Antibodies to c-Met and uses thereof

Abstract

The present application relates to an antibody or an antigen-binding fragment thereof capable of specifically binding to c-Met, as well as an immunoconjugate, pharmaceutical composition and kit comprising the same. The present application also relates to the use of the antibody or the antigen-binding fragment thereof in the preparation of a kit or drug. The antibody has a relatively high affinity for cells expressing or overexpressing c-Met, and also has some thermal stability. Furthermore, the antibody can inhibit the HGF-c-Met signal pathway, thereby causing cancer cells to undergo apoptosis, thus inhibiting the proliferation of cancer cells. The antibody has great potential for use in targeted therapy against tumors.

Description

[Technical field]

[0001] The present application belongs to the field of biomedical technology. More specifically, the present application relates to an antibody or an antigen-binding fragment thereof capable of specifically binding to c-Met, as well as to an immunoconjugate, pharmaceutical composition and kit containing the antibody or the antigen-binding fragment thereof. The present application also relates to the use of the antibody or the antigen-binding fragment thereof in the preparation of a kit or a medicament. [Background technology]

[0002] The c-Met protein is a receptor tyrosine kinase that is converted from a 170 kDa leader protein to a 50 kDa α subunit and a 145 kDa β subunit through post-translational modification. The c-Met protein is linked by disulfide bonds to form a transmembrane dimer. At present, the main known ligand of c-Met is hepatocyte growth factor (HGF). Upon stimulation by HGF, the intracellular domain of c-Met undergoes autophosphorylation at tyrosine residues Y1234 and Y1235, and then the phosphorylation signal is transmitted to Y1349 and Y1356, allowing the intracellular domain of c-Met to bind to adaptor proteins. The downstream signal activation pathways of c-Met include PI3K / Akt, Rac1 / Cdc42 and Erk / MAPK, which can significantly affect the associated appearances such as cell proliferation, migration, invasion and tubulogenesis. After the c-Met protein is activated by autophosphorylation, Cb1 ubiquitin ligase initiates the ubiquitination of this protein, which then enters into the degradation process, thus exerting negative regulation on the c-Met pathway.

[0003] Studies have shown that c-Met can promote cancer progression and is highly expressed in a variety of tumor tissues. This makes it a target for anti-cancer drug development. In addition to traditional small molecule RTK inhibitors, drug development of large molecule targeting antibodies has gradually advanced and deepened and entered clinical studies. Examples include the anti-c-Met monoclonal antibody MetMab. Another example is ADC molecules such as TR1801, which is complexed with the antitubulin toxin tesirin and is effective in intermediate and advanced mouse tumor PDX models with low MET expression. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, there is a need to develop novel anti-c-Met monoclonal antibodies and their humanized counterparts to improve affinity and facilitate production. [Means for solving the problem]

[0005] After many experiments and repeated explorations, the present inventors have provided an antibody or an antigen-binding fragment thereof that can specifically bind to c-Met. The antibody has high affinity with cells that express or overexpress c-Met and has a certain degree of thermal stability. In addition, the antibody can inhibit the HGF-c-Met signaling pathway, thereby inducing apoptosis of cancer cells and inhibiting the proliferation of cancer cells.

[0006] Thus, in a first aspect, the present application provides an antibody or antigen-binding fragment thereof capable of specifically binding to c-Met, the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs): (i) a VH CDR1 consisting of a sequence set forth in any one of SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, or 35, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; (ii) a VH CDR2 consisting of a sequence set forth in any one of SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, or 53, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (iii) A VH CDR3 consisting of a sequence set forth in any one of SEQ ID NOs: 55, 57, 59, 61, 63, 65, 67, 69, or 71, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto. and / or (b) a light chain variable region (VL) containing the following three complementarity determining regions (CDRs): (iv) a VL CDR1 consisting of a sequence set forth in any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, or 36, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; (v) a VL CDR2 consisting of a sequence set forth in any one of SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, or 54, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto; and (vi) A VL CDR3 consisting of a sequence set forth in any one of SEQ ID NOs: 56, 58, 60, 62, 64, 66, 68, 70, or 72, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared thereto. Includes.

[0007] In certain embodiments, the substitutions recited in any one of (i)-(vi) are conservative substitutions.

[0008] In certain embodiments, the CDRs set forth in any one of (i)-(vi) are defined according to the Kabat, IMGT or Chothia numbering systems.

[0009] In certain embodiments, the CDRs listed in any one of (i) to (vi) are defined according to the IMGT numbering system.

[0010] In certain embodiments, the above described antibodies or antigen-binding fragments thereof comprise the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO: 19, VH CDR2 set forth in SEQ ID NO: 37, VH CDR3 set forth in SEQ ID NO: 55, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO: 20, VL CDR2 set forth in SEQ ID NO: 38, VL CDR3 set forth in SEQ ID NO: 56.

