cells/well except for 160,000C 24-101 TYR/well. After two hours incubation with the test article on ice, the cells were washed twice (500. Mu.L/well FACS buffer each time (500 Xg, 3 min), and then incubated on ice for 30 minutes in the dark with 100. Mu.L of ice-cold FACS buffer containing 5. Mu.g/ml Alexa Fluor 488AffiniPure goat anti-alpaca IgG, VHH domain (Jackson ImmunoResearch, code: 128-545-230) secondary antibody. Cells were then washed twice (200. Mu.L/wash) with FACS buffer and resuspended in 100. Mu.L FACS buffer and collected on a Novocyte 2060 (s/n 45-1-1511-2036-9) flow cytometer using NovoExpress software version 1.5.0. The raw data (FCS 3.1 file) was analyzed to determine the geometric mean intensity (FlowJo v.10.8.1, windows) of Alexa Fluor-488 (in FITC channel). The mean of the mean Alexa Fluor-488 geometric intensities of the samples and the STDEV versus log concentration of the test article are plotted in GRAPHPAD PRISM (v 9.3.1). EC50 was calculated using the equation 'log (agonist) and response-variable slope (four parameters'). For visualization of the data, the binding curves for each test article were normalized in Prism, with the average of the lowest values for each test article set to 0% and the average highest value for each test article set to 100%. Binding affinities of the test article were measured by EC50 values after LNCaP cell flow staining, as shown in table 25. The biparatopic protein binds LNCaP cells in a dose dependent manner. The data indicate that an increased affinity is observed compared to the parent monomer compound, and thus binding of the biparatopic compound has an affinity effect.
TABLE 25 binding affinity in LNCaP cells
EXAMPLE 25 crosslinking of FSY-modified double paratope constructs with target cells
Crosslinking of C34-FSY and C36-FSY with PSMA-expressing cells was compared to monovalent C24-101 FSY. LNCaP cells in growth medium were seeded at a density of 250,000 cells/well in 12-well plates coated with poly-L-lysine. 48 hours after inoculation, the medium was removed and 0.4mL of medium containing the test compounds at the concentrations shown in Table 26 was added for 1 or 6 hours. Cells were washed twice with 0.5 mL/Kong Bingleng of 1xPBS and 0.15 mL/well RIPA buffer with 1x protease inhibitor cocktail was added. After incubation on ice for 10 minutes, the plates were scraped with a trimmed 200 μl pipette tip and cell lysates were collected. The lysate was transferred to a 1.5mL Eppendorf tube and centrifuged at 12,000rpm for 10 minutes at 4 ℃. After centrifugation, the supernatant was transferred to a new 1.5mL Eppendorf tube, and 0.2 mL/well of 0.25% EDTA-trypsin (Gibco, cat. No. 25200-056) was added.
Denatured samples were analyzed by electrophoresis and Western blotted with primary anti-human PSMA (Invitrogen, cat. No. 37-3900) or internal standard anti-GAPDH (CELL SIGNALING Technology, cat. No. CST-2118). The image was acquired by Azure Biosystem C600,600. GIMP 2.10.28 is used to process images. Band densities in Western blots were determined by means of ImageJ 1.51j8 (https:// Imagej. Nih. Gov/ij/docs/guide/146-30.Html#info box: densitome, section 30.13) as indicated. The percentage of crosslinked PSMA to total PSMA was calculated as follows: density of crosslinked PSMA/(density of crosslinked psma+density of uncrosslinked PSMA) x 100%. The density versus concentration profile for each test article (construct) is shown in fig. 24A and 24B.
Western blotting results are shown in FIGS. 25A and 25B. All 3 constructs tested were capable of crosslinking to PSMA. The double paratope construct C34-FSY showed higher apparent molecular weight migration than the monomer cross-bands, reflecting the increase in complex mass, while the C36-FSY construct showed higher apparent molecular weight cross-bands, indicating cross-linking of the two PSMA molecules. The intensity of the bands of all FSY compounds increased in a time and concentration dependent manner, indicating the presence of specific crosslinks. Quantification of crosslinking indicates that crosslinking of both biparatopic constructs with PSMA is more efficient and more powerful than single paratope constructs. (Table 26 and FIGS. 25A and 25B).
TABLE 26 kinetics of crosslinking in LNCaP cells
Example 26 double paratope conjugate with cytotoxic payload
Test preparations containing a cytotoxic payload conjugated to a dual paratope sdAb compound were prepared according to the methods generally described in examples 24 and 25, and modified as follows. To achieve cysteine-maleimide conjugation of the PEG8-VC-PABC-MMAE linker payload to the biparatopic compound, the plasmid sequence encoding the compound C34-FSY or C34-TYR was modified to contain cys residues at or near the C-terminus, which compound was expressed to incorporate FSY or TYR at position 101 for conjugation and purification.
To enable the PEG8-VC-PABC-MMAE linker payload to be sortag-mediated conjugation with a biparatopic compound, the sequence encoding the compound C34-FSY or C34-TYR was modified to contain the C-terminal sortase identification and His tag sequence, and the compound was expressed to incorporate FSY or TYR at position 101 for conjugation and purification.
PC3pip cell lines engineered to express PSMA and PC3flu PSMA negative cell lines were maintained, inoculated, and prepared for study as described in example 22. The dual paratope sdAb MMAE conjugate was diluted in growth medium to a final concentration of 50x, and then diluted in 3-fold increments to form a 10-point dilution series. Each test article dilution series was added to the wells in triplicate to achieve a final concentration (highest concentration) of 1x for each series.
