WO2024227426A1 - hROR1的抗原结合蛋白及其用途 - Google Patents

hROR1的抗原结合蛋白及其用途 Download PDF

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WO2024227426A1
WO2024227426A1 PCT/CN2024/090585 CN2024090585W WO2024227426A1 WO 2024227426 A1 WO2024227426 A1 WO 2024227426A1 CN 2024090585 W CN2024090585 W CN 2024090585W WO 2024227426 A1 WO2024227426 A1 WO 2024227426A1
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amino acid
acid sequence
binding protein
light chain
variable region
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PCT/CN2024/090585
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English (en)
French (fr)
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邹斌
钟琛
刘绍军
杜平
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泰诚思(上海)生物医药有限公司
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  • the present invention relates to an antigen-binding protein of hROR1, a bispecific antigen-binding protein, a nucleic acid molecule encoding the antigen-binding protein, a vector, a cell, a drug molecule, and uses thereof.
  • Receptor tyrosine kinase orphan receptor-1 ROR1
  • ROR2 family member receptor tyrosine kinase orphan receptor-2
  • ROR1 and ROR2 have similar structures and are both type I transmembrane proteins.
  • the mature full-length protein contains an extracellular region with three different domains (Ig-like, Frizzled and Kringle domains), a transmembrane region and an intracellular region.
  • the intracellular region contains a tyrosine kinase domain, two serine/threonine-rich domains and a proline-rich domain.
  • ROR1 and ROR2 are highly expressed in a variety of tumor tissues, but ROR2 is also expressed at low to moderate levels in some normal tissues. Although ROR1 can be detected in embryonic tissues, it is almost not expressed in normal adult tissues, except for very low levels of expression in the testis, uterus, lung, bladder and colon (Clin Cancer Res 2008; 14(2), doi:10.1158/1078-0432.CCR-07-1823), and is therefore considered a good tumor target.
  • ROR1 is widely expressed in both hematological tumors and solid tumors.
  • ROR1 is highly expressed in hematological tumors such as chronic lymphocytic leukemia (CLL), some acute lymphocytic leukemia (ALL), and chronic mantle cell lymphoma (CML), and ROR1 is highly expressed in a variety of solid tumors such as sarcoma, ovarian cancer, renal cell carcinoma, head and neck cancer, breast cancer, lung cancer, malignant mesothelioma, colon cancer, neuroblastoma, melanoma and other tumor tissues.
  • High expression of ROR1 is associated with accelerated course and poor prognosis of cancers such as CLL and ovarian cancer (Sci Rep.
  • ROR1 may promote the occurrence and development of tumors by participating in multiple signal transduction pathways. Reports have shown that ROR1 may mediate Wnt5a, Wnt5b and Wnt16 signals, enhance cell proliferation, inhibit cell apoptosis, induce epithelial-mesenchymal transition (EMT) and cancer cell migration and metastasis, and contribute to pit formation and promote endocytosis (Cells 2021, 10, 142, doi: 10.3390/cells10010142; Nat Commun 2016 Jan 4; 7: 10060. doi: 10.1038/ncomms10060).
  • EMT epithelial-mesenchymal transition
  • ROR1 is specifically expressed in tumors and may promote tumor growth, so ROR1 can be considered a good target for cancer therapy drugs, and work has been done to develop ROR1-specific antibody targets. Because ROR1 has high homology between different mammalian species, its extracellular domain amino acid sequence is exactly the same between humans and crab-eating monkeys, 96.8% identical between humans and mice, and 96.6% identical between humans and rats. Therefore, it is difficult to produce high-affinity antibodies against this target through standard techniques such as animal immunization.
  • the strategies targeting ROR1 mainly include monoclonal antibodies, bispecific antibodies, antibody-drug conjugates and CAR-T.
  • the extracellular region of ROR1 has three different domains.
  • VLS-101 and NBE002 target the Ig-like domain
  • LCB71 targets the Frizzled domain.
  • Specific recognition of different domains or different epitopes in the same domain may bring different affinities, endocytosis efficiencies or biological functions.
  • bispecific antibodies composed of different antibodies that recognize different epitopes may increase the functional properties of the antibodies.
  • existing ADC products have safety and drug resistance issues.
  • the main purpose of the present invention is to provide an antigen binding protein that can specifically bind to hROR1 and has good antigen binding activity.
  • the first aspect of the present invention relates to an isolated antigen binding protein capable of binding to hROR1, comprising any one of a to o:
  • a. having a first heavy chain variable region comprising HCDR1 of the amino acid sequence TYAMS, HCDR2 of the amino acid sequence SISSGGSTYYPDSVKG, and HCDR3 of the amino acid sequence YSLMVEAHWYFDV,
  • HCDR1 having an amino acid sequence of SYAMS
  • HCDR2 having an amino acid sequence of SISSGGSTYYPDSVKG
  • HCDR3 having an amino acid sequence of GYSNYVDWYFDV
  • HCDR1 with an amino acid sequence of DYTMH
  • HCDR2 with an amino acid sequence of GIDPNYGGTNYNEKFKD
  • HCDR3 with an amino acid sequence of DDGYYEDYFDY
  • d. having a fourth heavy chain variable region comprising HCDR1 of the amino acid sequence SYWIE, HCDR2 of the amino acid sequence EILPGSGNTQNNEKFKG, and HCDR3 of the amino acid sequence EGRRSHFFDY,
  • HCDR1 of the amino acid sequence RYAMS e. having a fifth heavy chain variable region, comprising HCDR1 of the amino acid sequence RYAMS, HCDR2 of the amino acid sequence SISSGGSTYYPDSVKG, and HCDR3 of the amino acid sequence GYYGNNDWYFDV,
  • LCDR1 having a first light chain variable region, LCDR1 containing an amino acid sequence of KASQDINSYLS, and an amino acid sequence of RANRLVD LCDR2, LCDR3 with the amino acid sequence LQYDEFPYT,
  • g. having a second light chain variable region comprising LCDR1 with an amino acid sequence of KATQDINSYLS, LCDR2 with an amino acid sequence of RPNRLVD, and LCDR3 with an amino acid sequence of LQYDEFPYT,
  • h. having a third light chain variable region comprising LCDR1 with an amino acid sequence of HASQGISSNIG, LCDR2 with an amino acid sequence of HGTNLED, and LCDR3 with an amino acid sequence of VQYAQFPYT,
  • LCDR1 having an amino acid sequence of KSSQSLLNSSNQKNYLA
  • LCDR2 having an amino acid sequence of FASTRES
  • LCDR3 having an amino acid sequence of QQHYSSPWT
  • j. having a fifth light chain variable region comprising LCDR1 with an amino acid sequence of KASQDINSYLS, LCDR2 with an amino acid sequence of RANSLVD, and LCDR3 with an amino acid sequence of LQFDEFPYT,
  • the framework region has a mouse-derived framework region or a human-derived framework region.
  • the framework region is an IgG framework region.
  • the human framework region has a back mutation at one or more sites relative to the mouse framework region.
  • a constant region is further present.
  • the constant region has a LALA mutation.
  • the heavy chain constant region of any one of a to e, k to o contains one or more mutations of C220S, A121C, V205C, K149C, D265C, S239C, A330C, and S442C.
  • the CDR sequences are determined according to the Kabat definition scheme.
  • the antigen binding protein comprises an antibody or an antigen binding fragment thereof.
  • the antibody is selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the antigen-binding fragment is selected from a Fab antibody, a single-chain antibody or a single-domain antibody.
  • the heavy chain variable region has a S62Q, S62A, S62N, S62E, S62V, S62Y, S62F or S62K mutation.
  • the light chain variable region has a G57A, G57S, G57N or G57L mutation.
  • the light chain variable region has a D56E, D56Q, D56T or D56Y mutation.
  • the light chain variable region has a S31A, S31Q, S31L, S31V, S31E, S31Y, S31F or S31K mutation.
  • the light chain variable region has a T57A, T57Q, T57L, T57V, T57E, T57Y, T57F or T57K mutation.
  • the light chain variable region has S27A, S27Q, S27L, S27V, S27E, S27Y, S27F, and S27K mutations.
  • the heavy chain has an amino acid sequence as shown in any of SEQ ID No. 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 65, 73, 75, 78, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 91, 92, 93, 134 or 135, and A light chain having an amino acid sequence as shown in any of SEQ ID No.
  • the antigen binding protein is selected from any one of antibodies Ab-1 to Ab-5, wherein:
  • Ab-1 has a heavy chain having an amino acid sequence as shown in SEQ ID NO.124, and a light chain as shown in SEQ ID No.129;
  • Ab-2 has a heavy chain having an amino acid sequence as shown in SEQ ID NO.125, and a light chain as shown in SEQ ID No.130;
  • Ab-3 has a heavy chain having an amino acid sequence as shown in SEQ ID NO.126, and a light chain as shown in SEQ ID No.131;
  • Ab-4 has a heavy chain having an amino acid sequence as shown in SEQ ID NO.127, and a light chain as shown in SEQ ID NO.132;
  • Ab-5 has a heavy chain with an amino acid sequence as shown in SEQ ID NO.128, and a light chain as shown in SEQ ID No.133.
  • a second aspect of the present invention relates to an isolated bispecific antigen binding protein capable of binding hROR1 having:
  • the first heavy chain variable region comprises a HCDR1 with an amino acid sequence of TYAMS, a HCDR2 with an amino acid sequence of SISSGGSTYYPDSVKG, and a HCDR3 with an amino acid sequence of YSLMVEAHWYFDV,
  • the first light chain variable region comprises LCDR1 with an amino acid sequence of KASQDINSYLS, LCDR2 with an amino acid sequence of RANRLVD, and LCDR3 with an amino acid sequence of LQYDEFPYT,
  • the fourth heavy chain variable region comprises a HCDR1 with an amino acid sequence of SYWIE, a HCDR2 with an amino acid sequence of EILPGSGNTQNNEKFKG, and a HCDR3 with an amino acid sequence of EGRRSHFFDY.
  • the four light chain variable regions contain LCDR1 with an amino acid sequence of KSSQSLLNSSNQKNYLA, LCDR2 with an amino acid sequence of FASTRES, and LCDR3 with an amino acid sequence of QQHYSSPWT.
  • the framework regions are of mouse origin or human origin.
  • the framework region is an IgG framework region.
  • the human framework region has a back mutation at one or more sites relative to the mouse framework region.
  • a constant region is further present.
  • the constant region has a LALA mutation.
  • the heavy chain constant region of the first antigen-binding fragment contains one or more mutations of C220S, A121C, V205C, K149C, D265C, S239C, A330C, and S442C.
  • the heavy chain constant region of the second antigen-binding fragment contains one or more mutations of C220S, A121C, V205C, K149C, D265C, S239C, A330C, and S442C.
  • the bispecific antigen binding protein is IgG-like or non-IgG-like.
  • the IgG-like antigen binding protein is selected from a DVD-Ig bispecific antigen binding protein, an IgG-scFv bispecific antigen binding protein or a CrossMAb bispecific antigen binding protein.
  • any one of p, q, r, s is present:
  • the bispecific antigen-binding protein is selected from any one of antibodies Ab-6 to 9, wherein Ab-6 is in the form of a DVD-Ig having a heavy chain with an amino acid sequence as shown in SEQ ID No. 45, and a light chain as shown in SEQ ID No. 49;
  • Ab-7 is in the form of IgG-scFv, having a heavy chain with an amino acid sequence as shown in SEQ ID No.46, and a light chain with an amino acid sequence as shown in SEQ ID No.50;
  • Ab-8 is in the form of crossmab, having a first heavy chain having an amino acid sequence as shown in SEQ ID NO.47, and a first light chain having an amino acid sequence as shown in SEQ ID No.67; a second heavy chain having an amino acid sequence as shown in SEQ ID NO.51, and a second light chain having an amino acid sequence as shown in SEQ ID No.69;
  • Ab-9 is in the form of DVD-Ig, having a heavy chain with an amino acid sequence as shown in SEQ ID NO.48, and a light chain as shown in SEQ ID No.52.
  • the third aspect of the present invention relates to a nucleic acid molecule for encoding any of the above-mentioned antigen-binding proteins or for encoding any of the above-mentioned bispecific antigen-binding proteins.
  • the fourth aspect of the present invention relates to a vector, characterized in that it comprises the nucleic acid molecule described above.
  • the fifth aspect of the present invention relates to a cell comprising the above-mentioned nucleic acid molecule, and/or the above-mentioned vector.
  • the sixth aspect of the present invention relates to a drug molecule, characterized by comprising any of the above-mentioned antigen-binding proteins or any of the above-mentioned bispecific antigen-binding proteins.
  • a therapeutic agent or a detectable marker is further included, wherein the therapeutic agent is selected from a cytotoxic agent, a cytostatic agent, a radioactive isotope, an anti-angiogenic agent or a liposome.
  • the cytotoxic agent is derived from plant, fungal or bacterial molecules or their derivatives.
  • the cytotoxic agent is selected from a peptide toxin, a protein toxin or an alkylating toxin.
  • the cytotoxic agent is selected from one or more of maytansine alkaloids, auristatins, eribulin, taxanes, calicheamicin, cemadotine, pyrrolobenzodiazepines, anthracyclines, camptothecin derivatives, ⁇ -amanitin and its derivatives, trabectedin and its derivatives, rubectedin and its derivatives.
  • the therapeutic agent or detectable marker is coupled to the antigen binding protein via a cleavable or non-cleavable linker.
  • the linker has one or more connectors.
  • the linker is selected from one or more of an oligopeptide linker, a hydrazine linker, a thiourea linker, a triggered self-degradation linker, a succinimidyl trans-4-(maleimidomethyl)cyclohexane-1-carboxylate linker, a maleimide linker, a disulfide linker, a thioether linker, and an olefin linker.
  • Ab is the antigen binding protein
  • L is a linker containing one or more linkers
  • D is the therapeutic agent or detectable marker
  • n is an integer selected from 1-20.
  • n is selected from integers of 2-8.
  • the seventh aspect of the present invention relates to a pharmaceutical composition, comprising any of the above-mentioned antigen-binding proteins, any of the above-mentioned bispecific antigen-binding proteins, the above-mentioned nucleic acid molecules, the above-mentioned vectors, the above-mentioned cells, and/or any of the above-mentioned drug molecules, and pharmaceutically acceptable carriers and/or additives.
  • the eighth aspect of the present invention relates to the use of any of the above-mentioned antigen binding proteins, any of the above-mentioned bispecific antigen binding proteins, any of the above-mentioned vectors, any of the above-mentioned cells, and/or any of the above-mentioned drug molecules in the preparation of drugs, wherein the drugs are used to diagnose, prevent and/or treat hROR1-related diseases and/or conditions.
  • the disease and/or condition comprises a tumor.
  • the tumor is chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic mantle cell lymphoma, sarcoma, ovarian cancer, renal cell carcinoma, head and neck cancer, breast cancer, lung cancer, malignant mesothelioma, colon cancer, neuroblastoma or melanoma.
  • the present invention provides novel different antibodies targeting the Ig-like domain and Kringle domain of hROR1 and bispecific antibodies based on these antibodies, and produces compositions based on these, such as ADCs combining different linkers and toxins. These antibodies and compositions provide new means for the diagnosis and treatment of tumors.
  • anti-hROR1 ADCs have good anti-tumor activity and have the potential to be used in tumor treatment.
  • FIG. 1 shows the extracellular regions of human ROR1 (hROR1) and human ROR2 (hROR2), including a signal peptide, an Ig-like domain, a Frizzled domain, and a Kringle domain.
  • Figure 2 shows the amino acid sequences of the variable immunoglobulin heavy and light chains of the novel mouse anti-hROR1 mAb.
  • Figure 3 shows the binding activity of chimeric mouse/human anti-hROR1 antibodies to the commercial mature hROR1 full-length extracellular region, three different domains of the hROR1 extracellular region, and the mature hROR2 full-length extracellular region as measured in Example 4.
  • Ab1 has a higher affinity for the hROR1 Ig-like domain
  • Ab4 is different from other antibodies and does not bind to the Ig-like domain but to the Kringle domain, indicating that it can recognize unique antigenic epitopes.