[0011] In certain embodiments, the above described antibodies or antigen-binding fragments thereof comprise the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO:21, VH CDR2 set forth in SEQ ID NO:39, VH CDR3 set forth in SEQ ID NO:57, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO:22, VL CDR2 set forth in SEQ ID NO:40, VL CDR3 set forth in SEQ ID NO:58.

[0012] In certain embodiments, the above described antibodies or antigen-binding fragments thereof comprise the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO:23, VH CDR2 set forth in SEQ ID NO:41, VH CDR3 set forth in SEQ ID NO:59, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO:24, VL CDR2 set forth in SEQ ID NO:42, VL CDR3 set forth in SEQ ID NO:60.

[0013] In certain embodiments, the above described antibodies or antigen-binding fragments thereof comprise the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO:25, VH CDR2 set forth in SEQ ID NO:43, VH CDR3 set forth in SEQ ID NO:61, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO:26, VL CDR2 set forth in SEQ ID NO:44, VL CDR3 set forth in SEQ ID NO:62.

[0014] In certain embodiments, the above-described antibody or antigen-binding fragment thereof comprises a VH CDR1 set forth in SEQ ID NO:27, a VH CDR2 set forth in SEQ ID NO:45, a VH CDR3 set forth in SEQ ID NO:63, and / or the following three light chain CDRs: a VL CDR1 set forth in SEQ ID NO:28, a VL CDR2 set forth in SEQ ID NO:46, and a VL CDR3 set forth in SEQ ID NO:64.

[0015] In certain embodiments, the above described antibodies or antigen-binding fragments thereof comprise the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO:29, VH CDR2 set forth in SEQ ID NO:47, VH CDR3 set forth in SEQ ID NO:65, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO:30, VL CDR2 set forth in SEQ ID NO:48, VL CDR3 set forth in SEQ ID NO:66.

[0016] In certain embodiments, the above described antibodies or antigen-binding fragments thereof comprise the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO: 31, VH CDR2 set forth in SEQ ID NO: 49, VH CDR3 set forth in SEQ ID NO: 67, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO: 32, VL CDR2 set forth in SEQ ID NO: 50, VL CDR3 set forth in SEQ ID NO: 68.

[0017] In certain embodiments, the above described antibodies or antigen-binding fragments thereof comprise the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO: 33, VH CDR2 set forth in SEQ ID NO: 51, VH CDR3 set forth in SEQ ID NO: 69, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO: 34, VL CDR2 set forth in SEQ ID NO: 52, VL CDR3 set forth in SEQ ID NO: 70.

[0018] In certain embodiments, the above described antibodies or antigen-binding fragments thereof comprise the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO: 35, VH CDR2 set forth in SEQ ID NO: 53, VH CDR3 set forth in SEQ ID NO: 71, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO: 36, VL CDR2 set forth in SEQ ID NO: 54, VL CDR3 set forth in SEQ ID NO: 72.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a human immunoglobulin framework region.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof described above comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the following: (i) a sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 or 17; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17. and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) a sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18 (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18; or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18. Includes.

[0021] In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0022] In certain embodiments, the above-mentioned antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:1 and a VL having the sequence set forth in SEQ ID NO:2.

[0023] In certain embodiments, the above-mentioned antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:3 and a VL having the sequence set forth in SEQ ID NO:4.

[0024] In certain embodiments, the above-mentioned antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:5 and a VL having the sequence set forth in SEQ ID NO:6.

[0025] In certain embodiments, the above-mentioned antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:7 and a VL having the sequence set forth in SEQ ID NO:8.

[0026] In certain embodiments, the above-mentioned antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:9 and a VL having the sequence set forth in SEQ ID NO:10.

[0027] In certain embodiments, the above-mentioned antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:11 and a VL having the sequence set forth in SEQ ID NO:12.

[0028] In certain embodiments, the above-mentioned antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:13 and a VL having the sequence set forth in SEQ ID NO:14.

[0029] In certain embodiments, the above-mentioned antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:15 and a VL having the sequence set forth in SEQ ID NO:16.

[0030] In certain embodiments, the above-mentioned antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:17 and a VL having the sequence set forth in SEQ ID NO:18.

[0031] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, the variant having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g., no more than 20, no more than 15, no more than 10 or no more than 5 amino acid substitutions, deletions or additions or any combination thereof; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); and / or (b) A human immunoglobulin light chain constant region (CL) or a variant thereof, the variant having one or several amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence from which it is derived (e.g., 20 or less, 15 or less, 10 or less, or 5 or less amino acid substitutions, deletions or additions, or any combination thereof; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions, or any combination thereof). Includes.

[0032] In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0033] In certain embodiments, the light chain constant region is a κ light chain constant region or a λ light chain constant region.

[0034] In certain embodiments, the heavy chain constant region has the sequence set forth in SEQ ID NO:121.