A set of plates was returned to the incubator for 5 hours, after which each well was gently rinsed once with 100 μl of growth medium to wash out the test article, and then the growth medium was replaced (100 μl/well) and the cells were cultured in a humidified environment of 5% CO 2 at 37 ℃ for 6 days. Another set of plates was incubated with the test article in a humidified environment of 5% CO 2 at 37 ℃ for an entire 6 days. On day 6, to assess cell viability, the growth medium of all plates was removed, CELLTITER GLO (Promega, G5737) was mixed with an equal volume of PBS and added to all plates at 100 μl/well, and the plates were then shaken at 1000rpm for one minute at room temperature. Luminescence is then read on a Victor X5 plate reader (PerkinElmer) or similar device and reported as Relative Luminescence Units (RLU). Viability was calculated in Excel or similar procedure as% viability=100 (treated RLU-background RLU/untreated RLU-background RLU). Data are plotted as% viability versus concentration of test articles in GRAPHPAD PRISM (V9.3.1) or similar procedure, and IC50 is calculated using log [ inhibitor ] and response-variable slope (four parameters).
EXAMPLE 27 mouse xenograft model for treatment of PSMA+ tumors
To evaluate the effect of tyrosine and FSY containing single and double paratope conjugates in controlling tumor growth in a mouse xenograft tumor model, cell lines LNCaP (psma+) and PC3 (PSMA-) were used. Cell lines were maintained, expanded and implanted into Balb/C nude mice as described in examples 20 and 21. When the tumor size reached about 200mm 3, the double paratope C34-TYR or C34-FSY conjugate (example 25), single paratope C26-54TYR or C26-54FSY conjugate (example 22) or vehicle, such as 5mg/kg per day, was injected through the tail vein at different doses and schedules for 7 days. Tumor size was measured daily via caliper measurements to track tumor growth over time. Animals with tumor volumes up to 1000mm 3 were sacrificed and survival times were recorded.
Example 28-EGFR-targeting double paratope construct
EGFR-targeting sdabs with FSY (C4-109FSY,SEQ ID NO:17) and dual paratope constructs with two EGFR-targeting sdabs linked by a linker (C4-109 TYR or FSY) -L1-C5, hereinafter referred to as C37-TYR or C37-FSY, SEQ ID NO:69, were constructed, cloned and expressed as described in example 2. The construct was synthesized with pelB leader sequence (SEQ ID NO: 15) cleaved from the mature protein and His-Tag purified using six C-terminal histidines. Double paratope construct C37 comprises a GGGGSGGGGS (SEQ ID NO: 14) linker between the first sdAb C4 and the second sdAb C5.
Kinetics of EGFR crosslinking of FSY modified sdabs were assessed. The protein was incubated with EGFR at a molar ratio of 8:1 (EGFR final concentration 0.125mg/mL, 1.25. Mu.M). Samples were taken at time points of 0-180 minutes and the percentage of EGFR cross-linking was assessed by SDS-PAGE. The percentage of cross-bands was calculated by quantifying sdAb EGFR cross-bands and EGFR band intensity was quantified using Image J.
Samples were collected from time zero to 360 minutes. The kinetics of the coupling are shown in FIGS. 26-27. The double paratope construct was compared to a single paratope construct comprising only sdAb C4 with FSY modification (C4-109 FSY). As shown in figure 27, the dual paratope construct C37-FSY coupled to EGFR faster than the single paratope C4-109FSY sequence, as measured by the reduced time of half maximum cross-linking compared to the single paratope construct.
Example 29: construction of hetero-multispecific binding conjugates
Heterobispecific FSY-containing compounds are constructed that target multiple different antigens with a target sdAb ("target sdAb 1"), such as a tumor cell antigen, linked by a linker to an sdAb that targets a different tumor cell antigen expressed on the same cell ("target sdAb 2"). In each case, target sdAb1, target sdAb2, or both, may comprise FSY. The bispecific construct was then cloned into the pBAD vector and expressed as described in example 2.
By ligating C4-109FSY (or variant thereof) from example 2 with Her 3-targeting sdAb (without FSY), compounds containing heterobispecific FSY targeting EGFR and Her3 extracellular domains were generated. Alternative constructs were generated that included Her 3-targeting sdAb (FSY-with) linked to C4 (FSY-free). Constructs were designed by adding a repeated linker containing the sequence GGGGS or similar sequences between two sdAb amino acid sequences. The bispecific construct was then cloned into the pBAD vector and expressed as described in example 2. Examples of bispecific constructs are expected to have the amino acid sequences shown below.
MKYLLPTAAAGLLLLAAQPAMAMGQVQLVQSGGGLVQAGGSLR
LSCAFSGRTFSMYTMGWFRQAPGKEREFVAANRGRGLSPDIADSVN
GRFTISRDNAKNTLYLQMDSLKPEDTAVYYCAADLQYGSSWPQRSS
AEYDYWGQGTTVTVSSGGGGSGGGGSGGGGSQVKLEESGGGSVQT
GGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGY
ADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYG
TLXEYDYWGQGTQVTVSSHHHHHH
X represents the position of the FSY incorporation site; the bold N-terminal residue is the pelB leader sequence cut from the mature protein. The C-terminal 6 histidines contained His-Tag for affinity purification. Other bispecific constructs for generation may include Her3-EGFR, EGFR-Her2, her2-Her3, her3-Her3 (same or different epitopes), EGFR-cMET, and EGFR-CEA.
Conjugates using bispecific FSY modified sdabs were prepared by engineering unique conjugation sites for chemical coupling of the linker/payload. The bispecific FSY modified construct described above is modified to contain free Cys residues at the C-terminus of the construct or other permissible sites within the construct that do not disrupt binding to the respective target upon modification. Alternative methods may include a linker/payload sortase-mediated or transglutaminase-mediated linkage, conjugation of NHS esters to lysine residues, or other methods common in the art. Conjugates with the linker payload are generated to attach the targeting domain (sdAb or other binding protein), the linker, and the payload, which may include toxic payloads, chelated radiometals, peptides, or protein toxins, or other functional cytotoxic compounds.
Bispecific FSY modified sdabs or conjugates thereof were evaluated for in vitro biochemical crosslinking with EGFR and Her3, respectively. The protein is incubated with EGFR or Her3 receptor domain in an excess stoichiometric ratio (such as 5:1 or 8:1). Samples were incubated, and the percentage of EGFR or Her3 crosslinked with sdAb FSY compound was assessed via gel displacement using SDS-PAGE and quantified by densitometry.