  • FIG. 4 shows FACS cell staining of human triple-negative breast cancer cells MDA-MB-231 using anti-human ROR1 antibodies Ab-1, Ab-2, Ab-3, Ab-4, and Ab-5 as described in Example 4.
  • FIG. 5 shows the binding activity of selected chimeric murine/human IgGs to endogenous hROR1 expressed on human breast cancer cells MDA-MB-231 measured by fluorescence activated cell sorting (FACS).
  • FIG6 shows the endocytosis of selected chimeric mouse/human IgG in human ovarian cancer cells PA-1 and human breast cancer cells MDA-MB-231 expressing endogenous hROR1.
  • A Endocytosis activity of selected antibodies on PA-1 cells.
  • B Endocytosis activity of selected antibodies on MDA-MB-231 cells. The selected antibodies showed significant endocytosis activity on both cell lines.
  • Figure 7 shows schematic diagrams of the structures of different bispecific antibodies based on selected chimeric mouse/human IgG.
  • B lists the full-length sequences of these antibodies.
  • FIG. 8 shows the binding activity of selected bispecific antibodies to endogenous hROR1 expressed on human breast cancer cells MDA-MB-231.
  • Figure 9 shows the binding activity of chimeric antibody mutants or humanized anti-hROR1 antibodies to the full-length extracellular region of mature hROR1.
  • the partially humanized antibody exhibited higher binding activity than the chimera.
  • FIG. 10(A) schematically illustrates how to produce the ADC disclosed in the present invention based on the cysteine corresponding to the interchain disulfide bond of the antibody coupled with different linker-payloads.
  • FIG. 10(B) schematically illustrates the main flow chart of producing ADC using the allene coupling technology.
  • FIG. 10(C) schematically illustrates how to produce the site-specific coupled ADC disclosed in the present invention based on the introduced cysteine mutation.
  • the cysteine mutation is introduced at the selected site by the method of point mutation and recombinant antibody expression, and then the site-specific coupled ADC is produced.
  • Figure 11 shows the cytotoxicity of selected anti-ROR1 ADCs in the concentration range of 0.01 to 1000 nM on human triple-negative breast cancer cells MDA-MB-231.
  • D shows the cytotoxicity of selected anti-ROR1 ADCs in the concentration range of 0.01 to 1000 nM on human sarcoma cells Saos2.
  • E shows the cytotoxicity of selected anti-ROR1 ADCs in the concentration range of 0.01 to 1000 nM on human soft tissue sarcoma cells SK-UT-1.
  • Figure 12 shows the efficacy of ADC41, DAR4 in a xenograft model in female NCG mice injected with hROR1 positive MDA-MB-231 cells.
  • the selected ADCs exhibited strong tumor inhibitory activity.
  • the present invention provides an antigen binding protein that can specifically bind to hROR1 and has good antigen binding activity.
  • the antigen binding protein of the present invention contains any one of a to o:
  • a. having a first heavy chain variable region comprising HCDR1 of the amino acid sequence TYAMS, HCDR2 of the amino acid sequence SISSGGSTYYPDSVKG, and HCDR3 of the amino acid sequence YSLMVEAHWYFDV,
  • HCDR1 having an amino acid sequence of SYAMS
  • HCDR2 having an amino acid sequence of SISSGGSTYYPDSVKG
  • HCDR3 having an amino acid sequence of GYSNYVDWYFDV
  • HCDR1 with an amino acid sequence of DYTMH
  • HCDR2 with an amino acid sequence of GIDPNYGGTNYNEKFKD
  • HCDR3 with an amino acid sequence of DDGYYEDYFDY
  • d. having a fourth heavy chain variable region comprising HCDR1 of the amino acid sequence SYWIE, HCDR2 of the amino acid sequence EILPGSGNTQNNEKFKG, and HCDR3 of the amino acid sequence EGRRSHFFDY,
  • HCDR1 of the amino acid sequence RYAMS e. having a fifth heavy chain variable region, comprising HCDR1 of the amino acid sequence RYAMS, HCDR2 of the amino acid sequence SISSGGSTYYPDSVKG, and HCDR3 of the amino acid sequence GYYGNNDWYFDV,
  • LCDR1 having an amino acid sequence of KASQDINSYLS
  • LCDR2 with an amino acid sequence of RANRLVD
  • LCDR3 with an amino acid sequence of LQYDEFPYT
  • g. having a second light chain variable region comprising LCDR1 with an amino acid sequence of KATQDINSYLS, LCDR2 with an amino acid sequence of RPNRLVD, and LCDR3 with an amino acid sequence of LQYDEFPYT,
  • h. having a third light chain variable region comprising LCDR1 with an amino acid sequence of HASQGISSNIG, LCDR2 with an amino acid sequence of HGTNLED, and LCDR3 with an amino acid sequence of VQYAQFPYT,
  • LCDR1 having an amino acid sequence of KSSQSLLNSSNQKNYLA
  • LCDR2 having an amino acid sequence of FASTRES
  • LCDR3 having an amino acid sequence of QQHYSSPWT
  • j. having a fifth light chain variable region comprising LCDR1 with an amino acid sequence of KASQDINSYLS, LCDR2 with an amino acid sequence of RANSLVD, and LCDR3 with an amino acid sequence of LQFDEFPYT,
  • antigen-binding proteins of hROR1 of the present invention include but are not limited to full-length antibodies, antibody fragments, immunoconjugates, antibody analogs, antibody derivatives, fusion proteins, etc.
  • Antibodies include but are not limited to chimeric antibodies and humanized antibodies.
  • Antibody fragments include but are not limited to Fab, Fab', F(ab)2, Fv, scFv, dsFv, VHH, etc.
  • Chimeric antibodies generally refer to antibodies having the V region (variable region) of a mouse monoclonal antibody and the C region (constant region) of a human immunoglobulin.
  • Humanized antibodies generally refer to antibodies formed by introducing the CDR sequences of mouse antibodies into the framework sequences of human VH and VL domains, respectively.
  • the hybridoma producing the monoclonal antibody of hROR1 of the present invention can be prepared using known techniques. For example, first, hROR1 protein, cells expressing hROR1, or genes encoding hROR1 are used as sensitizing antigens and immunized according to conventional immunization methods. The immune cells obtained from the immunized material are fused with known parent cells using conventional cell fusion methods to obtain hybridomas. Furthermore, cells producing the target antibody are screened from the hybridoma using conventional screening methods, thereby selecting hybridomas producing anti-hROR1 antibodies.
  • the preparation of monoclonal antibodies can be carried out as follows, for example.
  • the hROR1 gene by expressing the hROR1 gene, the hROR1 protein can be obtained and used as a sensitizing antigen for obtaining antibodies. That is, the gene sequence encoding hROR1 is included in a known expression vector, and after transforming an appropriate host cell, the target human hROR1 protein can be purified from this host cell or from the culture supernatant by a known method.
  • purified natural hROR1 protein can also be used in the same way. It can be generated by combining or using a variety of chromatography methods such as common ion chromatography or affinity chromatography once or multiple times.
  • a fusion protein can also be used as an immunogen, and the fusion protein is obtained by fusing the desired partial polypeptide of the hROR1 protein with different polypeptides.
  • an antibody Fc fragment or a peptide tag can be used in order to prepare a fusion protein as an immunogen.
  • the vector expressing the fusion protein can be prepared by fusing the genes encoding the desired two or more polypeptide fragments in frame, and the fusion gene is included in the expression vector as described above.
  • the hROR1 protein purified as described above can be used as a sensitizing antigen for immunization of mammals.
  • a partial peptide of hROR1 can also be used as a sensitizing antigen.
  • the mammal there is no particular limitation on the mammal to be immunized with the sensitizing antigen.
  • an immunized animal In order to obtain monoclonal antibodies by cell fusion, it is preferred to select an immunized animal in consideration of compatibility with the parent cells used in cell fusion.
  • rodents are preferred as immunized animals. Specifically, mice, rats, hamsters, or rabbits can be used as immunized animals. In addition, monkeys and the like can also be used as immunized animals.
  • the above-mentioned animals can be immunized with sensitizing antigens according to known methods.
  • mammals can be immunized by intraperitoneal or subcutaneous injection of sensitizing antigens.
  • the sensitizing antigen is administered to the mammal multiple times every 4 to 21 days.
  • the sensitizing antigen can be diluted with PBS (Phosphate-Buffered Saline) or physiological saline at an appropriate multiple for immunization.
  • the sensitizing antigen can be administered together with an adjuvant. For example, it can be mixed and emulsified with Freund's complete adjuvant to form a sensitizing antigen.
  • an appropriate carrier can be used for immunization with the sensitizing antigen.
  • a partial peptide with a small molecular weight is used as a sensitizing antigen, it is preferred to bind the sensitizing antigen peptide to a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.
  • immune cells are collected from the mammal and cell fusion is performed.
  • the immune cells spleen cells are particularly preferably used.
  • the screening and monocloning of the target antibody are preferably implemented by a screening method based on a known antigen-antibody reaction.
  • the antigen is bound to a carrier such as a small ball made of polystyrene or a commercially available 96-well microtiter plate, and reacted with the culture supernatant of the hybridoma. Then, after washing the carrier, an enzyme-labeled secondary antibody is reacted. If the culture supernatant contains a target antibody that reacts with the sensitizing antigen, the secondary antibody is bound to the carrier through this antibody. Finally, by detecting the secondary antibody bound to the carrier, it can be determined whether the target antibody is present in the culture supernatant.
  • a hybridoma that produces the desired antibody with antigen binding ability can be cloned by limiting dilution method, etc.
  • an antigen a protein used for immunization and a substantially identical hROR1 protein can be preferably used.
  • an oligopeptide containing the extracellular domain of hROR1 or a partial amino acid sequence constituting the region can be used as an antigen.
  • Chimeric antibodies and humanized antibodies can be produced by known methods.
  • the V region gene of 3 CDRs and 4 FRs is amplified in full length by primers that are annealed at the 5' end and 3' end and attached with appropriate restriction enzyme recognition sequences.
  • the DNA obtained as described above and the DNA encoding the human antibody C region are assembled into an expression vector to make them fuse in frame, and after the recombinant vector is introduced into a host to establish a recombinant cell, the recombinant cell is cultured to produce the antibody by expressing the DNA encoding the antibody.
  • Example 2 For details, please refer to Example 2.
  • the antibody forms include but are not limited to IgG, IgM, IgA, IgE, IgD, etc., preferably IgG. Including IgG1, IgG2, IgG3, IgG4, etc.
  • the constant regions used in chimeric antibodies and humanized antibodies include C ⁇ 1, C ⁇ 2, C ⁇ 3, C ⁇ 4, C ⁇ , C ⁇ , C ⁇ 1, C ⁇ 2, C ⁇ , etc. for the heavy chain, and C ⁇ , C ⁇ , etc. for the light chain.
  • LALA mutations can be introduced into the Fc region of the antibody to reduce the binding of the antibody to Fc ⁇ receptors and complement.
  • Mutations in the heavy chain constant region include, but are not limited to, one or more mutations in C220S, A121C, V205C, K149C, D265C, S239C, A330C, S442C.
  • CDR is used to represent the complementary determining region (or hypervariable loop) of an antibody.
  • the amino acid sequence in the CDR determines the chemical structure and characteristics of the antibody antigen binding site.
  • the CDR in the heavy chain variable region (VH) is represented by HCDR, and the three heavy chain CDR regions are named HCDR1, HCDR2 and HCDR3 respectively;
  • the CDR in the light chain variable region (VL) is represented by LCDR, and the three light chain CDR regions are named LCDR1, LCDR2 and LCDR3 respectively.
  • the CDR region in the antibody is generally predicted and identified by experiments.
  • the CDR sequences in the present invention are defined by the Kabat definition scheme, and the relevant amino acid numbers are determined by the Kabat numbering scheme. It should be understood that when different definition schemes are used to define CDR, the predicted CDR region sequences may only partially overlap, or be shortened or lengthened.
  • the CDR and FR gene sequences defined respectively can be concatenated and amplified to full length by known methods according to different definition schemes, thereby further preparing various antibodies or antibody fragments.
  • the antibody fragment Fab refers to the VH region of the heavy chain variable region, the CH1 functional region of the first constant region of the heavy chain, and the entire light chain connected by a disulfide bond, which mainly plays the antigen binding function of the antibody.
  • scFv is a single-chain antibody, specifically, the light chain variable region gene and the heavy chain variable region gene of the antibody are connected with a suitable oligonucleotide chain (linker) at the DNA level, so that it is expressed in the form of a single peptide chain in a suitable organism and folded into a new antibody consisting only of the heavy chain and light chain variable regions.
  • linker oligonucleotide chain
  • dsFv is a disulfide bond-stabilized antibody, specifically an antibody formed by mutating an amino acid residue in the heavy chain variable region (VH) and light chain variable region (VL) of the antibody to cysteine, and then linking VH and VL through an interchain disulfide bond.
  • VHH is a heavy chain single-domain antibody, specifically refers to a small molecule antibody that contains only the VH functional domain but not the VL functional domain.
  • Fusion protein refers to the product of fusing antibody molecule fragments with other proteins (such as antibody enzymes, immunotoxins, immunocytokines, immunoadhesins, etc.) using genetic engineering technology.
  • One method is to produce antibody fragments by treating antibodies with enzymes, and the enzymes used to produce antibody fragments include but are not limited to papain, pepsin, or plasmin.
  • Another method is to construct a gene encoding the above-mentioned antibody fragment, introduce it into an expression vector, and express it in an appropriate host cell to produce the antibody fragment.
  • the antibody derivatives in the present invention refer to products obtained by substituting, mutating, modifying, replacing, deleting and/or adding one or more amino acid residues in the antibodies of the present invention while retaining the endogenous functions.
  • CH is used to represent the heavy chain constant region of an antibody
  • CL is used to represent the light chain constant region of an antibody
  • the heavy chain constant region generally further includes CH1, CH2 and CH3 domains.
  • FR is used to represent the framework of an antibody variable region, wherein the framework region of the heavy chain variable region is represented by HFR, and the four heavy chain framework regions are named HFR1, HFR2, HFR3 and HFR4 respectively; the framework region of the light chain variable region is represented by LFR, and the four light chain framework regions are named LFR1, LFR2, LFR3 and LFR4 respectively.
  • the heavy chain variable region has a S62Q, S62A, S62N, S62E, S62V, S62Y, S62F or S62K mutation.
  • the light chain variable region has a G57A, G57S, G57N or G57L mutation.
  • the light chain variable region has a D56E, D56Q, D56T or D56Y mutation.
  • the light chain variable region has a S31A, S31Q, S31L, S31V, S31E, S31Y, S31F or S31K mutation.
  • the light chain variable region has a T57A, T57Q, T57L, T57V, T57E, T57Y, T57F or T57K mutation.
  • the light chain variable region has S27A, S27Q, S27L, S27V, S27E, S27Y, S27F, and S27K mutations.
  • the present invention also relates to a bispecific antigen binding protein capable of binding to HROR1, which comprises a first antigen binding fragment that binds to the Ig-like domain in hROR1 and a second antigen binding fragment that binds to the Kringle domain.
  • the first antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • the first heavy chain variable region comprises a HCDR1 with an amino acid sequence of TYAMS, a HCDR2 with an amino acid sequence of SISSGGSTYYPDSVKG, and a HCDR3 with an amino acid sequence of YSLMVEAHWYFDV,
  • the first light chain variable region comprises LCDR1 with an amino acid sequence of KASQDINSYLS and a LCDR2, and LCDR3 having the amino acid sequence LQYDEFPYT,
  • the second antigen-binding fragment has a fourth heavy chain variable region and a fourth light chain variable region, wherein:
  • the fourth heavy chain variable region comprises a HCDR1 with an amino acid sequence of SYWIE, a HCDR2 with an amino acid sequence of EILPGSGNTQNNEKFKG, and a HCDR3 with an amino acid sequence of EGRRSHFFDY.
  • the fourth light chain variable region contains LCDR1 with an amino acid sequence of KSSQSLLNSSNQKNYLA, LCDR2 with an amino acid sequence of FASTRES, and LCDR3 with an amino acid sequence of QQHYSSPWT.