[0035] In one particular embodiment, the light chain constant region has the sequence set forth in SEQ ID NO:122.

[0036] In certain embodiments, in the antibodies or antigen-binding fragments thereof described above, the antigen-binding fragment is Fab, Fab', (Fab') 2, Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb).

[0037] In certain embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a multispecific antibody.

[0038] In certain embodiments, the antigen-binding fragment is a Fab.

[0039] In certain embodiments, the antigen-binding fragment further comprises an Fc fragment (eg, an Fc fragment of human IgG1) or a variant thereof.

[0040] In certain embodiments, the Fc fragment has a LALA mutation and a knob mutation or the Fc fragment has a LALA mutation and a hole mutation.

[0041] In certain embodiments, the Fc fragment has the sequence set forth in SEQ ID NO:111 or 112.

[0042] In another aspect, the application provides an isolated nucleic acid molecule, which encodes an antibody or antigen-binding fragment thereof described above.

[0043] In another aspect, the present application provides a vector, which comprises the nucleic acid molecule described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0044] In another aspect, the present application provides a host cell, which comprises the nucleic acid molecule described above or the vector described above. In certain embodiments, the host cell is a mammalian cell.

[0045] In another aspect, the present application provides a method for preparing an antibody or antigen-binding fragment thereof as described above, comprising culturing a host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cells.

[0046] In another aspect, the application provides a multispecific molecule, which comprises an antibody or antigen-binding fragment thereof as described above.

[0047] In certain embodiments, a multispecific molecule is capable of specifically binding to c-Met and, in addition, is capable of specifically binding to one or more other targets.

[0048] In certain embodiments, the multispecific molecule is a bispecific molecule.

[0049] In certain embodiments, the bispecific molecule further comprises a molecule having a second binding specificity for a second target (eg, a second antibody).

[0050] In another aspect, the present application provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof as described above, or a multispecific molecule as described above, and a therapeutic agent linked to the antibody or antigen-binding fragment thereof or the multispecific molecule.

[0051] In certain embodiments, the therapeutic agent is selected from a cytotoxic agent.

[0052] In certain embodiments, the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, anticancer antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof.

[0053] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0054] In another aspect, the present application provides a pharmaceutical composition, comprising an antibody or antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or an immunoconjugate as described above, and a pharma- ceutically acceptable carrier and / or excipient.

[0055] In certain embodiments, the pharmaceutical composition further comprises an additional pharma- ceutical active agent.

[0056] In certain embodiments, the additional pharmacologic active agent is a drug with antitumor activity, such as an alkylating agent, a mitotic inhibitor, an anticancer antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide drug, a radiosensitizer, an antiangiogenic drug, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0057] In certain embodiments, the antibody or antigen-binding fragment thereof, multispecific molecule or immunoconjugate and the additional pharma- ceutical active agent are provided as separate components or as components of the same composition.

[0058] In another aspect, the present application provides a kit, which comprises the antibody or antigen-binding fragment thereof described above.

[0059] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (eg, a chemiluminescent material), or biotin.

[0060] In certain embodiments, the kit further comprises a second antibody capable of specifically recognizing the antibody or antigen-binding fragment thereof described above.

[0061] In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorochrome, a luminescent material (eg, a chemiluminescent material), or biotin.

[0062] In another aspect, the present application provides a chimeric antigen receptor, which comprises the antigen-binding domain of an antibody or antigen-binding fragment thereof described above.

[0063] In certain embodiments, the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment thereof described above.

[0064] In certain embodiments, the antigen binding domain is an scFv.

[0065] In certain embodiments, the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell).

[0066] In another aspect, the present application provides a method for inhibiting the growth of and / or killing tumor cells expressing c-Met, comprising contacting the tumor cells with an effective amount of the antibody or antigen-binding fragment thereof as described above, or the multispecific molecule as described above, or the immunoconjugate as described above, or the pharmaceutical composition as described above, or the chimeric antigen receptor as described above.

[0067] In another aspect, the present application provides the use of an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or an immunoconjugate as described above, or a pharmaceutical composition as described above, or a chimeric antigen receptor as described above, in the manufacture of a medicament for preventing and / or treating a tumor in a subject (e.g. a human).

[0068] In certain embodiments, the medicament further comprises an additional pharma- ceutical active agent.

[0069] In certain embodiments, the additional pharmacologic active agent is a drug with antitumor activity, such as an alkylating agent, a mitotic inhibitor, an anticancer antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide drug, a radiosensitizer, an antiangiogenic drug, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0070] In certain embodiments, the tumor expresses c-Met.

[0071] In certain embodiments, the tumor is associated with tumor cells that express c-Met, hi certain embodiments, c-Met is expressed on the surface of tumor cells.