ELISA was used to assess whether bispecific FSY modified sdabs or conjugates thereof bind to EGFR and Her3 simultaneously. The extracellular domain of Her3 or EGFR is adsorbed to the surface of the microtiter plate and extensively washed. The plate was blocked for non-specific interactions and bispecific FSY modified sdabs were added and incubated to allow binding. The wells are then washed to remove unbound material, and then a second receptor subunit is added and incubated to allow binding. Plates were again washed to remove unbound compounds and the second receptor was detected using anti-receptor antibodies. Alternatively, the second receptor subunit is added as a fusion or conjugate between the receptor domain and a detection system (such as HRP or fluorescent dye) and detected by standard colorimetric or fluorescent detection. As a specific control, the addition of the first receptor, bispecific compound or second receptor was omitted from the procedure. Binding of the anti-receptor antibody can be detected via an anti-species HRP secondary antibody, biotinylated anti-receptor antibody detected with streptavidin-HRP, a fluorescent dye conjugated secondary antibody, or other similar method standard of ELISA detection.
Surface Plasmon Resonance (SPR) was used to assess whether the bispecific FSY modified sdAb or conjugate thereof binds to EGFR and Her3 simultaneously. The extracellular domain of Her3 or EGFR receptor is immobilized on the surface of the SPR chip via NHS ester chemistry or other standard methods. Bispecific FSY modified sdAb compounds are then bound to the receptor 1 surface prior to washing to remove unbound material. To assemble and detect the ternary complex, the second receptor subunit is then injected and the signal is measured. As a specific control, the addition of the first receptor, bispecific compound or second receptor was omitted from the procedure, as some affinity was observed between the receptor subunits.
Specific binding of the bispecific FSY modified sdAb or conjugate thereof to EGFR and Her3 positive cells was assessed. A series of test preparations of different concentrations are applied to the cells and the dose dependent binding of the test preparations can be detected by flow cytometry or by cell-based ELISA or similar methods. To determine the contribution of each receptor specificity to the observed binding signal, a competitive antibody (cold competitor) was added in large stoichiometric excess to antagonize EGFR, her3, or the test article engagement of both receptors. After incubation and washing to remove unbound antagonist antibody, a concentration series of bispecific test preparations are added to the cells and allowed to bind. After incubation and washing to remove unbound test compounds, test preparations, such as anti-epitope tags or anti-sdAb antibodies or other methods, are detected with antibodies specific for the compounds. Binding of the anti-test sample antibody can be detected via flow cytometry, or by ELISA via anti-species HRP secondary antibodies, biotinylated anti-receptor antibodies detected with streptavidin HRP, fluorochrome conjugated secondary antibodies, or other similar method criteria for ELISA detection.
The dual specificity FSY modified sdAb or conjugate thereof is evaluated for the delivery of toxic payloads specifically to tumor cells expressing one or more target antigens in vitro. Constructs containing engineered C-terminal Cys residues are coupled to a cytotoxic payload, such as MMAE or other classes of highly cytotoxic compounds, using the general methods described above. Dose response curves were generated and samples were incubated with SKBR3 and Colo320DM cells in vitro. As a specific control, cross-reactive antibodies to the first receptor, the second receptor, or both are added to certain samples. After incubation for different periods of time, the cells are washed to remove free compounds and cell viability is measured using an assay such as Promega CytoxGreen, or other assays that measure cell viability via reporter compound detection, detecting living cells by prest Blue, CCK-8 or similar reagents and/or measuring apoptosis via annexin V-FITC, propidium iodide staining or similar reagents.
The dual specificity FSY modified sdAb or conjugate thereof was evaluated for delivery of the payload to tumor tissue in vivo. To compare tumor exposure of the construct to the non-covalent sdAb conjugate, a fluorophore label was used as a representative payload to enable tracking/biodistribution measurements over time. The sdAb construct with single residue substitution Tyr or FSY is conjugated to a chemical fluorophore (Alexa 680 or analog) via maleimide chemistry or other methods described above via engineered cysteine residues and purified as described above. The fluorophore-labeled sdADC conjugate molecules were administered by tail IV injection to male nude mice bearing tumor antigen + or-xenograft tumors and the biodistribution of the test article over time was observed via whole animal imaging using an AmiX, IVIS spectral imager or similar device. Tumor-specific and peripheral exposure were quantified via image densitometry and a comparison was made between FSY and Tyr versions of sdAb. In addition to imaging, tumor samples are collected and subjected to gel-based detection methods, such as western blot or fluorescence imaging, to detect time-varying cross-linked sdAb receptor complexes and free sdabs in tumor tissue following administration.
While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. The following claims are intended to define the scope of the present disclosure and thus cover methods and structures within the scope of these claims and their equivalents.

Claims (216)

1. A conjugate comprising a targeting domain and a payload, wherein the targeting domain comprises at least one Unnatural Amino Acid (UAA) residue, wherein the targeting domain is configured to bind to a target, and wherein the UAA residue is sufficiently close to form a covalent bond with the target when the targeting domain binds.
2. The conjugate of claim 1, wherein the payload is attached to an amino acid at position n+x relative to the UAA residue, wherein n is the position of the amino acid and x is at least 1.
3. The conjugate of claim 1, wherein the payload is attached to an amino acid at position n-x relative to the UAA residue, wherein n is the position of the amino acid and x is at least 1.
4. The conjugate of any one of claims 1 to 3, wherein the UAA residue is within 5-20 angstroms of the target when the targeting domain binds to the target.
5. The conjugate of claim 4, wherein the targeting domain binds to a cell surface molecule.
6. The conjugate of claim 5, wherein the cell surface molecule is a tumor associated antigen.
7. The conjugate of any one of claims 1-6, wherein the UAA residue is configured to form a covalent bond with a histidine, lysine, or tyrosine residue of the target.