  • Bispecific antibodies are divided into IgG-like bispecific antibodies containing Fc fragments and non-IgG-like antibodies without Fc fragments according to whether there is an Fc fragment.
  • Non-IgG-like antibodies include BiTE bispecific antibodies, DART bispecific antibodies, TandAb bispecific antibodies, and Bi-Nanobody bispecific antibodies.
  • Ig-like bispecific antibodies include Triomab quadroma bispecific antibodies, KIH IgG bispecific antibodies, CrossMAb bispecific antibodies, Orthodox-Fab IgG bispecific antibodies, YBODY bispecific antibodies, Two-in-One bispecific antibodies, DVD-Ig bispecific antibodies, IgG-ScFv and ScFv2-Fc bispecific antibodies, FIT-Ig bispecific antibodies, and Mebs-Ig bispecific antibodies.
  • the detection of the binding activity of the prepared antigen-binding protein adopts existing methods, such as flow cytometry.
  • the method of Example 4 can be used for detection.
  • the detection of the endocytic activity of the prepared antigen-binding protein can be carried out by methods known to those skilled in the art.
  • the antigen-binding protein to be tested containing a detectable marker is incubated with cells expressing hROR1, and the fluorescence intensity excited by entering the cell is detected by flow cytometry to analyze the endocytic activity.
  • flow cytometry to analyze the endocytic activity.
  • the vector of the present invention is used for the expression of replication, transcription and/or translation of DNA or RNA.
  • a desired expression product can be produced.
  • the selected vector is known, such as a plasmid.
  • the present invention also relates to a drug molecule containing the above antigen binding protein or bispecific antigen binding protein.
  • the drug molecule may also contain a therapeutic agent or a detectable marker.
  • the drug molecule may include any known drug form, including but not limited to proteins, peptides, small molecule drugs, antibody-drug conjugates, or combinations of drugs in various forms.
  • the antibody-drug conjugate in the present invention refers to an antigen-binding protein (usually an antibody or antibody fragment) coupled to a therapeutic agent through a linker.
  • the method for preparing an antibody-drug conjugate is known. Among them, the connection between the linker and the drug is usually produced by a known chemical synthesis method to form a linker-therapeutic agent structure that can bind to the antibody.
  • the antibody is coupled to one or more linker-therapeutic agent structures, and the coupling of the antibody and the linker-therapeutic agent structure is usually established for the side chain of the lysine or cysteine residue in the antibody.
  • the linker usually has N-hydroxysuccinimide, which can react with the ⁇ -amino group of the lysine to form a stable amide bond to form a coupling of the antibody and the therapeutic agent.
  • the linker usually has a functional group that can mediate the coupling reaction, such as a maleimide functional group, allenamide, etc., and the cysteine reduces the disulfide bond by a known method to produce a reactive thiol, thereby conjugating with maleimide to form a coupling of the antibody and the therapeutic agent.
  • a functional group that can mediate the coupling reaction such as a maleimide functional group, allenamide, etc.
  • the linkers that can be used include non-cleavable and cleavable ones.
  • the linkers may have one or more linkers, including but not limited to oligopeptide linkers (including cleavable and/or non-cleavable oligopeptide linkers), hydrazine linkers, thiourea linkers, self-immolative linkers, succinimidyl trans-4-(maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linkers, maleimide linkers, disulfide linkers, thioether linkers and/or alkene linkers.
  • oligopeptide linkers including cleavable and/or non-cleavable oligopeptide linkers
  • hydrazine linkers including cleavable and/or non-cleavable oligopeptide linkers
  • thiourea linkers include self-immolative linkers, succinimidyl trans-4-(maleimidomethyl)
  • the therapeutic agent that can be used can be an immunomodulator, such as a Sting agonist, a TLR7, TLR8 and/or TLR9 agonist, a PD-1 inhibitor, and the like.
  • an immunomodulator such as a Sting agonist, a TLR7, TLR8 and/or TLR9 agonist, a PD-1 inhibitor, and the like.
  • the therapeutic agent that can be used can be a cytotoxic agent, a cytostatic agent, etc.
  • Cytotoxic agents can be molecules or derivatives thereof from plants, fungi or bacteria, including but not limited to peptide toxins, protein toxins, alkylating agent toxins or other types of toxins.
  • Drug toxic agents can be selected from maytansine alkaloids (e.g., maytansinol or DM1 maytansine), auristatins (e.g., monomethyl auristatin E or monomethyl auristatin F), eribulin, taxanes, calicheamicin, cemaddin, pyrrolobenzodiazepines, anthracyclines, camptothecin derivatives, ⁇ -amanitin and its derivatives, trabectedin and its derivatives, rubectedin and its derivatives One or more.
  • the cytotoxic substance used in the present invention can be one, or two or more cytotoxic substances can be combined for use.
  • the therapeutic agent that can be used can also be a metabolite (e.g., an antifolate such as methotrexate, a fluoropyrimidine such as 5-fluorouracil, cytarabine, or a purine or adenosine analog); an intercalating agent (e.g., an anthracycline such as doxorubicin, nemorubicin, or preferably a derivative of PNU-159682), daunorubicin, epirubicin, idarubicin, mitomycin-C, actinomycin D, or mithramycin, or other intercalating agents.
  • a metabolite e.g., an antifolate such as methotrexate, a fluoropyrimidine such as 5-fluorouracil, cytarabine, or a purine or adenosine analog
  • an intercalating agent e.g., an anthracycline such as doxor
  • a pyrrolobenzodiazepine a DNA-reactive agent such as calicheamicin, tiancimycins, and other enediynes; a platinum derivative (e.g., cisplatin or carboplatin); an alkylating agent (e.g., nitrogen mustard, melphalan, chlorambucil, busulfan, cyclophosphamide, ifosfamide nitrosourea, or thiotepa); an RNA polymerase inhibitor such as ⁇ -amanitin; an antimitotic agent (e.g., a vinca alkaloid such as vincristine, or a taxane such as paclitaxel or docetaxel); a topoisomerase inhibitor (e.g., etoposide, teniposide, amsacrine, topotecan, exitecan); a cell cycle inhibitor (e.g., flavopyridol); or an antimicrotubule agent (e
  • the therapeutic agent can be a proteasome inhibitor or a topoisomerase inhibitor such as bortezomib, amsacrine, etoposide, etoposide phosphate, teniposide or doxorubicin, etc.
  • Therapeutic radioisotopes include iodine (131I), yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (At), rhenium (Re), bismuth (Bi or Bi) and rhodium (Rh), etc.
  • Anti-angiogenic agents include linomide, bevacizumab, angiostatin and razoxane.
  • Contrast agents can be radioisotope labels such as iodine (131I or 125I), indium (111In), technetium (99Tc), phosphorus (32P), carbon (14C), tritium (3H), other radioisotopes (e.g., radioactive ions), or therapeutic radioisotopes such as one of the therapeutic radioisotopes listed above.
  • contrast agents can include radiopaque materials, magnetic resonance imaging (MRI) agents, ultrasound imaging agents, and any other contrast agents suitable for detection by the device of the imaging animal body.
  • the detectable marker can also be a fluorescent marker, a bioactive enzyme marker, a luminescent marker, or a chromophore marker.
  • Ab is the antigen binding protein
  • L is a linker containing one or more linkers
  • D is the therapeutic agent or detectable marker
  • n is an integer selected from 1 to 20. Further, it can be an integer from 2 to 8.
  • the pharmaceutical composition of the present invention can be administered orally or non-orally.
  • Non-oral administration includes injection, nasal administration, pulmonary administration, transdermal administration, etc.
  • Injection can include intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.
  • the dosage can be specifically adjusted considering the patient's age and symptoms.
  • the dosage can be 0.0001 mg to 1000 mg per kg of body weight per administration.
  • each administration of each patient is 0.001 mg to 100000 mg. It should be noted that the above dosage is only used for illustration and is not used to limit the range of the dosage.
  • the pharmaceutical composition of the present invention can be formulated according to a common method, and can also contain pharmaceutically acceptable drug carriers and/or additives at the same time.
  • pharmaceutically acceptable drug carriers and/or additives for example, surfactants, excipients, colorants, spices, preservatives, stabilizers, buffers, suspending agents, isotonic agents, adhesives, disintegrants, lubricants, fluidity promoters, flavoring agents, etc. can be cited.
  • drug carriers include but are not limited to light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, etc.
  • the types of cancers treated and diagnosed by the present invention are not particularly limited, and are generally cancers expressing hROR1 protein, preferably chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic mantle cell lymphoma, sarcoma, ovarian cancer, renal cell carcinoma, head and neck cancer, breast cancer, lung cancer, malignant mesothelioma, colon cancer, neuroblastoma or melanoma.
  • hROR1 protein preferably chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic mantle cell lymphoma, sarcoma, ovarian cancer, renal cell carcinoma, head and neck cancer, breast cancer, lung cancer, malignant mesothelioma, colon cancer, neuroblastoma or melanoma.
  • the pharmaceutical composition of the present invention may be formulated with a variety of antibodies as needed.
  • the cytotoxicity to hROR1-expressing cells may be enhanced.
  • anti-hROR1 antibodies by formulating antibodies that recognize other tumor-associated antigens, the therapeutic effect may also be improved.
  • each CDR sequence in the present invention is defined by the Kabat definition scheme, and the relevant amino acid numbering is determined by the Kabat numbering scheme. It should be understood that when different definition schemes are used to define CDRs, the predicted CDR region sequences may only partially overlap, or be shortened or lengthened.
  • Human ovarian cancer cell line PA-1, human triple-negative breast cancer cell lines MDA-MB-231 and MCF-7, and human sarcoma cell line Saos-2 were all purchased from ATCC.
  • MDA-MB-231 cells were grown in DMEM supplemented with 10% (v/v) heat-inactivated FBS (Thermo Scientific; Logan, UT). (Thermo Scientific; Logan, UT).
  • Saos-2 cell line was grown in RPMI1640 (Thermo Scientific; Logan, UT) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Scientific; Logan, UT).
  • PA-1 and MCF-7 cell lines were grown in MEM (Thermo Scientific; Logan, UT) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Scientific; Logan, UT), 1% (v/v) nonessential amino acids, and 1 mM sodium pyruvate to support adherent culture.
  • Each culture medium was supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma Aldrich).
  • Human ROR1 (30-403)-His was used as an immunogen to prepare monoclonal antibodies against hROR1.
  • 15 mice (5 BALB/c mice, 5 C57bl/6 mice, and 5 A/J mice) were prepared.
  • 50 ⁇ g of human ROR1 (30-403)-His protein emulsified with Freund's complete adjuvant was used for subcutaneous injection for the first immunization, and the second immunization was performed two weeks later.
  • 25 ⁇ g of human ROR1 (30-403)-His protein emulsified with incomplete Freund's adjuvant was used for three immunizations.
  • the mice were immunized at intervals of two weeks by alternating intraperitoneal and subcutaneous injections. Seven days after the fourth immunization of the mice, blood was collected from the tail and the antigen binding ability was tested.
  • Antibody screening BALB/c mice with high serum titers were selected from 15 mice for cell fusion. The spleen cells and SP2/0 cells of the mice were electrofused, and the ratio of SP2/0 cells to spleen cells was 20:1. 7 to 10 days after fusion, the hybridoma supernatant specific antibodies were detected by indirect ELISA and FACS. Positive clones were selected for subcloning. After two subclones, monoclonal cell lines, i.e. hybridoma cell lines, were obtained. The positive hybridoma cell lines obtained by screening were 7F6D10, 33B10G8, 21A9A4, 84E3F11 and 94F8B4. The SBAClonetotyping system-HRP kit was used for subtype detection, and the subtypes of the selected antibodies were all IgG1 Kappa.
  • variable immunoglobulin heavy and light chains of mouse anti-hROR1 mAb are shown in Figure 2.
  • the figure shows the heavy chain variable domain sequences (SEQ ID NOs: 1, 5, 9, 13, 17, respectively) and light chain variable domain sequences (SEQ ID NOs: 21, 25, 29, 33, 37, respectively) of five antibodies named 7F6D10, 33B10G8, 21A9A4, 84E3F11 and 94F8B4.
  • variable region coding region of the antibody was prepared by whole gene synthesis using MHSSALLCCLVLLTGVRA as the leading signal peptide, and assembled with the human IgH- ⁇ 1 and IgL- ⁇ coding regions through NotI/XbaI restriction sites in the expression vector pcDNA3.4, which has a CMV promoter that can enhance the high-level expression of multiple genes in mammalian cells and contains an ampicillin resistance gene for selective markers.
  • CHO-K1 cells were selected as the host for antibody expression, and the cells were passaged three times under appropriate conditions (120rpm, 8% CO 2 , 37°C), the cells were collected and added to the electrotransfer solution and mixed evenly, and then the plasmid was added and mixed evenly and placed in an electrotransfer tube, and the pcDNA3.4 expression vector was transfected into the CHO-K1 cells by an electrotransfer instrument. After culturing for 24 hours at 37°C, 270rpm, 8% CO 2 , feed/sodium butyrate/double antibiotics were added and cultured for 3-7 days, and the harvested supernatant was filtered (0.22 ⁇ m) to remove the cells and used for subsequent purification.
  • the treated supernatant was purified by Protein A affinity chromatography column, then eluted with citrate buffer (pH 3.4), and the eluate was placed in a dialysis bag for buffer exchange, and finally 7F6D10, 33B10G8, 21A9A4, 84E3F11 and 94F8B4 chimeric mouse/human IgG1 antibodies were obtained.
  • concentration and quantity were determined by reading the A280nM absorbance by NanoDrop instrument, and the purity was detected by SDS-PAGE and SEC-HPLC, and the purity of the expressed antibody was>95%.
  • mice/human IgG1 chimeric antibodies used in the subsequent examples, including their concentrations and dilutions.
  • ELISA For ELISA, each well of a 96-well 3590 plate (Corning, NY) was coated with 100 ⁇ L of coating buffer (0.015 M Na 2 CO 3 , 0.035 M NaHCO 3 , pH 9.5) containing 50 ng of antigen protein at 4°C overnight. After blocking with 150 ⁇ L of PBS buffer containing 4% (w/v) skim milk powder per well at 37°C for 1 hour, the supernatant was discarded and the solution in the well was drained on a clean paper towel.
  • coating buffer 0.015 M Na 2 CO 3 , 0.035 M NaHCO 3 , pH 9.5
  • test antibody 7F6D10, 33B10G8, 21A9A4, 84E3F11 or 94F8B4
  • TMB colorimetric solution (Beyotime, Shanghai, CN) according to the manufacturer's instructions, incubated at room temperature in the dark for 15 minutes until the color was developed to the expected depth, and then TMB colorimetric stop solution was used.
  • the absorbance was measured at 450nm using a SpectraMax i3X microplate reader (Molecular Devices; Sunnyvale, CA).
  • hROR1_Ig-like, hROR1_Frizzled or hROR1_Kringle protein was directly coated, then incubated with the antibody to be tested, and then detected in the same way.
  • the cells to be tested were seeded in a 96-well round-bottom plate at a density of 40,000 cells per well, and the antibody diluted in gradients (starting from 100 nM, 4-fold dilution to 0.006 nM) was added as the primary antibody, and incubated at 4°C for 1 hour.
  • the cell wells incubated with the secondary antibody only without the primary antibody were used as blank controls. After incubation, the supernatant was discarded by centrifugation, and the cells were washed twice with 200 uL of ice-cold 1% (v/v) FBS per well in PBS.
  • FITC-labeled goat anti-human IgG-specific polyclonal antibody was used as the secondary antibody for incubation at 4°C for 1 hour, and the above washing steps were repeated. After discarding the supernatant, 100 uL of 1% FBS per well in PBS was added to resuspend. The staining after cell binding was analyzed using a Flow cytometer instrument (Agilent), and the data were analyzed using GraphPad 9.5.1.
  • Figure 3 shows the measured binding activity of chimeric mouse/human anti-hROR1 antibodies to the full-length extracellular region of commercial mature hROR1, three different domains of the hROR1 extracellular region, and the full-length extracellular region of mature hROR2.