[0072] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, B-cell rich lymphoma of T cell / histiocyte rich type, and other hematological malignancies, such as leukemia, lymphoma, myeloma, mycosis fungoides ... and EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, and central nervous system (CN) tumors, such as primary CNS lymphoma, spinal axis tumor, brain stem glioma, and the like.

[0073] In some embodiments, the subject is a mammal, such as a human.

[0074] Use of an antibody or antigen-binding fragment thereof as described above in the manufacture of a kit for determining whether a tumor can be treated by an anti-tumor therapy that targets c-Met. (1) contacting a sample containing tumor cells with an antibody or antigen-binding fragment thereof described above; (2) detecting the formation of a complex comprising the antibody or antigen-binding fragment thereof and c-Met.

[0075] In some embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label.

[0076] In some embodiments, the c-Met is mammalian (eg, human, monkey) c-Met.

[0077] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, and / or primary ovarian cancer. tumors of the central nervous system (CN), such as primary CNS lymphoma, spinal cord tumor, brain stem glioma, etc.; tumors of the pulmonary artery, bronchial, and pulmonary artery, such as pulmonary artery, pulmonary sarcoma, ...

[0078] In another aspect, the present application provides a method for preventing and / or treating a tumor in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof as described above, or a bispecific or multispecific molecule as described above, or an immunoconjugate as described above, or a pharmaceutical composition as described above, or a chimeric antigen receptor as described above, or a host cell as described above.

[0079] In certain embodiments, the tumor expresses c-Met.

[0080] In certain embodiments, the tumor is associated with tumor cells that express c-Met, hi certain embodiments, c-Met is expressed on the surface of tumor cells.

[0081] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, and / or primary ovarian cancer. tumors of the central nervous system (CN), such as primary CNS lymphoma, spinal cord tumor, brain stem glioma, etc.; tumors of the pulmonary artery, bronchial, and pulmonary artery, such as pulmonary artery, pulmonary sarcoma, ...

[0082] In certain embodiments, the subject is a mammal, such as a human.

[0083] In certain embodiments, the method further comprises administering an additional drug having anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anticancer antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide drug, a radiosensitizer, an antiangiogenic drug, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0084] In certain embodiments, the method further comprises administering an additional anti-tumor therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy, or palliative care.

[0085] In another aspect, the present application provides a method for determining whether a tumor can be treated by an anti-tumor therapy that targets c-Met, the method comprising the steps of: (1) contacting a sample containing tumor cells with an antibody or antigen-binding fragment thereof described above; (2) detecting the formation of a complex comprising the antibody or antigen-binding fragment thereof and c-Met.

[0086] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label.

[0087] In certain embodiments, the c-Met is mammalian (eg, human, monkey) c-Met.

[0088] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, and / or primary ovarian cancer. tumors of the central nervous system (CN), such as primary CNS lymphoma, spinal cord tumor, brain stem glioma, etc.; tumors of the pulmonary artery, bronchial, and pulmonary artery, such as pulmonary artery, pulmonary sarcoma, ...

[0089] In another aspect, the present application provides a method for determining the presence or amount of c-Met in a sample, the method comprising the steps of: (1) contacting the sample with an antibody or antigen-binding fragment thereof described above; (2) detecting the formation of a complex comprising the antibody or antigen-binding fragment thereof and c-Met or detecting the amount of a complex.

[0090] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label.

[0091] In certain embodiments, the c-Met is mammalian (eg, human, monkey) c-Met.

[0092] Definition of Terms In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA manipulation steps used herein are all common steps widely used in the corresponding fields.At the same time, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0093] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain (CL). The constant domains are not directly involved in binding of the antibody to an antigen, but exert various effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site, respectively. Assignment of amino acids to regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0094] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The heavy and light chain variable regions each contain three CDRs, designated CDR1, CDR2, and CDR3. The exact boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al. al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0095] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined according to the Kabat, Chothia or IMGT numbering system.

[0096] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0097] The term "antibody" is not limited to any particular method of producing the antibody. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be of various isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0098] As used herein, the terms "monoclonal antibody", "McAb" and "mAb" have the same meaning and are used interchangeably to refer to an antibody or antibody fragment derived from a population of highly homogeneous antibody molecules, i.e., identical except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high degree of specificity for a single epitope on an antigen. Polyclonal antibodies are described in comparison to monoclonal antibodies, which usually contain at least two or more different antibodies, and which usually recognize different epitopes on an antigen. In addition, the modifier "monoclonal" is intended only to indicate that the antibody is characterized as being obtained from a highly homogeneous population of antibodies, and should not be construed as requiring that the antibody be prepared by any particular method.