8. The conjugate of any one of claims 1-7, wherein the UAA residue comprises a fluorosulfate moiety.
9. The conjugate of any one of claims 1-7, wherein the UAA residue comprises an aryl fluorosulfate moiety.
10. The conjugate of claim 9, wherein the UAA residue comprises formula I:
11. the conjugate of claim 9, wherein the UAA of the UAA residue has the structure:
12. the conjugate of claim 9, wherein the UAA of the UAA residue has the structure:
13. the conjugate of claim 9, wherein the UAA residue comprises formula II:
14. the conjugate of claim 9, wherein the UAA of the UAA residue has the structure:
15. The conjugate of claim 9, wherein the UAA of the UAA residue has the structure:
16. The conjugate of claim 9, wherein the UAA residue comprises formula III:
17. the conjugate of claim 9, wherein the UAA of the UAA residue has the structure:
18. the conjugate of claim 9, wherein the UAA of the UAA residue has the structure:
19. the conjugate of any one of claims 1-7, wherein the UAA of the UAA residue has the structure of formula (IA):
wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is-O-or-NR-, m is 1; when Y is-n=m is 2.
20. The conjugate of claim 19, wherein the UAA of formula (IA) has formula (IA-a): Is a structure of (a).
21. The conjugate of claim 19, wherein the UAA of formula (IA) has formula (IA-b): Is a structure of (a).
22. The conjugate of claim 19, wherein the UAA of formula (IA) has the structure of formula (IB):
23. The conjugate of claim 19, wherein the UAA of formula (IA) has the structure of formula (IC):
24. The conjugate of claim 19, wherein the UAA of formula (IA) has the structure of formula (ID):
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
25. The conjugate of claim 19, wherein the UAA of formula (IA) has the structure of formula (IE):
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
26. The conjugate of claim 19, wherein the UAA of formula (IA) has the structure of formula (IIA):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
27. The conjugate of claim 19, wherein the UAA of formula (IA) has the structure of formula (IIB):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
28. The conjugate of any one of claims 1-7, wherein the UAA of the UAA residue is of formula (IV)Is characterized in that the structure of the (c) is that,
Wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
Ring a is a 5 to 6 membered aryl or heteroaryl group;
Each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; each R X is optionally substituted alkyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
29. The conjugate of any one of claims 1-28, further comprising at least one additional targeting domain.
30. The conjugate of any one of claims 1-29, wherein the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent.
31. The conjugate of claim 30, wherein the cytotoxic moiety comprises a small molecule drug or a chemotherapeutic drug.
32. The conjugate of claim 30, wherein the radioligand reagent is selected from 35S、3H、111In、112In、14C、186Re、188Re、32P、153Sm、177Lu、86Y、88Y、90Y、131I、123I、124I、125I、149Tb、211At、212Pb/212Bi、213Bi、223Ra、225Ac、64Cu、67Cu and 227 Th.
33. The conjugate of claim 31 or claim 32, wherein the radioligand reagent further comprises a chelator.
34. The conjugate of claim 30 or claim 33, wherein the radioligand reagent is selected from 99mTc、131I、201Tl、111 In and 67 Ga.
35. The conjugate of any one of claims 1-34, wherein the payload is attached to the conjugate with a linker.
36. The conjugate of claim 35, wherein the linker comprises a polymer.
37. The conjugate of claim 35, wherein the linker is a cleavable or non-cleavable linker.
38. The conjugate of claim 35, wherein the linker is 0.01kDa to 50kDa.
39. The conjugate of claim 35, wherein the linker is 0.01kDa to 10kDa.
40. The conjugate of any one of claims 35-39, wherein the linker is a linear, branched, multimeric, or dendrimer.
41. The conjugate of claim 40, wherein the linker is a bifunctional or polyfunctional linker or a bifunctional or polyfunctional polymer.
42. The conjugate of any one of claims 35-41, wherein the linker comprises a water-soluble polymer.
43. The conjugate of claim 42, wherein the water-soluble polymer is polyethylene glycol (PEG).
44. The conjugate of claim 43, wherein the molecular weight of the PEG is between 0.1kDa and 2.5 kDa.
45. The conjugate of claim 43, wherein the PEG comprises 1-8 monomers.
46. The conjugate of any one of claims 1-45, wherein the targeting domain comprises an antibody, antibody fragment, or antigen binding domain.
47. The conjugate of claim 46, wherein the targeting domain comprises an antigen binding domain, the conjugate comprises a CDR region, and the at least one UAA is within or near the CDR region.
48. The conjugate of claim 47, wherein the UAA is contained within the CDR regions.
49. The conjugate of any one of claims 46-48, wherein the targeting domain comprises a single domain antibody (sdAb).
50. The conjugate of any one of claims 5-49, wherein the cell surface molecule is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ephA2, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.
51. A conjugate comprising (i) a payload and (ii) an engineered single domain antibody (sdAb) comprising an sdAb having a CDR region and at least one Unnatural Amino Acid (UAA) residue within or near the CDR region, wherein the sdAb comprises any of SEQ ID NOs 1-4 or 16-64.
52. The conjugate of claim 51, wherein the UAA residue comprises a fluorosulfate moiety.
53. The conjugate of claim 52, wherein the UAA residue comprises an aryl fluorosulfate moiety.
54. The conjugate of claim 53, wherein the UAA residue comprises formula I:
55. the conjugate of claim 53, wherein the UAA of the UAA residue has the structure:
56. The conjugate of claim 53, wherein the UAA of the UAA residue has the structure:
57. the conjugate of claim 53, wherein the UAA residue comprises formula II:
58. the conjugate of claim 53, wherein the UAA of the UAA residue has the structure:
59. the conjugate of claim 53, wherein the UAA of the UAA residue has the structure:
60. The conjugate of claim 53, wherein the UAA residue comprises formula III:
61. the conjugate of claim 53, wherein the UAA of the UAA residue has the structure:
62. The conjugate of claim 53, wherein the UAA of the UAA residue has the structure:
63. the conjugate of claim 53, wherein the UAA of the UAA residue has the structure of formula (IA):
wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is-O-or-NR-, m is 1; when Y is-n=m is 2.