  • hROR1_ECD human hROR1 Ig-like domain
  • hROR1_Ig-like human hROR1 Ig-like domain
  • hROR1 Frizzled domain hROR1_Frizzled
  • hROR1 Kringle domain hROR1_Kringle
  • hROR2_ECD full-length extracellular region of mature hROR2
  • His-tagged hROR1_ECD, hROR1_Ig-like, hROR1_Frizzled, hROR1_Frizzled, or hROR2_ECD were captured by chimeric mouse/human anti-hROR1 antibody immobilized on the plate and then detected with goat anti-human antibody conjugated to HRP as secondary antibody.
  • FIG. 4 shows FACS cell staining of Ab-1 to 5 on human triple-negative breast cancer cells MDA-MB-231.
  • FIG. 5 shows the binding activity of selected chimeric mouse/human IgG to endogenous hROR1 expressed on human breast cancer cells MDA-MB-231 as determined by fluorescence activated cell sorting (FACS).
  • PA-1 and MDA-MB-231 cells were cultured overnight in a constant temperature incubator (37°C, 5% CO 2 ).
  • the selected antibodies Ab-1 to Ab-5 were labeled with pHrodo iFL Green human IgG reagent, and the antibody-pHrodo iFL Green human IgG conjugates were incubated with human ovarian cancer cells PA-1 and human breast cancer cells MDA-MB-231 for 24 hours.
  • the highest concentration of the antibody was set to 10 ⁇ g/ml, and a 4-fold gradient dilution was performed. After the incubation, the cells were washed with PBS and resuspended in culture medium.
  • the fluorescence intensity excited by pHrodo iFL Green entering the cells was measured on a NovoCyte flow cytometer to analyze the endocytic activity of the antibody.
  • Figure 6 shows the endocytosis of selected chimeric mouse/human IgG in human ovarian cancer cells PA-1 and human breast cancer cells MDA-MB-231 expressing endogenous hROR1.
  • Ab-1 to 5 are hROR1-specific mAbs, as well as control antibodies UC961 and C2E3.
  • A Endocytosis activity of selected antibodies on PA-1 cells.
  • B Endocytosis activity of selected antibodies on MDA-MB-231 cells.
  • Example 6 Expression and characterization of purified, recombinant anti-hROR1 bispecific antibodies
  • bispecific antibodies were constructed using 7F6D10 and 84E3F11 chimeric human IgG.
  • the expression and purification of bispecific antibodies were as described in Example 3.
  • the variable region coding regions of the bispecific antibodies were cloned, assembled on a pcDNA expression vector, and transfected into CHO-K1 cells for eukaryotic expression and purification.
  • the purified antibodies obtained were Ab-6 to Ab-9.
  • FIG. 7 shows schematic diagrams of the structures of different formats of bispecific antibodies based on selected chimeric mouse/human IgG.
  • the selected bispecific antibodies 84E3F11-7F6D10 and 7F6D10-84E3F11 are in DVD-Ig format; the selected bispecific antibody 84E3F11-7F6D10-VH-VL is in IgG-scFv format; the selected bispecific antibody 84E3F11-7F6D10 (cross) is in CrossMab format, as shown in Figure 7.
  • (A) is a schematic diagram of the structure of the selected bispecific antibody, and (B) is the sequence of the selected bispecific antibody.
  • Table 3 lists the final concentrations and buffers of selected mouse/human chimeric bispecific antibodies.
  • FIG. 8 shows the binding activity of selected bispecific antibodies to endogenous hROR1 expressed on human breast cancer cells MDA-MB-231.
  • Example 7 Expression and characterization of purified, recombinant chimeric antibody mutants or humanized anti-hROR1 antibodies
  • Table 4 Chimeric antibody mutants or humanized antibodies, including their concentrations and dilutions.
  • FIG. 9 shows the ELISA binding activity of chimeric antibody mutants or humanized anti-hROR1 antibodies to the full-length extracellular region of mature hROR1.
  • Example 8 Generation of ADC-1 to 56 by coupling using antibody interchain disulfide bonds and/or cysteine mutation sites
  • ADC-1 to 23 are prepared by coupling 1 to 20 anti-tumor toxin molecules through the interchain disulfide bond sites of the antibody:
  • the specific conjugation process for synthesizing ADC-1 to 23 is as follows: Take a 0.5 ml centrifuge tube, add 2 mg of antibody, add 30 mM His-HAC, (pH 5.5) buffer to dilute the antibody to 5 mg/ml, add 100 mM EDTA aqueous solution to a final concentration of 5 mM, and shake to mix.
  • the specific conjugation process for synthesizing ADC-24 is as follows: Take a 0.5 ml centrifuge tube, add 2 mg of antibody, add 25 mM Na 2 B 4 O 7 , 25 mM NaCl, 1 mM DTPA (pH 7.4) buffer to dilute the antibody to 10 mg/ml, add a certain volume of TCEP or DTT aqueous solution according to 2 to 15 times the molar number of the antibody, shake and mix, and incubate in a constant temperature mixer at 20°C for 2-24 hours.
  • 10(A) and 10(C) show the overall preparation method of antibody-drug conjugates, and ADC-25-56 as follows is prepared by coupling 1-20 anti-tumor toxin molecules through antibody interchain disulfide bonds and/or cysteine mutation sites.
  • 1.5 ⁇ 10 3 cells were seeded in a 96-well plate (excluding the edge wells filled with PBS).
  • the cells were cultured in 80 ⁇ L of DMEM supplemented with 10% FBS (v/v), 100 U/mL penicillin and 100 mg/mL streptomycin, and cultured overnight in a humidified incubator at 37°C and 5% CO 2. After incubation in a constant temperature incubator for one day, the diluted solution (20 ⁇ L per well) was added to each well so that the final ADC concentration range was 0.01 nM to 1000 nM. After incubation with the cells (37°C, 5% CO 2 ) for five days, the 96-well plate was removed from the incubator and equilibrated to room temperature.
  • Figure 11 shows the cytotoxicity of selected anti-ROR1 ADCs in the concentration range of 0.01 to 1000 nM on human triple-negative breast cancer cells MDA-MB-231.
  • D shows the cytotoxicity of selected anti-ROR1 ADCs in the concentration range of 0.01 to 1000 nM on human sarcoma cells Saos2.
  • E shows the cytotoxicity of selected anti-ROR1 ADCs in the concentration range of 0.01 to 1000 nM on sarcoma cells SK-UT-1.
  • Example 10 Evaluation of the in vivo efficacy of anti-hROR1 ADC in an ovarian cancer model established with hROR1-positive human ovarian cancer PA-1 cells.
  • ADC formulated in PBS was administered (iv) to groups of 6 mice at 3 mg/kg. PBS served as a negative vehicle control. All treatment groups were injected into the tail vein at a volume of 5 L/kg, once a week for three weeks. Tumor volume was measured to investigate whether tumor growth was inhibited, delayed or cured. Tumor size was measured twice a week with a caliper, and the average tumor volume of mice in each group was plotted over time.
  • V 0.5a ⁇ b 2
  • a and b are the long and short diameters of the tumor, respectively.
  • AALAC Laboratory Animal Care International
  • Figure 12 shows the antitumor efficacy of ADC-41, DAR4 in an ovarian cancer xenograft model in female balb/c nude mice injected with hROR1-positive PA-1 cells.

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Abstract

涉及hROR1的抗原结合蛋白及其用途，具体涉及hROR1的抗原结合蛋白、双特异性抗原结构蛋白、编码该抗原结合蛋白的核酸分子、载体、细胞、药物分子，以及它们的用途。提供新的靶向hROR1的Ig-like结构域或Kringle结构域的不同抗体和基于这些抗体的双抗，并在此基础上产生组合物，例如不同连接子和毒素组合ADC。这些抗体及组合物为肿瘤的诊疗提供了新的手段。特别是，抗hROR1ADC具有良好的抗肿瘤活性，具有用于肿瘤治疗的潜力。

Description

hROR1的抗原结合蛋白及其用途 技术领域
本发明涉及hROR1的抗原结合蛋白、双特异性抗原结合蛋白、编码该抗原结合蛋白的核酸分子、载体、细胞、药物分子，以及它们的用途。