[0099] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, e.g., Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, e.g., U.S. Patent Application No. 4,816,567), or phage antibody library technology (see, e.g., Clackson et al. Nature 352:624-628, 1991 or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0100] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specifically binding to an antigen, also referred to as an "antigen-binding moiety." See generally Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989) which is incorporated herein by reference in its entirety for any purpose. Antigen-binding fragments of antibodies can be obtained by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') and / or F(ab') fragments. 2 , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (Domantis technology), probodies and such polypeptides that contain at least a portion of an antibody sufficient to confer specificity to a polypeptide with antigen-binding ability. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0101] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains". In particular, a "full-length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in an N-terminal to C-terminal direction, and optionally further comprises a heavy chain constant region CH4 domain if the full-length antibody is an IgE isotype. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of a VH, a CH1, a HR, a CH2, and a CH3 in an N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in an N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked to each other by a disulfide bond between the CL and the CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be derived from a single species, such as human, and may also be a chimeric or humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites, each formed by a VH and VL pair, that specifically recognize / bind the same antigen.

[0102] As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains, the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)), the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains, and the term "F(ab') 2 The term "Fab' fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and the term "Fab' fragment" refers to an F(ab') 2 It refers to a fragment obtained by reducing the disulfide bond that links two heavy chain fragments in a fragment consisting of a complete light chain and an Fd fragment of the heavy chain (consisting of the VH and CH1 domains).

[0103] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0104] As used herein, the term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of a first heavy chain and the second and third constant regions of a second heavy chain of an antibody. The Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.

[0105] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, where the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers may consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, although variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. Optionally, there may further be a disulfide bond between the VH and VL of the scFv. In certain embodiments of the invention, the scFv may form a di-scFv, which refers to an antibody formed by linking two or more single scFvs in tandem. In certain embodiments of the invention, the scFv may form (scFv)2, which refers to an antibody formed by two or more single scFvs in parallel.

[0106] As used herein, the term "single domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), which retains the ability to specifically bind to the same antigen that the full-length antibody binds. Single domain antibodies are also called nanobodies.

[0107] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to antigen.

[0108] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of an antibody can be screened for specificity in the same way as an intact antibody.

[0109] As used herein, unless the context clearly indicates otherwise, when the term "antibody" is referred to it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0110] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light or / and heavy chain is derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass) and another portion of the light or / and heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), but in any case, the chimeric antibody still retains binding activity for the target antigen (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" may include an antibody in which the heavy and light chain variable regions of the antibody are derived from a first antibody and the heavy and light chain constant regions of the antibody are derived from a second antibody.

[0111] As used herein, the term "identity" refers to the sequence match between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions x 100%). In certain embodiments, the two sequences are the same length.

[0112] The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified by Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0113] As used herein, the term "variant" in reference to a polypeptide (including a polypeptide) also refers to a polypeptide or peptide that comprises an amino acid sequence that has been altered by the introduction of substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently attaching any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide can be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis with tunicamycin, etc. In addition, a variant has similar, identical, or improved function as the polypeptide or peptide from which the variant is derived.

[0114] As used herein, the term "specific binding" refers to a selective binding reaction between two molecules, such as the reaction between an antibody and an antigen to which the antibody is derived. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0115] The specific binding properties between two molecules can be determined using methods known in the art. Some methods involve measuring the rates of formation and dissociation of antigen-binding site / antigen complexes. Both the "association rate constant" (ka or k) and the "dissociation rate constant" (k or k) can be calculated from the concentrations and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k / k is equal to the dissociation constant K (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of K, k and k can be measured by any valid method. In certain embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in a Biacore. In addition, the dissociation constant can be measured by bioluminescence interferometry or Kinexa.

[0116] As used herein, a detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art and examples include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dyes). These include, but are not limited to, fluorescein derivatives (e.g., Cy7, Alexa750), luminescent materials (e.g., acridinium ester compounds, chemiluminescent materials such as luminol and its derivatives, ruthenium derivatives such as ruthenium terpyridine), magnetic beads (e.g., Dynabeads®), colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0117] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the element of genetic material carried by the vector is expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Vectors can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may also contain an origin of replication.

[0118] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0119] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., an amino acid residue that is physically or functionally similar (e.g., has similar size, shape, charge, chemical properties, etc., including the ability to form covalent or hydrogen bonds) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0120] The 20 common amino acids referred to herein are written according to conventional convention. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0121] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents for delaying absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers generally have the meaning understood by those skilled in the art and can stabilize the desired activity of the active ingredient of the drug, and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharma-ceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution).In certain exemplary embodiments, such a sterile injectable fluid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), a sodium chloride solution (e.g., 0.9% (w / v) NaCl), a glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0122] As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease, disorder or condition in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include (but are not limited to) alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., no longer worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and alleviation of symptoms (whether partial or total), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to expected survival (if not receiving treatment).

[0123] As used herein, the term "subject" refers to a mammal, such as a human, a cynomolgus monkey, or a mouse. In certain embodiments, the subject (e.g., a human, a cynomolgus monkey, or a mouse) is suffering from or at risk of suffering from a disease associated with c-Met.