64. The conjugate of claim 63, wherein the UAA of formula (IA) has formula (IA-a): Is a structure of (a).
65. The conjugate of claim 63, wherein the UAA of formula (IA) has formula (IA-b): Is a structure of (a).
66. The conjugate of claim 63, wherein the UAA of formula (IA) has the structure of formula (IB):
67. the conjugate of claim 63, wherein the UAA of formula (IA) has the structure of formula (IC):
68. the conjugate of claim 63, wherein the UAA of formula (IA) has the structure of formula (ID):
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
69. The conjugate of claim 62, wherein the UAA of formula (IA) has the structure of formula (IE):
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
70. The conjugate of claim 63, wherein the UAA of formula (IA) has the structure of formula (IIA):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
71. The conjugate of claim 63, wherein the UAA of formula (IA) has the structure of formula (IIB):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
72. The conjugate of claim 51, wherein the UAA of the UAA residue is of formula (IIV)Is characterized in that the structure of the (c) is that,
Wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
Ring a is a 5 to 6 membered aryl or heteroaryl group;
Each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; each R X is optionally substituted alkyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
73. The conjugate of any one of claims 51-72, wherein the engineered sdAb comprises SEQ ID No. 1, and wherein the UAA is comprised in any one of SEQ ID NOs 5, 6, and 7.
74. The conjugate of claim 73, wherein the UAA is present at an amino acid position selected from 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115 relative to SEQ ID No. 1.
75. The conjugate of any one of claims 51-72, wherein the engineered sdAb comprises SEQ ID No. 2, and wherein the UAA is comprised in any one of SEQ ID NOs 8, 9 and 10.
76. The conjugate of claim 75, wherein the UAA is present at an amino acid position selected from 50, 52, 53, 54, 56, 58, and 100 relative to SEQ ID No. 2.
77. The conjugate of any one of claims 51-72, wherein the engineered sdAb comprises SEQ ID No. 3, and wherein the UAA is comprised in any one of SEQ ID NOs 11, 12 and 13.
78. The conjugate of claim 77, wherein the UAA is present at an amino acid position selected from 58, 62, 101, 103, and 107 relative to SEQ ID No. 3.
79. The conjugate of any one of claims 51-72, wherein the engineered sdAb comprises SEQ ID No. 16, and wherein the UAA is present at an amino acid position of 109 relative to SEQ ID No. 16.
80. The conjugate of any one of claims 51-72, wherein the engineered sdAb comprises SEQ ID No. 25, and wherein the UAA is present at an amino acid position selected from 52, 53, 54, 55, 56, 58, 60, 62, and 64 relative to SEQ ID No. 23.
81. The conjugate of any one of claims 51-72, wherein the engineered sdAb comprises SEQ ID No. 26, and wherein the UAA is present at an amino acid position selected from 53, 55, 56, 57, 58, 60, 64, and 67 relative to SEQ ID No. 24.
82. The conjugate of any one of claims 51-81, wherein the payload is not linked via the UAA side chain.
83. The conjugate of any one of claims 51-82, wherein the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent.
84. The conjugate of claim 83, wherein the cytotoxic moiety comprises a small molecule drug or a chemotherapeutic drug.
85. The conjugate of claim 83, wherein the radioligand reagent is selected from 35S、3H、111In、112In、14C、186Re、188Re、32P、153Sm、177Lu、86Y、88Y、90Y、131I、123I、124I、125I、149Tb、211At、212Pb/212Bi、213Bi、223Ra、225Ac、64Cu、67Cu and 227 Th.
86. The conjugate of claim 83 or claim 85, wherein the radioligand reagent further comprises a chelator.
87. The conjugate of claim 83, wherein the imaging agent comprises a fluorophore or a radioligand reagent selected from 99mTc、131I、201Tl、111 In and 67 Ga.
88. A method comprising administering the conjugate of any one of claims 1-87, wherein the conjugate covalently binds to a target on the surface of a cell.
89. The method of claim 88, wherein the cells comprise tumor cells.
90. The method of claim 89, wherein the conjugate kills the tumor cell or inhibits growth of the tumor cell.
91. The method of any one of claims 88-90, wherein the target is a tumor associated antigen.
92. The method of any one of claims 88-91, wherein the target is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ephA2, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.
93. A method of treating a disease or condition comprising administering the conjugate of any one of claims 1-92 to a subject in need thereof.
94. The method of claim 93, wherein the method comprises, wherein the disease comprises PRAD (prostate cancer), mCRPC (metastatic castration resistant prostate cancer), solid tumor (neovasculature), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), HNSC (head and neck squamous cell carcinoma, THCA (thyroid cancer), ESCA (esophagus cancer), STAD (stomach adenocarcinoma), GIST (gastrointestinal stromal tumor), COAD (colon adenocarcinoma), READ (rectal adenocarcinoma), SARC (sarcoma), SCLC (small cell lung cancer), MESO (mesothelioma), PAAD (pancreatic adenocarcinoma), B-cell malignancy, T-cell malignancy, ALL (acute lymphoblastic leukemia), NHL (non-Hodgkin's lymphoma) HL (hodgkin's lymphoma), CLL (chronic lymphocytic leukemia), AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), MSI-high tumor, SKCM (cutaneous melanoma), UVM (uveal melanoma), DLBC (diffuse large B-cell lymphoma), endometrial cancer, CESC (cervical cancer), bone cancer, BLCA (bladder urothelial cancer), BRCA (invasive breast cancer), TNBC (triple negative breast cancer), LIHC (hepatocellular carcinoma), OV (ovarian serous cystic carcinoma), UCEC (endometrial carcinoma), NE-PCa (neuroendocrine prostate cancer), UCEC (endometrial carcinoma), one or more of GBM (glioblastoma multiforme) and KIRC (renal clear cell carcinoma).