背景技术
受体酪氨酸激酶孤儿受体-1(ROR1)与同家族成员受体酪氨酸激酶孤儿受体-2(ROR2)隶属于受体酪氨酸激酶(RTKs)家族，都是近期受关注的肿瘤靶点。ROR1和ROR2结构相似，均为I型跨膜蛋白，成熟的全长蛋白包含由3个不同结构域(Ig-like、Frizzled和Kringle结构域)的胞外区、跨膜区和胞内区组成，胞内区包含一个酪氨酸激酶结构域、两个富含丝氨酸/苏氨酸的结构域和一个富含脯氨酸的结构域。ROR1和ROR2都在多种肿瘤组织中高表达，但ROR2在一些正常组织中也有低中程度的表达，而ROR1尽管在胚胎组织中可检测，在成年人正常组织中几乎不表达，除了睾丸、子宫、肺、膀胱和结肠里有很低水平的表达(Clin Cancer Res 2008；14(2)，doi:10.1158/1078-0432.CCR-07-1823)，因而被认为是一个很好的肿瘤靶点。
ROR1在血液瘤和实体瘤中都有较为广泛的表达。例如，ROR1在慢性淋巴细胞白血病(CLL)、一些急性淋巴细胞白血病(ALL)、和慢性套细胞淋巴瘤(CML)等血液瘤中高表达，且ROR1在多种实体瘤中如肉瘤、卵巢癌、肾细胞癌、头颈癌、乳腺癌、肺癌、恶性间皮瘤、结肠癌、神经母细胞瘤、黑色素瘤和其它肿瘤组织中高表达。ROR1高表达与癌症如CLL和卵巢癌病程加快及预后不良相关(Sci Rep.2014 Jul24；4:5811.doi:10.1038/srep05811；Blood.2016 Dec 22；128(25):2931–2940.10.1182/blood-2016-04-712562)。ROR1可能通过参与多种信号转导通路促进肿瘤的发生发展。有报道显示ROR1可能介导Wnt5a，Wnt5b和Wnt16信号，增强细胞增殖、抑制细胞凋亡，诱导上皮-间充质转换(EMT)和癌细胞迁移和转移，并且有助于小窝形成促进内吞(Cells 2021,10,142,doi:10.3390/cells10010142；Nat Commun 2016Jan 4；7:10060.doi:10.1038/ncomms10060)。
如前所述，ROR1在肿瘤中有特异性表达且有可能促进肿瘤生长，因而ROR1可视为较好的癌症疗法药物靶标，已有工作开发ROR1特异性抗体的靶标。因ROR1在不同哺乳动物物种之间具有高同源性，其胞外区氨基酸序列人和食蟹猴完全相同，在人和小鼠之间96.8％相同，人和大鼠之间96.6％相同。因而，通过标准技术如动物免疫产生针对这个靶标的高亲和力抗体具有一定的困难。
靶向ROR1的策略主要包括单抗、双抗、抗体偶联药物和CAR-T，目前尚无靶向ROR1的产品得到临床确证。此外，ROR1的胞外区具有三个不同的结构域，进入临床研究阶段的三个靶向ROR1抗体偶联药物中VLS-101和NBE002靶向Ig-like结构域，LCB71靶向Frizzled结构域。而特异性识别不同结构域或同一结构域的不同表位有可能带来不一样的亲和力、内吞效率或生物性功能，特别是识别不同表位的不同抗体组成的双抗也许会增加抗体的功能特性。另外，现有ADC产品都有安全性和耐药性问题。
发明内容
本发明主要目的在于提供能够与hROR1中特异性结合的抗原结合蛋白，并且具有很好的抗原结合活性。
本发明的第一方面涉及分离的抗原结合蛋白，能够结合hROR1，含有a～o任一项：
a.具有第一重链可变区，含有氨基酸序列为TYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，氨基酸序列为YSLMVEAHWYFDV的HCDR3，
b.具有第二重链可变区，含有氨基酸序列为SYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，氨基酸序列为GYSNYVDWYFDV的HCDR3，
c.具有第三重链可变区，含有氨基酸序列为DYTMH的HCDR1，氨基酸序列为GIDPNYGGTNYNEKFKD的HCDR2，氨基酸序列为DDGYYEDYFDY的HCDR3，
d.具有第四重链可变区，含有氨基酸序列为SYWIE的HCDR1，氨基酸序列为EILPGSGNTQNNEKFKG的HCDR2，氨基酸序列为EGRRSHFFDY的HCDR3，
e.具有第五重链可变区，含有氨基酸序列为RYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，氨基酸序列为GYYGNNDWYFDV的HCDR3，
f.具有第一轻链可变区，含有氨基酸序列为KASQDINSYLS的LCDR1，氨基酸序列为RANRLVD的 LCDR2，氨基酸序列为LQYDEFPYT的LCDR3，
g.具有第二轻链可变区，含有氨基酸序列为KATQDINSYLS的LCDR1，氨基酸序列为RPNRLVD的LCDR2，氨基酸序列为LQYDEFPYT的LCDR3，
h.具有第三轻链可变区，含有氨基酸序列为HASQGISSNIG的LCDR1，氨基酸序列为HGTNLED的LCDR2，氨基酸序列为VQYAQFPYT的LCDR3，
i.具有第四轻链可变区，含有氨基酸序列为KSSQSLLNSSNQKNYLA的LCDR1，氨基酸序列为FASTRES的LCDR2，氨基酸序列为QQHYSSPWT的LCDR3，
j.具有第五轻链可变区，含有氨基酸序列为KASQDINSYLS的LCDR1，氨基酸序列为RANSLVD的LCDR2，氨基酸序列为LQFDEFPYT的LCDR3，
k.具有a项所述的第一重链可变区和具有f项所述的第一轻链可变区，
l.具有b项所述的第二重链可变区和具有g项所述的第二轻链可变区，
m.具有c项所述的第三重链可变区和具有h项所述的第三轻链可变区，
n.具有d项所述的第四重链可变区和具有i项所述的第四轻链可变区，
o.具有e项所述的第五重链可变区和具有j项所述的第五轻链可变区。
本发明第一方面的一些实施例中，具有鼠源的框架区或人源的框架区。
本发明第一方面的一些实施例中，所述的框架区为IgG框架区。
本发明第一方面的一些实施例中，所述的人源的框架区具有相对于鼠源的框架区的一个或多个位点的回复突变。
本发明第一方面的一些实施例中，还具有恒定区。
本发明第一方面的一些实施例中，所述的恒定区具有LALA突变。
本发明第一方面的一些实施例中，所述的a～e、k～o中的任一项的重链恒定区含有C220S、A121C、V205C、K149C、D265C、S239C、A330C、S442C中的一个或多个突变。
本发明第一方面的一些实施例中，CDR序列根据Kabat定义方案确定。
本发明第一方面的一些实施例中，所述的抗原结合蛋白包括抗体或其抗原结合片段。
本发明第一方面的一些实施例中，所述的抗体选自单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体或全人源抗体。
本发明第一方面的一些实施例中，所述的抗原结合片段选自Fab抗体、单链抗体或单域抗体。
本发明第一方面的一些实施例中，所述的重链可变区具有S62Q、S62A、S62N、S62E、S62V、S62Y、S62F或S62K突变。
本发明第一方面的一些实施例中，所述的轻链可变区具有G57A、G57S、G57N或G57L突变。
本发明第一方面的一些实施例中，所述的轻链可变区具有D56E、D56Q、D56T或D56Y突变。
本发明第一方面的一些实施例中，所述的轻链可变区具有S31A、S31Q、S31L、S31V、S31E、S31Y、S31F或S31K突变。
本发明第一方面的一些实施例中，所述的轻链可变区具有T57A、T57Q、T57L、T57V、T57E、T57Y、T57F或T57K突变。
本发明第一方面的一些实施例中，所述的轻链可变区具有S27A、S27Q、S27L、S27V、S27E、S27Y、S27F、S27K突变。
本发明第一方面的一些实施例中，具有氨基酸序列如SEQ ID No.53、54、55、56、57、58、59、60、62、63、65、73、75、78、77、78、79、80、81、82、83、84、85、86、87、88、90、91、92、93、134或135任一所示的重链，和氨基酸序列如SEQ ID No.64、66、67、68、70、71、94、95、96、97、98、99、100、101、102、103、105、106、108、109、110、112、113、114、115、116、117、119、120、121、122或123任一所示的轻链。
本发明第一方面的一些实施例中，所述的抗原结合蛋白，选自抗体Ab-1～5中的任一项，其中，
Ab-1具有氨基酸序列如SEQ ID NO.124所示的重链，和如SEQ ID No.129所示的轻链；
Ab-2具有氨基酸序列如SEQ ID NO.125所示的重链，和如SEQ ID No.130所示的轻链；
Ab-3具有氨基酸序列如SEQ ID NO.126所示的重链，和如SEQ ID No.131所示的轻链；
Ab-4具有氨基酸序列如SEQ ID NO.127所示的重链，和如SEQ ID NO.132所示的轻链；
Ab-5具有氨基酸序列如SEQ ID NO.128所示的重链，和如SEQ ID No.133所示的轻链。
本发明的第二方面涉及分离的双特异性抗原结合蛋白，能够结合hROR1，具有：
结合hROR1中Ig-like结构域的第一抗原结合片段，所述的第一抗原结合片段具有第一重链可变区和第一轻链可变区，其中，
所述的第一重链可变区含有氨基酸序列为TYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，和氨基酸序列为YSLMVEAHWYFDV的HCDR3，
所述的第一轻链可变区含有氨基酸序列为KASQDINSYLS的LCDR1，氨基酸序列为RANRLVD的LCDR2，和氨基酸序列为LQYDEFPYT的LCDR3，
结合hROR1中Kringle结构域的第二抗原结合片段，所述的第二抗原结合片段具有第四重链可变区和第四轻链可变区，其中，
所述的第四重链可变区含有氨基酸序列为SYWIE的HCDR1，氨基酸序列为EILPGSGNTQNNEKFKG的HCDR2，氨基酸序列为EGRRSHFFDY的HCDR3，
所述的四轻链可变区含有氨基酸序列为KSSQSLLNSSNQKNYLA的LCDR1，氨基酸序列为FASTRES的LCDR2，氨基酸序列为QQHYSSPWT的LCDR3。
本发明的第二方面的一些实施例中，具有鼠源的框架区或人源的框架区。
本发明的第二方面的一些实施例中，所述的框架区为IgG框架区。
本发明的第二方面的一些实施例中，所述的人源的框架区具有相对于鼠源的框架区的一个或多个位点的回复突变。
本发明的第二方面的一些实施例中，还具有恒定区。
本发明的第二方面的一些实施例中，所述的恒定区具有LALA突变。
本发明的第二方面的一些实施例中，所述的第一抗原结合片段的重链恒定区含有C220S、A121C、V205C、K149C、D265C、S239C、A330C、S442C中的一个或多个突变。
本发明的第二方面的一些实施例中，所述的第二抗原结合片段的重链恒定区含有C220S、A121C、V205C、K149C、D265C、S239C、A330C、S442C中的一个或多个突变。
本发明的第二方面的一些实施例中，所述的双特异性抗原结合蛋白为IgG样或非IgG样。
本发明的第二方面的一些实施例中，所述的IgG样抗原结合蛋白选自DVD-Ig双特异性抗原结合蛋白、IgG-scFv双特异性抗原结合蛋白或CrossMAb双特异性抗原结合蛋白。
本发明的第二方面的一些实施例中，具有p、q、r、s中的任一项：
p.氨基酸序列如61所示的第一重链，和氨基酸序列如140所示的第二重链，氨基酸序列如69所示的第一轻链，氨基酸序列如141所示的第二轻链；
q.氨基酸序列如72所示的第一重链，和氨基酸序列如137所示的第二重链，氨基酸序列如69所示的第一轻链，氨基酸序列如141所示的第二轻链；
r.氨基酸序列如89所示的第一重链，和氨基酸序列如138所示的第二重链，氨基酸序列如69所示的第一轻链，氨基酸序列如139所示的第二轻链；
s.氨基酸序列如136所示的第一重链，和氨基酸序列如142所示的第二重链，氨基酸序列如69所示的第一轻链，氨基酸序列如139所示的第二轻链。
本发明的第二方面的一些实施例中，所述的双特异性抗原结合蛋白选自抗体Ab-6～9中的任一项，其中，Ab-6形式为DVD-Ig，具有氨基酸序列如SEQ ID NO.45所示的重链，和如SEQ ID No.49所示的轻链；
Ab-7形式为IgG-scFv，具有氨基酸序列如SEQ ID NO.46所示的重链，和如SEQ ID No.50所示的轻链；
Ab-8形式为crossmab，具有氨基酸序列如SEQ ID NO.47所示的第一重链，和如SEQ ID No.67所示的第一轻链；氨基酸序列如SEQ ID NO.51所示的第二重链，和如SEQ ID No.69所示的第二轻链；
Ab-9形式为DVD-Ig，具有氨基酸序列如SEQ ID NO.48所示的重链，和如SEQ ID No.52所示的轻链。
本发明的第三方面涉及核酸分子，用于编码以上任一所述的抗原结合蛋白或用于编码以上任一所述的双特异性抗原结合蛋白。
本发明的第四方面涉及载体，其特征在于包含以上所述的核酸分子。
本发明的第五方面涉及细胞，包含以上所述的核酸分子，和/或以上所述的载体。
本发明的第六方面涉及药物分子，其特征在于包含以上任一所述的抗原结合蛋白或以上任一所述的双特异性抗原结合蛋白。
本发明第六方面的一些实施例中，还包括治疗剂或可检测标记物，所述的治疗剂选自细胞毒性剂、细胞抑制剂、放射性同位素、抗血管生成剂或脂质体。
本发明第六方面的一些实施例中，所述的细胞毒性剂来自植物、真菌或细菌的分子或其衍生物。
本发明第六方面的一些实施例中，所述的细胞毒性剂选自肽毒素、蛋白毒素或烷化剂类毒素。
本发明第六方面的一些实施例中，所述的细胞毒性剂选自美登素生物碱类、澳瑞他汀类、艾瑞布林、紫杉烷、卡奇霉素、西马多丁、吡咯苯并二氮杂卓、蒽环类药物、喜树碱衍生物、α-鹅膏蕈碱及其衍生物、曲贝替定及其衍生物、鲁贝替定及其衍生物中的一种或多种。
本发明第六方面的一些实施例中，所述的治疗剂或可检测标记物通过可切割或不可切割的连接子与所述的抗原结合蛋白偶联。
本发明第六方面的一些实施例中，所述的连接子具有一个或多个接头。
本发明第六方面的一些实施例中，所述的接头选自寡肽接头、肼接头、硫脲接头、触发自降解接头、琥珀酰亚胺基反式-4-(马来酰亚胺甲基)环己烷-1-羧酸酯接头、马来酰亚胺接头、二硫化物接头、硫醚接头、连烯接头中的一种或多种。
本发明第六方面的一些实施例中，结构如式I所示：
Ab为所述的抗原结合蛋白，L是含一个或多个接头的连接子，D为所述的治疗剂或可检测标记物，n选自1-20的整数。
本发明第六方面的一些实施例中，n选自2～8的整数。
本发明第七方面涉及药物组合物，包含以上任一所述的抗原结合蛋白、以上任一所述的双特异性抗原结合蛋白，以上所述的核酸分子、以上所述的载体、以上所述的细胞、和/或以上任一所述的药物分子，以及药学上可接受的载体和/或添加物。
本发明的第八方面涉及以上任一所述的抗原结合蛋白、以上任一所述的双特异性抗原结合蛋白，以上所述的载体、以上所述的细胞、和/或以上任一所述的药物分子，在制备药物中的应用，所述的药物用于诊断、预防和/或治疗hROR1相关的疾病和/或病症。
本发明的第八方面的一些实施例中，所述的疾病和/或病症包括肿瘤。
本发明的第八方面的一些实施例中，所述的肿瘤为慢性淋巴细胞白血病、急性淋巴细胞白血病、慢性套细胞淋巴瘤、肉瘤、卵巢癌、肾细胞癌、头颈癌、乳腺癌、肺癌、恶性间皮瘤、结肠癌、神经母细胞瘤或黑色素瘤。
本发明提供新的靶向hROR1的Ig-like结构域和Kringle结构域的不同抗体和基于这些抗体的双抗，并在此基础上产生组合物，例如不同连接子和毒素组合ADC。这些抗体及组合物为肿瘤的诊疗提供了新的手段。特别是，抗hROR1 ADC具有良好的抗肿瘤活性，具有用于肿瘤治疗的潜力。
附图说明
图1示出人ROR1(hROR1)和人ROR2(hROR2)的胞外区，包含信号肽、Ig-like结构域、Frizzled结构域和Kringle结构域。
图2示出新的鼠抗hROR1 mAb的可变免疫球蛋白重链和轻链的氨基酸序列。
图3示出如实施例4所述测出的嵌合鼠/人抗hROR1抗体对商业化的成熟hROR1全长胞外区、hROR1胞外区三个不同结构域、以及成熟hROR2全长胞外区的结合活性。其中，Ab1对hROR1 Ig-like结构域具有较高的亲和力，而Ab4与其它抗体不同，不结合Ig-like结构域而结合Kringle结构域，说明能识别独特的抗原表位。
图4示出如实施例4所述用抗人ROR1抗体Ab-1、Ab-2、Ab-3、Ab-4、Ab-5在人三阴性乳腺癌细胞MDA-MB-231上的FACS细胞染色。
图5示出荧光激活细胞分选术(FACS)测量的所选嵌合鼠/人IgG对人乳腺癌细胞MDA-MB-231上表达的内源hROR1的结合活性。