[0124] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, an effective amount for preventing a disease refers to an amount sufficient to prevent, inhibit or delay the onset of the disease, and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of a person skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the general condition of the patient, such as age, weight and sex, the method of administration of the drug, and other treatments administered at the same time, etc.

[0125] As used herein, the term "single-armed antibody" refers to an antigen-binding fragment comprising a Fab segment and an Fc segment, where the Fab segment generally comprises a heavy chain (e.g., VH and CH1) and a light chain (e.g., VL and CL), and the Fc segment comprises a constant region (e.g., CH2 and CH3). The Fab and Fc segments may or may not be connected by a linker. The single-armed antibodies of the present invention can be prepared or synthesized by a variety of methods, for example, by constructing sequences encoding a Fab heavy chain and sequences encoding an Fc into the same vector and constructing sequences encoding a Fab light chain into another vector and transforming the two vectors separately into a host cell to obtain a single-armed antibody.

[0126] As used herein, the term "bispecific antibody" refers to a complex formed by a first antibody (or fragment thereof) and a second antibody (or fragment thereof) or antibody analogue through coupling arms, the coupling methods including, but not limited to, chemical reaction, gene fusion and enzymatic. Bispecific antibodies can be linked or generated by a variety of methods, see for example the methods of Songsivilai et al. (Clin. Exp. Immunol., 79:315-321 (1990)) and Kostelny et al. (J. Immunol., 148:1547-1553 (1992)).

[0127] Beneficial Effects of the Invention Compared with the prior art, the antibody or its antigen-binding fragment prepared in the present application can specifically bind to c-Met, and then specifically recognize or bind to cells expressing c-Met. The antibody has high affinity with cells expressing or overexpressing c-Met, and has a certain degree of thermal stability. In addition, the antibody can inhibit the HGF-c-Met signaling pathway, thereby causing apoptosis of cancer cells and inhibiting the proliferation of cancer cells, and has great potential for use in targeted tumor therapy.

[0128] Although the embodiments of the present invention will be described in detail below with drawings and examples, those skilled in the art will understand that the following drawings and examples are used only to illustrate the present invention, rather than to limit the scope of the present invention. From the drawings and the following detailed description of the preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art. [Brief description of the drawings]

[0129] [Figure 1] FIG. 1 shows a schematic diagram of the structure of the antibody constructed in Example 2 of the present application. [Figure 2A]Figure 2 shows the binding activity of anti-c-Met antibodies of the present application to cells overexpressing c-Met. Figure 2A shows the binding activity of antibodies of the present application (antibodies 22, 23, 74, 111, and 136) and a control antibody to HEK293-Hu c-Met cells. [Figure 2B] FIG. 2B shows the binding activity of the antibodies of the present application (antibodies 187, 216, 221, 223) and control antibodies to HEK293-Hu c-Met cells. [Figure 2C] FIG. 2C shows the binding activity of the antibodies of the present application (antibodies 22, 23, 74, 111, 136) and a control antibody to HEK293-Rhe c-Met cells. [Figure 2D] FIG. 2D shows the binding activity of the antibodies of the present application (antibodies 187, 216, 221, 223) and control antibodies to HEK293-Rhe c-Met cells. [Diagram 3] FIG. 3 shows the inhibitory activity of the anti-c-Met antibodies of the present application (antibodies 22, 23, 74, 111, 136, 187, 216, 221, and 223) and control antibodies against HCC827 cells. [Figure 4] FIG. 4 shows the inhibition of the anti-c-Met antibodies of the present application (antibodies 136, 187) and control antibodies against H596 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0130] Sequence information Some sequence information relevant to the present invention is provided in Table 1 below.

[0131] [Table 1]

[0132] [Table 2]

[0133] [Table 3]

[0134] [Table 4]

[0135] [Table 5]

[0136] [Table 6]

[0137] [Table 7]

[0138] [Table 8]

[0139] Specific Models for Carrying Out the Invention The invention will now be described with reference to the following examples which are intended to illustrate, but not to limit, the invention.

[0140] Unless otherwise stated, the experiments and methods described in the examples are essentially carried out according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FMA Usubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL METHOD.APPROACH) (MJ MacPherson, BD Hames and GR Tailor, ed. (1995)) and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0141] In addition, unless specific conditions are specified in the examples, the examples were carried out according to normal conditions or conditions recommended by the manufacturer. All reagents or equipment used without indicating the manufacturer were normal products that could be obtained commercially. It is known to those skilled in the art that the examples are illustrative and are not intended to limit the scope of protection of the present invention. All disclosures and other references mentioned herein are incorporated herein in their entirety by reference. EXAMPLES