95. A method of making the conjugate of any one of claims 1-87, comprising:
(a) Generating the targeting domain comprising at least one unnatural amino acid; and
(B) The targeting domain is conjugated to the payload, optionally via a linker.
96. The method of claim 95, wherein the targeting domain comprising at least one unnatural amino acid is synthesized in vivo.
97. The method of claim 96, wherein generating comprises using an orthogonal tRNA synthetase/suppressor tRNA pair.
98. The method of claim 97, wherein generating comprises the orthogonal tRNA synthetase/suppressor tRNA pair that is derived from a pyrrolysine tRNA synthetase/tRNA Pyl.
99. The method of claim 97, wherein the orthogonal tRNA synthetase comprises SEQ ID NOs 84, 87, 92 or a variant thereof.
100. A method of delivering a cytotoxic payload to a cell comprising administering the conjugate of any one of claims 30-33, 35-50, or 83-86, wherein the conjugate covalently binds to a target on the surface of the cell, thereby delivering the cytotoxic payload.
101. The method of claim 100, wherein the cell is a tumor cell.
102. The method of claim 100, wherein the cell is contained within a tumor microenvironment.
103. The method of claim 100, wherein the cell is contained within a mammalian subject.
104. The method of claim 100, wherein the cell is contained within a human subject.
105. The method of any one of claims 100-104, wherein the conjugate kills the tumor cell or inhibits growth of the tumor cell.
106. The method of any one of claims 100-105, wherein the target is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.
107. The method of any one of claims 100-106, wherein the human subject has or is diagnosed with a disease or condition selected from PCa (prostate cancer), CRPCa (castration-resistant prostate cancer), solid tumors (neovasculature), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal cancer), GC (gastric cancer), CRC (colorectal cancer), SCLC (small cell lung cancer), MPM (mesothelioma), PDAC (ductal pancreatic adenocarcinoma), ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), MSI-high tumors, melanoma, DLBCL (diffuse large B-cell lymphoma), endometrial cancer, cervical cancer, bladder cancer, brCa (breast cancer), TNBC (triple negative breast cancer), NE-PCa (neuroendocrine prostate cancer), GBM (glioblastoma), and RCC (renal cell carcinoma).
108. A conjugate comprising (i) a first targeting domain, (ii) a second targeting domain, and optionally (iii) a payload, wherein the first targeting domain comprises at least one first Unnatural Amino Acid (UAA), whereby the first targeting domain is capable of covalently binding to a first target at the site of the UAA, and the second targeting domain is configured to bind to a second target.
109. The conjugate of claim 108, wherein the first target and the second target are located on the same cell.
110. The conjugate of claim 108 or claim 109, wherein the first targeting domain comprises an antibody, antibody fragment or antigen binding domain.
111. The conjugate of claim 110, wherein the first targeting domain comprises a single domain antibody (sdAb).
112. The conjugate of any one of claims 108-111, wherein the first UAA is contained within or near a region of the first targeting domain that interfaces with the first target.
113. The conjugate of any one of claims 108-112, wherein the second targeting domain comprises an antibody, antibody fragment, or antigen binding domain.
114. The conjugate of claim 113, wherein the second targeting domain comprises a single domain antibody (sdAb).
115. The conjugate of any one of claims 108-114, wherein the first targeting domain and the second targeting domain are linked to form a fusion protein.
116. The conjugate of any one of claims 108-114, wherein the first targeting domain and the second targeting domain are linked by chemical conjugation.
117. The conjugate of claim 115 or claim 116, wherein the first targeting domain and the second domain are linked by a linker.
118. The conjugate of any one of claims 108-117, wherein the first targeting domain and the second targeting domain bind to the same target.
119. The conjugate of claim 118, wherein the first targeting domain and the second targeting domain bind to different epitopes of the same target.
120. The conjugate of claim 118, wherein the first targeting domain and the second targeting domain bind to the same epitope of the same target.
121. The conjugate of any one of claims 118-120, wherein the same target is a monomer.
122. The conjugate of any one of claims 118-120, wherein the same target is a multimeric molecule.
123. The conjugate of any one of claims 108-117, wherein the first targeting domain and the second targeting domain bind different targets.
124. The conjugate of any one of claims 108-123, wherein the first target is a first cell surface molecule.
125. The conjugate of any one of claims 108-124, wherein the second target is a second cell surface molecule.
126. The conjugate of any one of claims 108-125, wherein the at least one first UAA comprises a fluorosulfate moiety.
127. The conjugate of any one of claims 108-125, wherein the at least one UAA comprises an aryl fluoro sulfate moiety.
128. The conjugate of claim 127, wherein the at least one first UAA comprises formula I:
129. the conjugate of claim 127, wherein the at least one first UAA has the structure:
130. The conjugate of claim 127, wherein the at least one first UAA has the structure:
131. the conjugate of claim 127, wherein the at least one first UAA comprises formula II:
132. the conjugate of claim 127, wherein the at least one first UAA has the structure:
133. The conjugate of claim 127, wherein the at least one first UAA has the structure:
134. The conjugate of claim 127, wherein the at least one first UAA comprises formula III:
135. the conjugate of claim 127, wherein the at least one first UAA has the structure:
136. the conjugate of claim 127, wherein the at least one first UAA has the structure:
137. the conjugate of any one of claims 108-125, wherein the at least one first UAA has the structure of formula (IA):
wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is-O-or-NR-, m is 1; when Y is-n=m is 2.