图6示出所选嵌合鼠/人IgG在表达内源hROR1的人卵巢癌细胞PA-1和人乳腺癌细胞MDA-MB-231的内吞。(A)所选抗体在PA-1细胞上的内吞活性。(B)所选抗体在MDA-MB-231细胞上的内吞活性。所选抗体在这两株细胞上都表现出明显的内吞活性。
图7(A)示出基于所选嵌合鼠/人IgG的不同形式的双特异性抗体的结构示意图。(B)列出这些抗体的全长序列。
图8示出所选双特异性抗体所选对人乳腺癌细胞MDA-MB-231上表达的内源hROR1的结合活性。
图9示出嵌合抗体突变体或人源化抗hROR1抗体与成熟hROR1全长胞外区的结合活性。部分人源化抗体展示出比嵌合体更高的结合活性。
图10(A)示意性地示出怎样产生本发明公开的基于抗体链间二硫键对应的半胱氨酸与不同linker-payload偶联的ADC。图10(B)示意性地示出利用连烯偶联技术产生ADC的主要流程图。10(C)示意性地示出基于引入的半胱氨酸突变怎样产生本发明公开的位点特异性偶联的ADC。通过点突变和重组抗体表达的方法在所选位点引入半胱氨酸突变，之后产生位点特异性偶联的ADC。
图11(A-C)示出浓度范围为0.01～1000nM的所选抗ROR1 ADC在人三阴性乳腺癌细胞MDA-MB-231上的细胞毒性。(D)示出浓度范围为0.01～1000nM的所选抗ROR1 ADC在人肉瘤细胞Saos2上的细胞毒性。(E)示出浓度范围为0.01～1000nM的所选抗ROR1 ADC在人软组织肉瘤细胞SK-UT-1上的细胞毒性。
图12示出在用hROR1阳性MDA-MB-231细胞注射的雌性NCG小鼠中的异种移植模型中ADC41,DAR4的效力。所选ADC展示了很强的抑瘤活性。
具体实施方式
除非另有定义，本文使用的所有技术和科学术语的含义与本领域普通技术人员通常理解的含义相同。如果发生冲突，则以本文的描述、定义为准。
有鉴于现有技术中存在的问题，本发明提供能够与hROR1中特异性结合的抗原结合蛋白，并且具有很好的抗原结合活性，本发明的抗原结合蛋白中，含有a～o任一项：
a.具有第一重链可变区，含有氨基酸序列为TYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，氨基酸序列为YSLMVEAHWYFDV的HCDR3，
b.具有第二重链可变区，含有氨基酸序列为SYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，氨基酸序列为GYSNYVDWYFDV的HCDR3，
c.具有第三重链可变区，含有氨基酸序列为DYTMH的HCDR1，氨基酸序列为GIDPNYGGTNYNEKFKD的HCDR2，氨基酸序列为DDGYYEDYFDY的HCDR3，
d.具有第四重链可变区，含有氨基酸序列为SYWIE的HCDR1，氨基酸序列为EILPGSGNTQNNEKFKG的HCDR2，氨基酸序列为EGRRSHFFDY的HCDR3，
e.具有第五重链可变区，含有氨基酸序列为RYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，氨基酸序列为GYYGNNDWYFDV的HCDR3，
f.具有第一轻链可变区，含有氨基酸序列为KASQDINSYLS的LCDR1，氨基酸序列为RANRLVD的LCDR2，氨基酸序列为LQYDEFPYT的LCDR3，
g.具有第二轻链可变区，含有氨基酸序列为KATQDINSYLS的LCDR1，氨基酸序列为RPNRLVD的LCDR2，氨基酸序列为LQYDEFPYT的LCDR3，
h.具有第三轻链可变区，含有氨基酸序列为HASQGISSNIG的LCDR1，氨基酸序列为HGTNLED的LCDR2，氨基酸序列为VQYAQFPYT的LCDR3，
i.具有第四轻链可变区，含有氨基酸序列为KSSQSLLNSSNQKNYLA的LCDR1，氨基酸序列为FASTRES的LCDR2，氨基酸序列为QQHYSSPWT的LCDR3，
j.具有第五轻链可变区，含有氨基酸序列为KASQDINSYLS的LCDR1，氨基酸序列为RANSLVD的LCDR2，氨基酸序列为LQFDEFPYT的LCDR3，
k.具有a项所述的第一重链可变区和具有f项所述的第一轻链可变区，
l.具有b项所述的第二重链可变区和具有g项所述的第二轻链可变区，
m.具有c项所述的第三重链可变区和具有h项所述的第三轻链可变区，
n.具有d项所述的第四重链可变区和具有i项所述的第四轻链可变区，
o.具有e项所述的第五重链可变区和具有j项所述的第五轻链可变区。本发明的hROR1的抗原结合蛋白种类包括但不限于全长抗体、抗体片段、免疫缀合物、抗体类似物、抗体衍生物、融合蛋白等。抗体包括但不限于嵌合抗体、人源化抗体。抗体片段包括但不限于Fab、Fab’、F(ab)2、Fv、scFv、dsFv、VHH等等。
嵌合抗体通常是指具有小鼠单克隆抗体的V区(可变区)和人免疫球蛋白的C区(恒定区)的抗体。
人源化抗体通常是指小鼠抗体的CDR序列分别引入人VH和VL结构域的框架序列所形成的抗体。
产生本发明的hROR1的单克隆抗体的杂交瘤可以使用公知技术制作。如，首先，使用hROR1蛋白质、表达hROR1的细胞或编码hROR1的基因作为敏化抗原，按照通常的免疫方法将其免疫。采用通常的细胞融合法使从免疫功物中得到的免疫细胞与公知的亲细胞融合，得到杂交瘤。进而，利用通常的筛选法，从此杂交瘤中筛选产生目标抗体的细胞，由此可以选择出产生抗hROR1抗体的杂交瘤。
具体而言，单克隆抗体的制作例如可以如下所示进行。首先，通过表达hROR1基因，可以得到hROR1蛋白质，用作获得抗体的敏化抗原。即，将编码hROR1的基因序列括入公知的表达载体中，转化适当的宿主细胞后，可以采用公知的方法从此宿主细胞中或培养上清液中纯化目标人hROR1蛋白质。另外，纯化的天然的hROR1蛋白质也可以同样地使用。可以通过组合或者单独使用1次或者多次通常的离子层析或亲和层析等多种层析法进行生成。另外，也可以将融合蛋白质用作免疫原，所述融合蛋白质是将hROR1蛋白质的所期望的部分多肽与不同的多肽融合得到的。为了制备为免疫原的融合蛋白质，例如可以使用抗体的Fc片段或肽标记等。表达融合蛋白质的载体，可以通过使编码所期望的二种或者二种以上的多肽片段的基因框内融合，并如上所述地将该融合基因括入表达载体中，进行制作。
如上所示纯化的hROR1蛋白质可以用作对哺乳动物进行免疫时使用的敏化抗原。另外，hROR1的部分肽也可以用作敏化抗原。
用该敏化抗原免疫的哺乳动物没有特殊的限定。为了通过细胞融合法得到单克隆抗体，考虑到与细胞融合中使用的亲细胞的相容性，优选选择免疫动物。一般情况下，作为免疫动物优选啮齿动物。具体而言，可以将小鼠、大鼠、仓鼠、或者家兔用作免疫动物。此外，也可以将猴等用作免疫动物。
上述动物可以按照公知的方法通过敏化抗原免疫。例如，作为一般的方法，可以通过腹腔内或皮下注射敏化抗原对哺乳动物进行免疫。具体而言，每隔4至21日向哺乳动物多次给与该敏化抗原。敏化抗原可以用PBS(Phosphate-Buffered Saline)或生理盐水等以适当的倍率稀释，用于免疫。并且，可以与佐剂一起给与敏化抗原。例如可以与弗式完全佐剂(Freund's complete adjuvant)混合、乳化，形成敏化抗原。另外，用敏化抗原免疫时可以使用适当的载体。特别是分子量小的部分肽用作敏化抗原的情况下，优选使该敏化抗原肽与白蛋白、匙孔血蓝蛋白(keyhole limpet hemocyanin)等载体蛋白质结合进行免疫。
如上所述将哺乳动物免疫，确认血清中的所期望的抗体量升高后，从哺乳动物中采取免疫细胞，进行细胞融合。作为免疫细胞，特别优选使用脾细胞。
目标抗体的筛选及单克隆优选采用基于公知的抗原抗体反应的筛选方法实施。例如，使抗原与用聚苯乙烯等制成的小球或市售96孔微量滴定板等载体结合，与杂交瘤的培养上清液反应。接着，清洗载体后，使酶标记的二次抗体等反应。如果培养上清液中含有与敏化抗原反应的目标抗体，则二次抗体通过此抗体与载体结合。最终通过检测与载体结合的二次抗体，能够确定在培养上清液中是否存在目标抗体。可以通过有限稀释法等克隆产生具有抗原结合能力的所期望的抗体的杂交瘤。此时，作为抗原，可以优选使用用于免疫的蛋白质以及实质相同的hROR1蛋白质。例如包含hROR1的胞外域、或者构成该区域的部分氨基酸序列的寡肽可以作为抗原使用。
嵌合抗体和人源化抗体可以采用已知的方法制造。如将3个CDR和4个FR连结的V区基因，通过在5'末端和3'末端返火且附着适当的限制酶识别序列的引物，将其全长扩增。将如上所述得到的DNA和编码人抗体C区的DNA组装入表达载体中使其框内融合，将该重组载体导入宿主建立重组细胞后，培养该重组细胞，通过使编码该抗体的DNA表达以产生抗体。具体的，可参考实施例2。
所述的抗体形式包括但不限于IgG、IgM、IgA，IgE、IgD等，优选使用IgG。进一步的，所述IgG包 括IgG1、IgG2、IgG3、IgG4等。嵌合抗体和人源化抗体所使用的恒定区中，重链可以使用包括Cγ1，Cγ2，Cγ3，Cγ4、Cμ、Cδ、Cα1、Cα2、Cε等，轻链可以使用包括Cκ、Cλ等。
在抗体的Fc区中可以引入LALA突变以降低抗体与Fcγ受体和补体的结合。重链恒定区的突变包括但不限于C220S、A121C、V205C、K149C、D265C、S239C、A330C、S442C中的一个或多个突变。
本发明中，CDR用于表示抗体的互补决定区(或称高变环)，CDR中的氨基酸序列决定了抗体抗原结合位点的化学结构和特性。重链可变区(VH)中的CDR以HCDR表示，对于三个重链CDR区分别命名为HCDR1，HCDR2和HCDR3；轻链可变区(VL)的CDR以LCDR表示，对于三个轻链CDR区分别命名为LCDR1、LCDR2和LCDR3。需要说明的是，在抗体中CDR区域一般通过实验进行预测识别，如无特别说明，本发明中的各CDR序列由Kabat定义方案界定，相关的氨基酸编号由Kabat编号方案确定。应当理解，当采用不同的定义方案界定CDR时，所预测的CDR区序列可能仅存在部分的重叠，或发生缩短或加长。
在抗体制备中，可根据不同的定义方案，将各自定义的CDR和FR基因序列通过已知的方法连结扩增至全长，从而进一步制备各种抗体或抗体片段。
抗体片段Fab是指，由重链可变区的VH区和重链第一恒定区CH1功能区以及通过二硫键连接的整个轻链组成，主要发挥抗体的抗原结合功能。
scFv为单链抗体，具体是指将抗体的轻链可变区基因和重链可变区基因在DNA水平上与合适的寡核苷酸链(linker)连接起来，使其在合适的生物体中以单肽链的形式表达，并折叠成仅由重链和轻链可变区组成的新抗体。连结的linker没有特殊的限制。例如可以使用包含3至25个残基左右的任意的单链肽作为连接体。
dsFv为二硫键稳定抗体，具体是将抗体的重链可变区(VH)和轻链可变区(VL)中的一个氨基酸残疾突变为半胱氨酸，然后通过链间二硫键连接VH和VL而形成的抗体。
VHH为重链单域抗体，具体是指只含有VH功能域而不含有VL功能域的小分子抗体。
融合蛋白是指，利用基因工程技术将抗体分子片段与其他蛋白质(如抗体酶、免疫毒素、免疫细胞因子、免疫粘附素等)融合的产物。
制备上述各种抗体片段的方法是已知的。其中一种方法为通过酶处理抗体以产生抗体片段，用于产生抗体片段的酶包括但不限于木瓜蛋白酶、胃蛋白酶、或者纤溶酶等。另一种方法为构筑编码上述抗体片段的基因，将其导入表达载体后，使其在适当的宿主细胞中表达以产生抗体片段。
本发明中的抗体衍生物是指在保留内源功能的前提下，对本发明的抗体中一个或多个氨基酸残疾进行的取代、变异、修饰、替换、缺失和/或添加所得到的产物。
本发明中，CH用于表示抗体的重链恒定区，CL用于表示抗体的轻链恒定区，重链恒定区通常进一步包括了CH1、CH2和CH3结构域。FR用于表示抗体可变区的框架，其中，重链可变区的框架区以HFR表示，对于四个重链框架区分别命名为HFR1、HFR2、HFR3和HFR4；轻链可变区的框架区以LFR表示，对于四个轻链框架区分别命名为LFR1、LFR2、LFR3和LFR4。
重链可变区具有S62Q、S62A、S62N、S62E、S62V、S62Y、S62F或S62K突变。
轻链可变区具有G57A、G57S、G57N或G57L突变。
轻链可变区具有D56E、D56Q、D56T或D56Y突变。
轻链可变区具有S31A、S31Q、S31L、S31V、S31E、S31Y、S31F或S31K突变。
轻链可变区具有T57A、T57Q、T57L、T57V、T57E、T57Y、T57F或T57K突变。
轻链可变区具有S27A、S27Q、S27L、S27V、S27E、S27Y、S27F、S27K突变。
本发明还涉及了能够结合HROR1的双特异性抗原结合蛋白，其具有结合hROR1中Ig-like结构域的第一抗原结合片段以及Kringle结构域的第二抗原结合片段，
所述的第一抗原结合片段具有第一重链可变区和第一轻链可变区，其中，
所述的第一重链可变区含有氨基酸序列为TYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，和氨基酸序列为YSLMVEAHWYFDV的HCDR3，
所述的第一轻链可变区含有氨基酸序列为KASQDINSYLS的LCDR1，氨基酸序列为RANRLVD的 LCDR2，和氨基酸序列为LQYDEFPYT的LCDR3，
所述的第二抗原结合片段具有第四重链可变区和第四轻链可变区，其中，
所述的第四重链可变区含有氨基酸序列为SYWIE的HCDR1，氨基酸序列为EILPGSGNTQNNEKFKG的HCDR2，氨基酸序列为EGRRSHFFDY的HCDR3，
所述的第四轻链可变区含有氨基酸序列为KSSQSLLNSSNQKNYLA的LCDR1，氨基酸序列为FASTRES的LCDR2，氨基酸序列为QQHYSSPWT的LCDR3。
双特异性的抗体根据是否存在Fc片段，分为含Fc片段的IgG样双特异性抗体，不含Fc片段的非IgG样抗体。非IgG样抗体包括了BiTE双特异性抗体、DART双特异性抗体、TandAb双特异性抗体、Bi-Nanobody双特异性抗体。Ig样双特异性抗体包括了Triomab quadroma双特异性抗体、KIH IgG双特异性抗体，CrossMAb双特异性抗体、Orthodox-Fab IgG双特异性抗体，YBODY双特异性抗体，Two-in-One双特异性抗体，DVD-Ig双特异性抗体、IgG-ScFv及ScFv2-Fc双特异性抗体、FIT-Ig双特异性抗体，Mebs-Ig双特异性抗体。本申请中对于所制备的抗原结合蛋白结合活性的检测采用的是现有的方法，如流式细胞术。如可参考实施例4的方法进行检测。
本申请中对于所制备的抗原结合蛋白内吞活性的检测可以采用本领域技术人员公知的方法进行。如使用含有可检测标记物的待测抗原结合蛋白，使其与表达有hROR1的细胞共孵育，并通过流式细胞术检测其进入细胞内激发的荧光强度，分析内吞活性。可参考实施例5。
本发明中的载体用于DNA或RNA的复制、转录和/或翻译的表达。通过培养包含所述载体的细胞，可以产生期望的表达产物。所选用的载体是已知，如可以为质粒。
此外，本发明还涉及了含有上述抗原结合蛋白或双特异性抗原结合蛋白的药物分子。
所述的药物分子中还可以包含治疗剂或可检测标记物。
药物分子可以包含任何已知的药物形式。其形式包括但不限于蛋白、多肽、小分子药物、抗体偶联药物或各种形态药物的结合物等。
本发明中的抗体偶联药物是指抗原结合蛋白(通常为抗体或抗体片段)通过连接子与治疗剂偶联。制备抗体偶联药物的方法是已知的。其中，连接子与药物之间的连接通常通过已知的化学合成方法产生，形成能够与抗体结合的连接子-治疗剂结构，在抗体偶联药物的制备过程中，抗体偶联一个或多个连接子-治疗剂结构，抗体与连接子-治疗剂结构的偶联通常针对抗体中的赖氨酸或半胱氨酸残基的侧链进行建立。其中，对于赖氨酸的缀合，连接子中通常具有N-羟基琥珀酰亚胺，可以与所述的赖氨酸的ε-氨基反应形成稳定的酰胺键，形成抗体与治疗剂的偶联。对于半胱氨酸的缀合，连接子中通常具有可介导偶联反应的官能团如马来酰亚胺官能团、连烯酰胺(allenamide)等，所述的半胱氨酸通过已知的方法将二硫键还原，以产生反应性硫醇，从而与马来酰亚胺缀合，形成抗体与治疗剂的偶联。如可参考本发明的实施例8中所示例的方法。
可采用的连接子包括不可切割的和可切割的。连接子中可以具有一个或多个接头，包括但不限于寡肽接头(包括可切割和/或不可切割的寡肽接头)、肼接头、硫脲接头、触发自降解(self-immolative)接头、琥珀酰亚胺基反式-4-(马来酰亚胺甲基)环己烷-1-羧酸酯(SMCC)接头、马来酰亚胺接头、二硫化物接头、硫醚接头和/或连烯接头。
可采用的治疗剂可以是免疫调节剂，例如Sting激动剂，TLR7、TLR8和/或TLR9激动剂，PD-1抑制剂等。