[0142] Example 1: Screening for anti-c-Met antibodies 1.1. Construction of antibody library Immunization of animals 1 mg of cellular mesenchymal epithelial transition factor (c-Met) antigen (purchased from AcroBiosystems) was mixed with an equal amount of Freund's adjuvant and used to immunize five humanized mice (purchased from Alloy Company), immunized once a week for a total of four times, to stimulate B cells to produce antigen-specific antibodies. After four immunizations, the spleens of the mice were harvested and total RNA was extracted using the RNA extraction reagent Trizol (purchased from Invitrogen). The cDNA of the fully humanized mice was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

[0143] Antibody gene amplification First PCR: The sequences of the heavy and light chain variable regions of the fully humanized antibody were amplified from cDNA using heavy chain upstream primers 1 to 4 (SEQ ID NO: 73 to 76), heavy chain downstream primers 1 to 2 (SEQ ID NO: 77 and SEQ ID NO: 78), light chain upstream primers 1 to 3 (SEQ ID NO: 79 to 81), and light chain downstream primer 1 (SEQ ID NO: 82) shown in Table 1.

[0144] The PCR products were subjected to agarose gel electrophoresis to determine the size and uniformity of the bands. The target fragments were 400bp to 500bp.

[0145] Second PCR: Heavy chain upstream primers 5 to 16 (SEQ ID NO: 83 to SEQ ID NO: 94), heavy chain downstream primers 3 to 5 (SEQ ID NO: 95 to SEQ ID NO: 97), light chain upstream primers 4 to 13 (SEQ ID NO: 98 to SEQ ID NO: 107), and light chain downstream primers 2 to 4 (SEQ ID NO: 108 to SEQ ID NO: 110) shown in Table 1 were used, and the product of the first PCR was used as a template for the second sequence amplification to add arms homologous to the heavy and light chain genes.

[0146] The target fragment was recovered using a PCR purification kit (purchased from QIAGEN).

[0147] Library Construction The linearized yeast display vector and the product of the second PCR were mixed and electrotransformed into Saccharomyces cerevisiae (purchased from ATCC) to construct a complete humanized anti-c-Met antibody library derived from five mice, and the amount of the library was determined. The amount of the library was 3 × 10 7 It was.

[0148] 1.2. Screening for c-Met antibodies Biotinylation labeling of c-Met protein An appropriate volume of double distilled water was measured to dissolve human c-Met protein (purchased from AcroBiosystems). According to the instructions of the biotin labeling kit (purchased from Thermo), biotin was dissolved and mixed with the protein solution, then incubated at 4°C for 2 hours. Excess biotin was removed using a desalting column (purchased from Thermo), and all operations of pretreatment with the desalting column and sample collection were performed according to the steps in the product instructions.

[0149] Magnetic bead sorting (MACS) to enrich for yeast capable of specifically binding human c-Met The antibody library constructed in Example 1.1 was inoculated into SD-CAA amplification medium (1L of SD-CAA amplification medium contained 6.7g YNB, 5g tyrosine, 13.62g Na2HPO4·12H2O, 7.44g NaH2PO4 and 2% glucose) and cultured overnight at 30℃ and 225 rpm. An appropriate amount of yeast cells was measured and centrifuged at 3000 rpm×5 min (the same centrifugation procedure below) to remove the culture medium, and then the yeast cells were resuspended in SD-CAA induction medium and induced overnight. The concentration of the library after induction was determined, and an appropriate amount of yeast cells was harvested and centrifuged to remove the medium. The yeast cells were resuspended in 50ml of PBS and centrifuged to remove the supernatant. The yeast cells were resuspended in 10ml of PBS.

[0150] Biotin-labeled human c-Met protein (its final concentration was 100 nM) was added and incubated at room temperature for 30 min, and the yeast cells were collected by centrifugation and washed three times with 50 ml of PBS. The yeast cells were resuspended in 5 ml of washing solution, 200 μl of SA magnetic beads were added (purchased from Miltenyi Biotec), and the sample was incubated for 10 min. The yeast and magnetic beads mixture was washed three times with PBS, and the mixture was added to an LS purification column (purchased from Miltenyi Biotec). The LS purification column was placed on a magnetic stand and washed with PBS to remove non-specifically bound yeast cells. The purification column was removed from the magnetic stand and PBS was added to elute the yeast cells. The eluted yeast cells were centrifuged and transferred to SD-CAA amplification medium for amplification.

[0151] Flow cytometric sorting (FACS) to obtain high affinity yeast cells Yeast cells enriched by MACS were seeded in SD-CAA amplification medium and grown overnight at 30°C and 225 rpm in a shake flask. Yeast cells were resuspended in SD-CAA induction medium and induced overnight. Anti-c-Myc mouse antibody (purchased from Thermo) and 100 nM biotin-labeled c-Met antigen were added and incubated for 10 minutes. Yeast cells were washed three times with PBS, and goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (purchased from Invitrogen) and streptavidin APC conjugated fluorescent antibody (purchased from Invitrogen) were added and incubated for 15 minutes. Cells were resuspended in PBS and sorted using a BD AriaIII machine to obtain yeast with high binding ability to c-Met antigen.