138. The conjugate of claim 137, wherein the at least one first UAA has the formula (IAa): Is a structure of (a).
139. The conjugate of claim 137, wherein the at least one first UAA has the formula (IAb): Is a structure of (a).
140. The conjugate of claim 137, wherein the at least one first UAA has the structure of formula (IB):
141. the conjugate of claim 137, wherein the at least one first UAA has the structure of formula (IC):
142. the conjugate of claim 137, wherein the at least one first UAA has the structure of formula (ID):
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
143. The conjugate of claim 137, wherein the at least one first UAA has the structure of formula (IE):
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
144. The conjugate of claim 137, wherein the at least one first UAA has the structure of formula (IIA):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
145. The conjugate of claim 137, wherein the at least one first UAA has the structure of formula (IIB):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
146. The conjugate of any one of claims 108-125, wherein the at least one first UAA has formula (IV)Is characterized in that the structure of the (c) is that,
Wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
Ring a is a 5 to 6 membered aryl or heteroaryl group;
Each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; p is 0, 1,2,3 or 4; each R X is optionally substituted alkyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
147. The conjugate of any one of claims 124-146, wherein the first cell surface molecule is selected from PSMA, EGFR, HER2, HER3, PD-L1, ephA4, fibronectin ED-B、EpCAM、CCR4、CD25、VEGF、VEGFR2、endo180、LIV-1、PTK7、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、DLK1、Muc16、cMET、CEA、LRP5, and LRP6.
148. The conjugate of any one of claims 125-147, wherein the second cell surface molecule is selected from PSMA, EGFR, HER2, HER3, PD-L1, ephA4, fibronectin ED-B、EpCAM、CCR4、CD25、VEGF、VEGFR2、endo180、LIV-1、PTK7、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、DLK1、Muc16、cMET、CEA、LRP5, and LRP6.
149. The conjugate of any of claims 108-148, wherein the second targeting domain comprises a second UAA, whereby the second targeting domain is capable of covalently binding to the second target at the site of the second UAA.
150. The conjugate of any of claims 149, wherein the second UAA is different from the at least one UAA contained in the first targeting domain.
151. The conjugate of claim 149, wherein the second UAA is identical to the at least one UAA contained in the first targeting domain.
152. The conjugate of any of claims 149-151, wherein the second UAA comprises a fluorosulfate moiety.
153. The conjugate of any of claims 149-151, wherein the second UAA comprises an aryl fluorosulfate moiety.
154. The conjugate of claim 153, wherein the second UAA comprises formula I:
155. The conjugate of claim 153, wherein the second UAA has the structure:
156. The conjugate of claim 153, wherein the second UAA has the structure:
157. the conjugate of claim 153, wherein the second UAA comprises formula II:
158. The conjugate of claim 153, wherein the second UAA has the structure:
159. The conjugate of claim 153, wherein the second UAA has the structure:
160. The conjugate of claim 153, wherein the second UAA comprises formula III:
161. The conjugate of claim 153, wherein the second UAA has the structure:
162. The conjugate of claim 153, wherein the second UAA has the structure:
163. the conjugate of any one of claims 149-151, wherein the second UAA has the structure of formula (IA):
wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is-O-or-NR-, m is 1; when Y is-n=m is 2.
164. The conjugate of claim 163, wherein the second UAA has the formula (IA-a): Is a structure of (a).
165. The conjugate of claim 163, wherein the second UAA has the formula (IA-b): Is a structure of (a).
166. The conjugate of claim 163, wherein the second UAA has the structure of formula (IB):
167. the conjugate of claim 163, wherein the second UAA has the structure of formula (IC):
168. The conjugate of claim 163, wherein the second UAA has the structure of formula (ID):
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
169. The conjugate of claim 163, wherein the second UAA has the structure of formula (IE):
Wherein:
each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
R 1 is hydrogen, fluorine or iodine; r 2 is hydrogen or methyl; and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
170. The conjugate of claim 163, wherein the second UAA has the structure of formula (IIA):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
171. The conjugate of claim 163, wherein the second UAA has the structure of formula (IIB):
Wherein:
x is independently O or NR'; and
When present, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.
172. The conjugate of any one of claims 149-151, wherein the second UAA is of formula (IV)Is characterized in that the structure of the (c) is that,
Wherein,
Each X is independently O or NR';
y is a bond, -O-, -NR-, or-N=;
A is a bond or- (CH 2)n -; m is 1 or 2;n is an integer from 1 to 4;
When present, each R and R' is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl;
Ring a is a 5 to 6 membered aryl or heteroaryl group;
Each R A is independently-OH, -OR X, halogen, NHR X、N(RX)2, OR optionally substituted alkyl; p is 0, 1,2,3 or 4; each R X is optionally substituted alkyl;
L is- (CH 2)p -or-C (O) NH- (CH 2)p -; p is an integer from 1 to 6), and
Wherein when Y is a bond, -O-, or-NR-, m is 1; when Y is-n=m is 2.
173. The conjugate of any one of claims 108-172, wherein the first targeting domain comprises any one of SEQ ID NOs 1-4 or 16-64.
174. The conjugate of any one of claims 108-172, wherein the first targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs 1-4 or 16-64.
175. The conjugate of any one of claims 108-173, wherein the second targeting domain comprises any one of SEQ ID NOs 1-4, 16-64.
176. The conjugate of any one of claims 108-173, wherein the second targeting domain comprises a sequence having at least 70% identity to any one of SEQ ID NOs 1-4, 16-64.
177. The conjugate of any one of claims 1-87 or 108-172, wherein the conjugate comprises SEQ ID NOs 65-72.
178. The conjugate of any one of claims 1-87 or 108-172, wherein the conjugate comprises a sequence having at least 70% sequence identity to SEQ ID NOs 65-72.
179. The conjugate of any one of claims 1-87 or 108-172, wherein the conjugate comprises any one of SEQ ID NOs 1-4, 16-64.
180. The conjugate of any one of claims 1-87 or 108-172, wherein the conjugate comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs 1-4, 16-64.
181. The conjugate of any one of claims 1-87 or 108-172, wherein the conjugate comprises a sequence having at least 80% sequence identity to SEQ ID NOs 65-72.
182. The conjugate of any one of claims 108-172, wherein at least one of the first targeting domain and the second targeting domain comprises SEQ ID No. 1.