可采用的治疗剂可以是细胞毒性剂、细胞抑制剂等。细胞毒性剂可以是来自植物、真菌或细菌的分子或其衍生物，包括但不限于肽毒素，蛋白毒素，烷化剂类毒素或其他类型毒素。药物毒性剂可以选自美登素生物碱类(例如，美登醇(maytansinol)或DM1美登素)、澳瑞他汀类(例如，一甲基澳瑞他汀E或一甲基澳瑞他汀F)、艾瑞布林、紫杉烷、卡奇霉素、西马多丁、吡咯苯并二氮杂卓、蒽环类药物、喜树碱衍生物、α-鹅膏蕈碱及其衍生物、曲贝替定及其衍生物、鲁贝替定及其衍生物中的一种或多种。本发明中使用的细胞毒性物质可以为一种，也可以组合两种以上细胞毒性物质进行使用。
可采用的治疗剂还可以是代谢药(例如，抗叶酸剂如甲氨蝶呤，氟嘧啶如5-氟尿嘧啶，阿糖胞苷，或者嘌呤或腺苷的类似物)；插入剂(例如，蒽环类如多柔比星，奈莫柔比星，或者优选PNU-159682的衍生物)，道诺霉素，表柔比星(epirabicin)，伊达比星，丝裂霉素-C，放线菌素D，或光神霉素，或者其他插入 剂如吡咯苯并二氮杂卓；DNA-反应剂如卡奇霉素、天赐米星类(tiancimycins)和其他烯二炔；铂衍生物(例如，顺铂或卡铂)；烷化剂(例如，氮芥、美法仑、苯丁酸氮芥、白消安、环磷酰胺、异环磷酰胺亚硝基脲或塞替派)；RNA聚合酶抑制剂如α-鹅膏蕈碱；抗有丝分裂剂(例如，长春花生物碱如长春新碱，或者紫杉烷类如紫杉醇或多西他赛)；拓扑异构酶抑制剂(例如，依托泊苷、替尼泊苷、安吖啶、托泊替康、依喜替康)；细胞周期抑制剂(例如，flavopyridol)；或者抗微管剂(例如，埃坡霉素、tubulysine、pre-tubulysine、淅皮海绵内酯类似物或艾榴素类似物)。治疗剂可以是蛋白酶体抑制剂或拓扑异构酶抑制剂如硼替佐米、安吖啶、依托泊苷、磷酸依托泊苷、替尼泊苷或多柔比星等。治疗性放射性同位素包括碘(131I)、钇(90Y)、镥(177Lu)、锕(225Ac)、镨、砹(At)、铼(Re)、铋(Bi或Bi)和铑(Rh)等。抗血管生成剂包括利诺胺、贝伐单抗、血管抑素和雷佐生。
含有可检测标记物的药物分子可以用于诊断。如可以包括造影剂。造影剂可以是放射性同位素标记如碘(131I或125I)、铟(111In)、锝(99Tc)、磷(32P)、碳(14C)、氚(3H)、其他放射性同位素(例如放射性离子)，或者治疗性放射性同位素如上文列出的治疗性放射性同位素之一。此外，造影剂可以包括不透射线材料、磁共振成像(MRI)剂、超声显影剂以及适合通过成像动物体的装置检测的任何其他造影剂。所述的可检测标记物还可以是荧光标记、生物活性酶标记、发光标记或发色团标记。
药物分子的结构如式I所示：
Ab为所述的抗原结合蛋白，L是含一个或多个接头的连接子，D为所述的治疗剂或可检测标记物，n选自1-20的整数。进一步，可以是自2-8的整数。
本发明的药物组合物的给药方式可以是通过口服或非口服。非口服的给药方式包括如注射给药、经鼻给药、经肺给药、经皮给药等等。注射给药又可以包括静脉注射、肌肉注射、腹腔内注射、皮下注射等。除此以外，给药量可以考虑患者的年龄、症状进行具体的调整。作为给药量，如，给药量可以是每1kg体重每次给药0.0001mg～1000mg。又如，在每位患者的每次给药0.001mg～100000mg。需要说明的是，上述给药量仅用于举例说明，而不用于限定给药量的范围。
本发明的药物组合物可以按照常用方法进行制剂化，也可以同时含有药学上可接受的药物载体和/或添加物。例如可以举出表面活性剂、赋形剂、着色剂、香料、防腐剂、稳定剂、缓冲剂、悬浮剂、等渗剂、粘合剂、崩解剂、润滑剂、流动性促进剂、矫味剂等。并且，并不限定于此，可以适当地使用其它常用的药物载体。具体而言，作为药物载体包括但不限于轻质无水硅酸、乳糖、结晶纤维素、甘露醇、淀粉、羧甲基纤维素钙、羧甲基纤维素纳、羟丙基纤维素、羟丙基甲基纤维素、聚乙烯醇缩乙醛二乙氨基乙酸酯、聚乙烯吡咯烷酮、明胶、中链脂肪酸三甘油酯、聚氧乙烯氢化蓖麻油60、白糖、竣甲基纤维素、玉米淀粉、无机盐类等。
本发明治疗和诊断的癌症的种类没有特殊的限制，通常为hROR1蛋白质表达的癌症，优选为慢性淋巴细胞白血病、急性淋巴细胞白血病、慢性套细胞淋巴瘤、肉瘤、卵巢癌、肾细胞癌、头颈癌、乳腺癌、肺癌、恶性间皮瘤、结肠癌、神经母细胞瘤或黑色素瘤。
并且，本发明的药物组合物中，根据需要可以配合多种抗体。例如，通过制成多种抗hROR1抗体的混合物，可能能够增强对hROR1表达细胞的细胞毒性。或者，除抗hROR1抗体之外，通过配合识别其它肿瘤相关抗原的抗体，也可以提高治疗效果。
如无特别说明，本发明中的各CDR序列由Kabat定义方案界定，相关的氨基酸编号由Kabat编号方案确定。应当理解，当采用不同的定义方案界定CDR时，所预测的CDR区序列可能仅存在部分的重叠，或发生缩短或加长。
以下，通过实施例对于本发明作进一步说明。
实施例1.细胞系
人卵巢癌细胞PA-1，人三阴性乳腺癌细胞系MDA-MB-231和MCF-7，人肉瘤细胞系Saos-2，均购自ATCC。
MDA-MB-231细胞系在补充了10％(v/v)热灭活的FBS(Thermo Scientific；Logan,UT)的DMEM (Thermo Scientific；Logan,UT)中生长。
Saos-2细胞系在补充了10％(v/v)热灭活的FBS(Thermo Scientific；Logan,UT)的RPMI1640(Thermo Scientific；Logan,UT)中生长。
PA-1和MCF-7细胞系在补充了10％(v/v)热灭活的FBS(Thermo Scientific；Logan,UT)1％(v/v)非必须氨基酸，以及1mM丙酮酸钠中的MEM(Thermo Scientific；Logan,UT)生长，以支持贴壁培养。
每种培养基中均添加100U/mL青霉素和100mg/mL链霉素(SigmaAldrich)。
实施例2.小鼠免疫和抗体筛选
免疫原的制备与小鼠免疫：采用human ROR1(30-403)-His作为免疫原制备抗hROR1的单克隆抗体。准备15只小鼠(5只BALB/c小鼠、5只C57bl/6小鼠,5只A/J小鼠)，使用弗氏完全佐剂乳化的50μg human ROR1(30-403)-His蛋白进行皮下注射初次免疫，两周后进行第二次免疫，初次免疫后使用不完全弗氏佐剂乳化的25μg human ROR1(30-403)-His蛋白进行3次免疫，采用腹腔-皮下交替的注射方式，以两周为间隔免疫小鼠。小鼠第四次免疫后7天，采用尾部取血的方式进行采血并进行抗原结合能力的检测。
抗体筛选：从15只小鼠中选择血清效价高的BALB/c小鼠进行细胞融合。取小鼠的脾细胞和SP2/0细胞采用电融合的方式进行细胞融合，SP2/0细胞与脾细胞的比例为20:1。融合后7～10天后，采用间接ELISA和FACS的方法进行杂交瘤上清特异性抗体的检测。挑选阳性克隆进行亚克隆，经过两次亚克隆后得到单克隆细胞株，即杂交瘤细胞株，筛选所得到的阳性杂交瘤细胞株分别为7F6D10、33B10G8、21A9A4、84E3F11和94F8B4。采用SBAClonotyping system-HRP kit进行亚型检测，所选抗体的亚型均为IgG1 Kappa。
鼠抗hROR1 mAb的可变免疫球蛋白重链和轻链的氨基酸序列，如图2所示。
利用Kabat定义的框架区(FR)和互补决定区(CDR)，图中示出命名为7F6D10、33B10G8、21A9A4、84E3F11和94F8B4的5个抗体的重链可变结构域序列(分别为SEQ ID NO:1、5、9、13、17)和轻链可变结构域序列(分别为SEQ ID NO:21、25、29、33、37)。
实施例3.嵌合鼠/人IgG抗体的表达和纯化
利用MHSSALLCCLVLLTGVRA作为前导信号肽通过全基因合成制备抗体的可变区编码区，并与人IgH-γ1以及IgL-κ编码区通过NotI/XbaI酶切位点组装在表达载体pcDNA3.4，该载体具有CMV启动子可增强多种基因在哺乳细胞中的高水平表达，并含有氨苄抗性基因用于选择性标记。选择CHO-K1细胞作为抗体表达的宿主，细胞在合适的条件下(120rpm，8％CO2，37℃)下传代三次，收集细胞加入电转液混合均匀，随后添加质粒并混合均匀后至于电转管中，经电转仪将pcDNA3.4表达载体转染至CHO-K1细胞中。于37℃，270rpm，8％CO2条件下培养24h后，添加补料/丁酸钠/双抗生素后培养3-7天，将收获的上清过滤(0.22μm)除去细胞后用于后续纯化。将处理好的上清用Protein A亲和层析柱纯化，随后用柠檬酸缓冲液(pH 3.4)洗脱，将洗脱液置于透析袋中进行缓冲液换液，最终得到7F6D10、33B10G8、21A9A4、84E3F11和94F8B4嵌合鼠/人IgG1抗体。通过NanoDrop仪器读取A280nM吸光度确定其浓度和数量，并通过SDS-PAGE和SEC-HPLC进行纯度检测，所表达抗体的纯度为>95％。
下表中列出了后续实施例中使用的鼠/人IgG1嵌合抗体，包括它们的浓度和稀释液
实施例4.抗体结合活性的检测
ELISA：对于ELISA，将96孔3590板(Corning，NY)的每个孔分别用100μL含50ng抗原蛋白的包被缓冲液(0.015MNa2CO3，0.035MNaHCO3，pH 9.5)在4℃条件下包被过夜。用150μL每孔含4％(w/v)脱脂奶粉的PBS缓冲液在37℃下封闭1h之后，弃去上清并在干净的纸巾上排干孔内溶液。然后在37℃下于每孔中加入4倍梯度稀释后的待测抗体(7F6D10、33B10G8、21A9A4、84E3F11或94F8B4)作为一抗，孵育1小时后，用1％PBST清洗三遍后弃去上清。再加入100uL每孔PBS缓冲液，其中含1.25ng/mL辣根过氧化物酶(HRP)偶联的山羊抗人IgG Fc(Invitrogen，California，CA)以及4％(w/v)脱脂奶粉在37℃条件下孵育45分钟。用1％PBST清洗三遍后，并且利用TMB显色液(Beyotime，Shanghai，CN)根据制造商的指导进行显色反应，室温避光孵育15分钟，直至显色至预期深浅，后利用TMB显色终止液。利用SpectraMax i3X酶标仪(Molecular Devices；Sunnyvale，CA)在450nm下测量吸光度。为了确定表位范围，直接包被hROR1_Ig-like、hROR1_Frizzled或hROR1_Kringle蛋白，然后用待测抗体温育，之后用同样方式检测。
流式细胞术：将待测细胞按照40000细胞每孔种在96孔圆底板中，加入用梯度稀释(100nM起，4倍稀释到0.006nM)后的抗体作为一抗，在4℃的条件下孵育1小时。将不用一抗孵育，只用二抗孵育的细胞孔作为空白对照。孵育后离心弃去上清，用200uL每孔冰冷的1％(v/v)FBS的PBS洗涤两次。之后用FITC标记的山羊抗人IgG特异性多克隆抗体作为二抗在4℃条件下孵育1小时后重复以上洗涤步骤，弃去上清后重新加入100uL每孔1％FBS的PBS重悬。利用Flow cytometer仪器(Agilent)分析细胞结合后的染色情况，并且利用GraphPad 9.5.1分析数据。
图3示出测出的嵌合鼠/人抗hROR1抗体对商业化的成熟hROR1全长胞外区、hROR1胞外区三个不同结构域、以及成熟hROR2全长胞外区的结合活性。通过ELISA分析每种嵌合鼠/人Fab结合至与成熟hROR1全长胞外区(hROR1_ECD)、人hROR1 Ig相似结构域(hROR1_Ig-like)、hROR1 Frizzled结构域(hROR1_Frizzled)、hROR1 Kringle结构域(hROR1_Kringle)、以及成熟hROR2全长胞外区(hROR2_ECD)。这些商业化的抗原已被验证可用于抗人ROR1或/ROR2抗体的ELISA实验以检测抗体的结合活性。通过固定在板上的嵌合鼠/人抗hROR1抗体捕获带His标签的hROR1_ECD、hROR1_Ig-like、hROR1_Frizzled、hROR1_Frizzled或hROR2_ECD，然后用HRP的山羊抗人抗体作为二抗进行检测。
图4示出Ab-1～5在在人三阴性乳腺癌细胞MDA-MB-231上的FACS细胞染色。
图5示出荧光激活细胞分选术(FACS)测定的所选嵌合鼠/人IgG对人乳腺癌细胞MDA-MB-231上表达的内源hROR1的结合活性。
实施例5.利用pHrodoiFLgreen对抗体内吞活性的检测
将PA-1和MDA-MB-231细胞在恒温培养箱(37℃，5％CO2)中培养过夜。采用pHrodo iFL Green human IgG试剂分别标记所选抗体Ab-1～5，将抗体-pHrodo iFL Green human IgG缀合物分别与人卵巢癌细胞PA-1和人乳腺癌细胞MDA-MB-231共孵育24小时。抗体最高浓度设置为10μg/ml，并进行4倍梯度稀释。孵育完成后，用PBS清洗并用培养基重悬细胞，在NovoCyte流式细胞仪上测定pHrodo iFL Green进入细胞内激发的荧光强度，分析抗体的内吞活性。
图6示出所选嵌合鼠/人IgG在表达内源hROR1的人卵巢癌细胞PA-1和人乳腺癌细胞MDA-MB-231的内吞。Ab-1～5为hROR1特异性mAb，以及对照抗体UC961和C2E3。(A)所选抗体在PA-1细胞上的内吞活性。(B)所选抗体在MDA-MB-231细胞上的内吞活性。
实施例6.纯化的、重组抗hROR1双抗的表达和表征
使用7F6D10和84E3F11嵌合人IgG构建四个双抗，双抗的表达纯化参照实施例3。克隆双抗的可变区编码区，组装于pcDNA表达载体上，并转染至CHO-K1细胞中，进行真核表达并纯化。所得的纯化抗体分别为Ab-6～9。
图7示出基于所选嵌合鼠/人IgG的不同形式的双特异性抗体的结构示意图。
所选双特异性抗体84E3F11-7F6D10和7F6D10-84E3F11为DVD-Ig形式；所选双特异性抗体84E3F11-7F6D10-VH-VL为IgG-scFv形式；所选双特异性抗体84E3F11-7F6D10(cross)为CrossMab形式，如图7所示。(A)为所选双特异性抗体的结构示意图，(B)为所选双特异性抗体的序列。
表3列出了所选鼠/人嵌合双特异性抗体的终浓度和缓冲液。
图8示出所选双特异性抗体所选对人乳腺癌细胞MDA-MB-231上表达的内源hROR1的结合活性。
实施例7.纯化的、重组嵌合抗体突变体或人源化抗hROR1抗体的表达和表征
嵌合抗体突变体或人源化抗体的表达纯化参照实施例3。
表4嵌合抗体突变体或人源化抗体，包括它们的浓度和稀释液。



图9示出嵌合抗体突变体或人源化抗hROR1抗体与成熟hROR1全长胞外区的ELISA结合活性。
实施例8.利用抗体链间二硫键和/或半胱氨酸突变位点偶联产生ADC-1～56
通过抗体链间二硫键位点偶联1-20个抗肿瘤毒素分子制备如下的ADC-1～23：






结合图10(A)所示出利用抗体链间二硫键位点对应的半胱氨酸和马来酰亚胺接头制备抗体偶联药物的总体方法，具体合成ADC-1～23时的偶联过程如下：取一个0.5ml离心管，加入2mg抗体，加入30mM His-HAC，(pH5.5)缓冲液将抗体稀释至5mg/ml，加入100mM EDTA水溶液至终浓度5mM，震荡混匀。按TCEP与抗体摩尔比3:1加入浓度为2mg/ml TCEP水溶液,震荡混匀后于恒温混匀仪内20℃孵育1.5小时。按化合物与抗体摩尔比12:1加入浓度为10mg/ml的连接子-毒素DMSO溶液，并补充DMSO至最终反应体积的20％。震荡混匀后于恒温混匀仪内20℃孵育1.5小时。完成偶联之后，使用300mg/mL的葡聚糖包被活性炭(厂家：Sigma)去除游离小分子。最后用超滤管将ADC储存缓冲液置换为50mM PBS(pH7.2)，-80℃冷冻保存备用。
结合图10(B)所示出利用抗体链间二硫键位点对应的半胱氨酸和连烯接头制备抗体偶联药物的总体方法，具体合成ADC-24时的偶联过程如下：取一个0.5ml离心管，加入2mg抗体，加入25mM Na2B4O7，25mM NaCl，1mM DTPA(pH7.4)缓冲液将抗体稀释至10mg/ml，按抗体摩尔数的2～15倍加入一定体积的TCEP或DTT水溶液，震荡混匀后于恒温混匀仪内20℃孵育2-24小时。按抗体摩尔数的4～24倍加入一定体积的毒素-连接子溶液。震荡混匀后于恒温混匀仪内20℃孵育0.5-24小时。完成偶联之后，停止反应并纯化ADC。
结合图10(A)和图10(C)示出抗体偶联药物的总体制备方法，通过抗体链间二硫键和/或半胱氨酸突变位点偶联1-20个抗肿瘤毒素分子制备如下的ADC-25～56。
取一个0.5ml离心管，加入2mg抗体，加入30mM His-HAC，(pH5.5)缓冲液将抗体稀释至5mg/ml，加入100mM EDTA水溶液至终浓度5mM，震荡混匀。按TCEP与抗体摩尔比3∶1加入浓度为2mg/ml TCEP水溶液，震荡混匀后于恒温混匀仪内20℃孵育1.5小时。或用脱盐柱将抗体缓冲液置换为2mM EDTA， 100mM Tris-HCl(pH 8.0)。取一个1.5ml离心管，加入1mg抗体，按照DTT与抗体摩尔比20:1-100:1加入浓度为100mM DTT水溶液，移液器吹打混匀后于恒温混匀仪内22℃孵育过夜。按化合物与抗体摩尔比12:1加入浓度为10mg/ml的连接子-毒素DMSO溶液，并补充DMSO至最终反应体积的20％。震荡混匀后于恒温混匀仪内20℃孵育1.5小时。完成偶联之后，使用300mg/mL的葡聚糖包被活性炭(厂家：Sigma)去除游离小分子。最后用超滤管将ADC储存缓冲液置换为50mM PBS(pH7.2)，-80℃冷冻保存备用。
或用脱盐柱将抗体缓冲液置换为2mM EDTA，100mM Tris-HCl(pH 8.0)。取一个1.5ml离心管，加入1mg抗体，按照DTT与抗体摩尔比100:1加入浓度为100mM DTT水溶液，移液器吹打混匀后于恒温混匀仪内22℃孵育过夜。用脱盐柱将抗体缓冲液置换为2mM EDTA，150mM NaCl,50mM Tris-HCl(pH 7.5)。按DHAA与抗体摩尔比20:1加入浓度为100mM DHAADMA溶液，移液器吹打混匀后于恒温混匀仪内22℃孵育2小时后用脱盐柱除去DHAA,将抗体缓冲液置换为150mM NaCl，50mM Tris-HCl(pH 7.5)。按化合物与抗体摩尔比6∶1加入浓度为10mg/ml的连接子-毒素DMA溶液，并补充DMA至最终反应体积的10％。震荡混匀后于恒温混匀仪内20℃孵育4小时。完成偶联之后，用脱盐柱除去游离小分子，将ADC储存缓冲液调整为50mM His-HAC(pH5.5)，-80℃冷冻保存备用。








实施例9.基于抗ROR1抗体的ADC的体外细胞毒性检测
将1.5×103个细胞接种在96孔板里(排除PBS填充的边缘孔)。细胞培养在80μL补充了10％FBS(v/v)、100U/mL青霉素和100mg/mL链霉素的DMEM中，并且在37℃、5％CO2的加湿培养箱中过夜培养。在恒温培养箱中孵育一天后，取稀释好的溶液(每孔20μl)添加至各孔，使最终ADC浓度范围为0.01nM～1000nM。与细胞共孵育(37℃，5％CO2)五天后，将96孔板从培养箱中取出并平衡至室温。大约30min之后，在每个孔加入40μL的(Promega,G7572)。将板在450rpm振荡5min，不振荡平衡10min之后，使用SpectraMax i3X酶标仪测定荧光值。用GraphpadPrism软件拟合发光对ADC浓度(nM)的曲线。利用Prism软件的内置“log(抑制剂)对反应-可变斜率(4个参数)”功能确定IC50值。
图11(A-C)示出浓度范围为0.01～1000nM的所选抗ROR1 ADC在人三阴性乳腺癌细胞MDA-MB-231上的细胞毒性。(D)示出浓度范围为0.01～1000nM的所选抗ROR1 ADC在人肉瘤细胞Saos2上的细胞毒性。(E)示出浓度范围为0.01～1000nM的所选抗ROR1 ADC在肉瘤细胞SK-UT-1上的细胞毒性。
实施例10.在hROR1阳性人卵巢癌PA-1细胞建立的卵巢癌模型中评价抗hROR1 ADC的体内效力。
在研究第0天将每只称重至少20g的8-12周龄小鼠，用PA-1卵巢癌肿瘤细胞接种(5×106个细胞/动物，1:1添加基质胶，在100μL PBS中)。在转接后的第21天将配制于PBS中的ADC以3mg/kg施用(i.v.)至6只小鼠的组。PBS做为阴性载体对照(vehicle control)。所有处理组均为尾静脉注射，注射体积为5L/kg，每周一次，共三周。通过测量肿瘤体积大小，考察肿瘤生长是否被抑制、延缓或治愈。每周用卡尺测量2次肿瘤大小，将各组中小鼠的平均肿瘤体积随时间作图。体积单位用mm3表示，公式如下:V＝0.5a×b2，其中a和b分别为肿瘤的长径和短径。实验动物的使用及福利将遵照国际实验动物评估和认可委员会(AAALAC)的规则执行。
图12示出在用hROR1阳性PA-1细胞注射的雌性balb/c nude小鼠中的卵巢癌异种移植模型中ADC-41，DAR4的抗肿瘤效力。
本发明中的实施例仅用于对本发明进行说明，并不构成对权利要求范围的限制，本领域内技术人员可以想到的其他实质上等同的替代，均在本发明保护范围内。

Claims (39)

  1. 分离的抗原结合蛋白，能够结合hROR1，其特征在于含有a～o任一项：
    a.具有第一重链可变区，含有氨基酸序列为TYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，氨基酸序列为YSLMVEAHWYFDV的HCDR3，
    b.具有第二重链可变区，含有氨基酸序列为SYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，氨基酸序列为GYSNYVDWYFDV的HCDR3，
    c.具有第三重链可变区，含有氨基酸序列为DYTMH的HCDR1，氨基酸序列为GIDPNYGGTNYNEKFKD的HCDR2，氨基酸序列为DDGYYEDYFDY的HCDR3，
    d.具有第四重链可变区，含有氨基酸序列为SYWIE的HCDR1，氨基酸序列为EILPGSGNTQNNEKFKG的HCDR2，氨基酸序列为EGRRSHFFDY的HCDR3，
    e.具有第五重链可变区，含有氨基酸序列为RYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，氨基酸序列为GYYGNNDWYFDV的HCDR3，
    f.具有第一轻链可变区，含有氨基酸序列为KASQDINSYLS的LCDR1，氨基酸序列为RANRLVD的LCDR2，氨基酸序列为LQYDEFPYT的LCDR3，
    g.具有第二轻链可变区，含有氨基酸序列为KATQDINSYLS的LCDR1，氨基酸序列为RPNRLVD的LCDR2，氨基酸序列为LQYDEFPYT的LCDR3，
    h.具有第三轻链可变区，含有氨基酸序列为HASQGISSNIG的LCDR1，氨基酸序列为HGTNLED的LCDR2，氨基酸序列为VQYAQFPYT的LCDR3，
    i.具有第四轻链可变区，含有氨基酸序列为KSSQSLLNSSNQKNYLA的LCDR1，氨基酸序列为FASTRES的LCDR2，氨基酸序列为QQHYSSPWT的LCDR3，
    j.具有第五轻链可变区，含有氨基酸序列为KASQDINSYLS的LCDR1，氨基酸序列为RANSLVD的LCDR2，氨基酸序列为LQFDEFPYT的LCDR3，
    k.具有a项所述的第一重链可变区和具有f项所述的第一轻链可变区，
    l.具有b项所述的第二重链可变区和具有g项所述的第二轻链可变区，
    m.具有c项所述的第三重链可变区和具有h项所述的第三轻链可变区，
    n.具有d项所述的第四重链可变区和具有i项所述的第四轻链可变区，
    o.具有e项所述的第五重链可变区和具有j项所述的第五轻链可变区。
  2. 如权利要求1所述的抗原结合蛋白，其特征在于具有鼠源的框架区或人源的框架区。
  3. 如权利要求2所述的抗原结合蛋白，其特征在于所述的框架区为IgG框架区。
  4. 如权利要求2所述的抗原结合蛋白，其特征在于所述的人源的框架区具有相对于鼠源的框架区的一个或多个位点的回复突变。
  5. 如权利要求1所述的抗原结合蛋白，其特征在于还具有恒定区。
  6. 如权利要求5所述的抗原结合蛋白，其特征在于所述的a～e、k～o中的任一项的重链恒定区含有C220S、A121C、V205C、K149C、D265C、S239C、A330C、S442C中的一个或多个突变。
  7. 如权利要求1所述的抗原结合蛋白，其特征在于所述的抗原结合蛋白包括抗体或其抗原结合片段。
  8. 如权利要求1所述的抗原结合蛋白，其特征在于所述的重链可变区具有S62Q、S62A、S62N、S62E、S62V、S62Y、S62F或S62K突变。
  9. 如权利要求1所述的抗原结合蛋白，其特征在于所述的轻链可变区具有G57A、G57S、G57N或G57L突变。
  10. 如权利要求1所述的抗原结合蛋白，其特征在于所述的轻链可变区具有D56E、D56Q、D56T或D56Y突变。
  11. 如权利要求1所述的抗原结合蛋白，其特征在于所述的轻链可变区具有S31A、S31Q、S31L、S31V、S31E、S31Y、S31F或S31K突变。
  12. 如权利要求1所述的抗原结合蛋白，其特征在于所述的轻链可变区具有T57A、T57Q、T57L、T57V、T57E、T57Y、T57F或T57K突变。
  13. 如权利要求1所述的抗原结合蛋白，其特征在于所述的轻链可变区具有S27A、S27Q、S27L、S27V、S27E、S27Y、S27F、S27K突变。
  14. 如权利要求1所述的抗原结合蛋白，其特征在于具有氨基酸序列如SEQ ID No.53、54、55、56、57、58、59、60、62、63、65、73、75、78、77、78、79、80、81、82、83、84、85、86、87、88、90、91、92、93、134或135任一所示的重链，和氨基酸序列如SEQ ID No.64、66、67、68、70、71、94、95、96、97、98、99、100、101、102、103、105、106、108、109、110、112、113、114、115、116、117、119、120、121、122或123任一所示的轻链。
  15. 如权利要求1所述的抗原结合蛋白，其特征在于选自抗体Ab-1～5中的任一项，其中，
    Ab-1具有氨基酸序列如SEQ ID NO.124所示的重链，和如SEQ ID No.129所示的轻链；
    Ab-2具有氨基酸序列如SEQ ID NO.125所示的重链，和如SEQ ID No.130所示的轻链；
    Ab-3具有氨基酸序列如SEQ ID NO.126所示的重链，和如SEQ ID No.131所示的轻链；
    Ab-4具有氨基酸序列如SEQ ID NO.127所示的重链，和如SEQ ID NO.132所示的轻链；
    Ab-5具有氨基酸序列如SEQ ID NO.128所示的重链，和如SEQ ID No.133所示的轻链。
  16. 分离的双特异性抗原结合蛋白，能够结合hROR1，其特征在于具有：
    结合hROR1中Ig-like结构域的第一抗原结合片段，所述的第一抗原结合片段具有第一重链可变区和第一轻链可变区，其中，
    所述的第一重链可变区含有氨基酸序列为TYAMS的HCDR1，氨基酸序列为SISSGGSTYYPDSVKG的HCDR2，和氨基酸序列为YSLMVEAHWYFDV的HCDR3，
    所述的第一轻链可变区含有氨基酸序列为KASQDINSYLS的LCDR1，氨基酸序列为RANRLVD的LCDR2，和氨基酸序列为LQYDEFPYT的LCDR3，
    结合hROR1中Kringle结构域的第二抗原结合片段，所述的第二抗原结合片段具有第四重链可变区和第四轻链可变区，其中，
    所述的第四重链可变区含有氨基酸序列为SYWIE的HCDR1，氨基酸序列为EILPGSGNTQNNEKFKG的HCDR2，氨基酸序列为EGRRSHFFDY的HCDR3，
    所述的第四轻链可变区含有氨基酸序列为KSSQSLLNSSNQKNYLA的LCDR1，氨基酸序列为FASTRES的LCDR2，氨基酸序列为QQHYSSPWT的LCDR3。
  17. 如权利要求16所述的双特异性抗原结合蛋白，其特征在于具有鼠源的框架区或人源的框架区。
  18. 如权利要求17所述的双特异性抗原结合蛋白，其特征在于所述的框架区为IgG框架区。
  19. 如权利要求17所述的双特异性抗原结合蛋白，其特征在于所述的人源的框架区具有相对于鼠源的框架区的一个或多个位点的回复突变。
  20. 如权利要求16所述的双特异性抗原结合蛋白，其特征在于还具有恒定区。
  21. 如权利要求20所述的双特异性抗原结合蛋白，其特征在于所述的第一抗原结合片段的重链恒定区含有C220S、A121C、V205C、K149C、D265C、S239C、A330C、S442C中的一个或多个突变。
  22. 如权利要求20所述的双特异性抗原结合蛋白，其特征在于所述的第二抗原结合片段的重链恒定区含有C220S、A121C、V205C、K149C、D265C、S239C、A330C、S442C中的一个或多个突变。
  23. 如权利要求16所述的双特异性抗原结合蛋白，其特征在于所述的双特异性抗原结合蛋白为IgG样或非IgG样。
  24. 如权利要求23述的双特异性抗原结合蛋白，其特征在于所述的IgG样抗原结合蛋白选自DVD-Ig双特异性抗原结合蛋白、IgG-scFv双特异性抗原结合蛋白或CrossMAb双特异性抗原结合蛋白。
  25. 如权利要求16所述的双特异性抗原结合蛋白，其特征在于具有p、q、r、s中的任一项：
    p.氨基酸序列如61所示的第一重链，和氨基酸序列如140所示的第二重链，氨基酸序列如69所示的第一轻链，氨基酸序列如141所示的第二轻链；
    q.氨基酸序列如72所示的第一重链，和氨基酸序列如137所示的第二重链，氨基酸序列如69所示的第一轻链，氨基酸序列如141所示的第二轻链；
    r.氨基酸序列如89所示的第一重链，和氨基酸序列如138所示的第二重链，氨基酸序列如69所示的第一轻链，氨基酸序列如139所示的第二轻链；
    s.氨基酸序列如136所示的第一重链，和氨基酸序列如142所示的第二重链，氨基酸序列如69所示的第一轻链，氨基酸序列如139所示的第二轻链。
  26. 如权利要求16所述的双特异性抗原结合蛋白，其特征在于选自抗体Ab-6～9中的任一项，其中，
    Ab-6形式为DVD-Ig，具有氨基酸序列如SEQ ID NO.45所示的重链，和如SEQ ID No.49所示的轻链；
    Ab-7形式为IgG-scFv，具有氨基酸序列如SEQ ID NO.46所示的重链，和如SEQ ID No.50所示的轻链；
    Ab-8形式为crossmab，具有氨基酸序列如SEQ ID NO.47所示的第一重链，和如SEQ ID No.67所示的第一轻链；氨基酸序列如SEQ ID NO.51所示的第二重链，和如SEQ ID No.69所示的第二轻链；
    Ab-9形式为DVD-Ig，具有氨基酸序列如SEQ ID NO.48所示的重链，和如SEQ ID No.52所示的轻链。
  27. 核酸分子，其特征在于用于编码权利要求1～15任一所述的抗原结合蛋白或用于编码权利要求16～26任一所述的双特异性抗原结合蛋白。
  28. 载体，其特征在于包含权利要求27所述的核酸分子。
  29. 细胞，包含权利要求27所述的核酸分子，和/或权利要求28所述的载体。
  30. 药物分子，其特征在于包含权利要求1～16任一所述的抗原结合蛋白或权利要求17～26任一所述的双特异性抗原结合蛋白。
  31. 如权利要求30所述的药物分子，其特征在于结构如式I所示：
    Ab为所述的抗原结合蛋白，L是含一个或多个接头的连接子，D为所述的治疗剂或可检测标记物，n选自1-20的整数。
  32. 如权利要求31任一所述的药物分子，其特征在于n选自2～8的整数。
  33. 如权利要求31所述的药物分子，其特征在于还包括治疗剂或可检测标记物，所述的治疗剂选自细胞毒性剂、细胞抑制剂、放射性同位素、抗血管生成剂、免疫调节剂或脂质体。
  34. 如权利要求33所述的药物分子，其特征在于所述的细胞毒性剂选自美登素生物碱类、澳瑞他汀类、艾瑞布林、紫杉烷、卡奇霉素、西马多丁、吡咯苯并二氮杂卓、蒽环类药物、喜树碱衍生物、α-鹅膏蕈碱及其衍生物、曲贝替定及其衍生物、鲁贝替定及其衍生物中的一种或多种。
  35. 如权利要求31所述的药物分子，其特征在于所述的接头选自寡肽接头、肼接头、硫脲接头、触发自降解接头、琥珀酰亚胺基反式-4-(马来酰亚胺甲基)环己烷-1-羧酸酯接头、马来酰亚胺接头、二硫化物接头、硫醚接头、连烯接头中的一种或多种。
  36. 如权利要求31所述的药物分子，其特征在于选自ADC1～56的任一项：















  37. 权利要求1～15任一所述的抗原结合蛋白、权利要求16～26任一所述的双特异性抗原结合蛋白，权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞、和/或权利要求30～35任一所述的药物分子，在制备药物中的应用，所述的药物用于诊断、预防和/或治疗hROR1相关的疾病和/或病症。
  38. 如权利要求37所述的应用，其特征在于所述的疾病和/或病症包括肿瘤。
  39. 如权利要求38所述的应用，其特征在于所述的肿瘤为慢性淋巴细胞白血病、急性淋巴细胞白血病、慢性套细胞淋巴瘤、肉瘤、卵巢癌、肾细胞癌、头颈癌、乳腺癌、肺癌、恶性间皮瘤、结肠癌、神经母细胞瘤或黑色素瘤。
PCT/CN2024/090585 2023-04-30 2024-04-29 hROR1的抗原结合蛋白及其用途 WO2024227426A1 (zh)

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