183. The conjugate of claim 182, wherein the UAA is present at an amino acid position selected from 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115 relative to SEQ ID No. 1.
184. The conjugate of any one of claims 108-172, wherein at least one of the first targeting domain and the second targeting domain comprises SEQ ID No. 2.
185. The conjugate of claim 184, wherein the UAA is present at an amino acid position selected from 50, 52, 53, 54, 56, 58, and 100 relative to SEQ ID No. 2.
186. The conjugate of any one of claims 108-172, wherein at least one of the first targeting domain and the second targeting domain comprises SEQ ID No. 3.
187. The conjugate of claim 186, wherein the UAA is present at an amino acid position selected from 58, 62, 101, 103, and 107 relative to SEQ ID No. 3.
188. The conjugate of any one of claims 108-172, wherein at least one of the first targeting domain and the second targeting domain comprises SEQ ID No. 16.
189. The conjugate of claim 188, wherein the at least one of the first targeting domain and the second targeting domain comprising SEQ ID No. 16 further comprises an unnatural amino acid at position 109 relative to SEQ ID No. 16.
190. The conjugate of claims 108-172, wherein at least one of the first targeting domain and the second targeting domain comprises SEQ ID No. 18.
191. The conjugate of any one of claims 108-190, wherein the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent.
192. The conjugate of claim 191, wherein the cytotoxic moiety comprises a small molecule drug or a chemotherapeutic drug.
193. The conjugate of claim 191, wherein the radioligand reagent is selected from 35S、3H、111In、112In、14C、186Re、188Re、32P、153Sm、177Lu、86Y、88Y、90Y、131I、123I、124I、125I、149Tb、211At、212Pb/212Bi、213Bi、223Ra、225Ac、64Cu、67Cu and 227 Th.
194. The conjugate of claim 192 or claim 193, wherein the radioligand reagent further comprises a chelator.
195. The conjugate of claim 192, wherein the radioligand reagent is selected from 99mTc、131I、201Tl、111 In and 67 Ga.
196. The conjugate of any one of claims 108-195, wherein the payload is attached to the conjugate with a linker.
197. A method comprising administering the conjugate of any one of claims 108-196, wherein the conjugate covalently binds to the first target on the surface of a first cell.
198. The method of claim 197, wherein the conjugate binds to the second target on the surface of the first cell.
199. The method of claim 197 or claim 198, wherein the first targeting domain and the second targeting domain bind to the same target.
200. The method of claim 199, wherein the first targeting domain and the second targeting domain bind to the same epitope of the same target.
201. The method of claim 199, wherein the first targeting domain and the second targeting domain bind to different epitopes of the same target.
202. The method of claim 197 or claim 198, wherein the first targeting domain and the second targeting domain bind to different targets on the surface of the first cell.
203. The method of any of claims 197-202, wherein the second domain comprises a second UAA, and wherein the second UAA is covalently bound to the second target.
204. The method of any one of claims 197-203, wherein the first cell is a tumor cell.
205. The method of claim 204, wherein the conjugate, when bound to the first target, kills the tumor cell or inhibits growth of the tumor cell.
206. The method of claim 204, wherein the conjugate, when bound to the second target, kills the tumor cell or inhibits growth of the tumor cell.
207. The method of claim 204, wherein the conjugate, when bound to the first target and the second target, kills the tumor cell or inhibits growth of the tumor cell.
208. The method of any one of claims 197-207, wherein the first target is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ephA2, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.
209. The method of any one of claims 197-208, wherein the second target is selected from PSMA、EGFR、EGFRviii、MSLN、CEA、DLL3、FAP、CD33、HER3、PD-L1、EphA2、EphA4、HER2、SIRPa、DLK1、Muc16、LRP5、LRP6、endo180、LIV-1、SLAMF7、PTK7、GPR20、CDH6、CSP-1、CD71、PRLR、SEZ6、DLL1、NOTCH3 rec、NaPi2b、CD16、GCC、SSTR2、CAIX、CAXII、MC1R、CXCR4、B1R、GRPR、STEAP1、CD70、CD46、CD166、CLL-1、ADAM9、cKIT、CD36、CD73、ITGaVb3、ITGaVb6、GPC-1、CD38、CD51、FGFR3、Ly6E、CD44v6、ENPP3、CXCR3、CXCR5、FcRH5、VEGF、VEGFR2、CD45、CCR4、CD25、5T4、ROR1、TROP-2、NECTIN4、cMET、CD19、CD22、CD30、CD33、CD123、BCMA、CD79b、AXL、RON、B7-H3、B7-H4、KAAG1、Muc1、ADAM-9、GPNMB、EDB fibronectin, tissue factor, GPNMB, folRa, ephA2, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.
210. The method of any of claims 197-209, wherein the payload is a radiolabeling agent or a cytotoxic agent.
211. The method of any one of claims 198-209, wherein the payload is an imaging agent.
212. The method of claim 211, wherein the method images or identifies the first cell when the conjugate binds to the first target and the second target on the first cell.
213. A method of making the conjugate of any one of claims 108-196, comprising:
(a) Synthesizing in vivo the first targeting domain comprising at least one unnatural amino acid; and
(B) The payload is conjugated to the first targeting domain or the second targeting domain, optionally via a linker.
214. The method of claim 213, further comprising synthesizing the second targeting domain in vivo as a fusion protein with the first targeting domain.
215. The method of claim 213 or claim 214, wherein synthesizing comprises using an orthogonal tRNA synthetase/suppressor tRNA pair.
216. The method of claim 215, wherein synthesizing comprises the orthogonal tRNA synthetase/suppressor tRNA pair that is derived from a pyrrolysine tRNA synthetase/tRNA Pyl.
CN202280092390.2A 2021-12-22 2022-12-22 Cross-linked antibodies Pending CN118922209A (en)

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US202263388072P 2022-07-11 2022-07-11
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PCT/US2022/082277 WO2023122753A1 (en) 2021-12-22 2022-12-22 Crosslinking antibodies

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