[0152] Search for c-Met antibody candidate molecules and antibody genes The yeast solution with high binding ability to human c-Met antigen obtained by enrichment with MACS and FACS was coated on a solid culture plate of SD-CAA, then a single clone was selected and cultured in SD-CAA amplification medium at 30°C and 225 rpm overnight. The amplified single clone was treated with 0.1% SDS, centrifuged, and the supernatant was used as a template for PCR amplification. The PCR product was sequenced to obtain the gene sequence. A total of nine antibodies were obtained, named antibodies 22, 23, 74, 111, 136, 187, 216, 221 and 223, whose specific sequences are shown in Table 1, and the CDR sequences of the antibodies were determined by the IMGT numbering system (Lefranc et al., Dev. Comparat.Immunol. 27:55-77, 2003).

[0153] Example 2: Construction, expression and purification of single-arm antibodies 2.1. Construction of antibody genes into pCDNA3.1 expression vector The nucleotide sequence encoding the heavy chain variable region (described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17) was ligated to the nucleotide sequence encoding the heavy chain constant region 1 (described in SEQ ID NO: 121) and the nucleotide sequence encoding the human IgG1-Fc (LALA mutation, knob mutation) segment (SEQ ID NO: 111) and cloned using the homologous recombination system into a linearized pCDNA3.1 vector digested on both sides with EcoRI and NotI restriction enzymes (purchased from Vazyme). The nucleotide sequence encoding the light chain variable region (described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18) and the nucleotide sequence encoding the light chain constant region (described in SEQ ID NO: 122) were cloned into an EcoR I / Xhol I double-sided digested linearized pCDNA3.1 vector. The nucleotide sequence encoding the Fc-LALA-hole (SEQ ID NO: 112) was cloned into an EcoR I / Xhol I double-sided digested linearized pCDNA3.1 vector. The process was carried out as per the product manual. The homologous recombination products were transformed into Top10 competent cells, plated on ampicillin-resistant plates, then incubated overnight at 37°C, and single colonies were picked for sequencing and plasmids were extracted.

[0154] 2.2. Cell transfection and protein purification Using the ExpiCHO™ Expression System Kit (purchased from Thermo), the heavy chain (Fc-LALA-knob), light chain and Fc-LALA-hole (SEQ ID NO: 112) plasmids extracted above were transfected simultaneously into Expi-CHO cells to produce an antibody with a single Fab structure (Figure 1). The transfection method was performed as described in the product manual. After 5 days of cell culture, the supernatant was collected and the target protein was purified by Protein A magnetic bead (purchased from GenScript) sorting method. The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1-4 times the volume of the magnetic beads) and added to the sample for purification, and incubated with gentle shaking at room temperature for 1 hour. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. Add elution buffer (0.1 M sodium citrate, pH 3.2) 3-5 times the volume of magnetic beads, shake at room temperature for 5-10 min, place back on the magnetic stand, collect the elution buffer, transfer to a collection tube with neutralization buffer (1 M Tris, pH 8.54), and mix thoroughly to obtain the target protein.

[0155] Example 3: Determination of c-Met antibody affinity Affinity determination by ForteBio was performed according to existing methods (Estep, P et al., Determination of antibody-antigen affinity and epitope binding based on high-throughput methods. MAbs, 2013.5(2):p.270-8). Briefly, the sensor was equilibrated offline in assay buffer for 30 minutes, then the test was performed online for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor. The sensor was then placed in 100 nM human c-Met antigen for 5 minutes, then transferred to PBS for 5 minutes for dissociation. Kinetic analysis was performed using a 1:1 binding model.

[0156] [Table 9]

[0157] Candidate molecules were also tested for cross-reactivity with proteins from various species, including proteins from cynomolgus monkeys (purchased from AcroBiosystems).

[0158] [Table 10]

[0159] The experimental results are shown in Tables 2 and 3. All nine antibodies of the present application had some affinity with mammalian (eg, human, monkey) c-Met protein.

[0160] The protein construction, expression and purification methods were the same as in Example 2.2, and the protein purity was obtained by HPLC detection. The HPLC method was as follows: mobile phase: 150 mM Na2HPO4·12H2O, pH 7.0; chromatographic conditions: detection wavelength: 280 nm, column temperature: 25°C, flow rate: 0.35 ml / min, detection time: 20 min, Zenix-C SEC-300 chromatography column (SEPAX 4.6×300 mm, 3 μm).

[0161] [Table 11]

[0162] The experimental results are shown in Table 4. All nine antibodies in this application had relatively high purity, i.e., all were above 94%.

[0163] Example 4. Thermostability of anti-c-Met candidates DSC (Differential Scanning Calorimetry) was used to detect the thermal stability of various antibodies. The samples were concentrated and diluted to 1mg / ml with PBS. 5000x fluorescent colorimetric reagent Cypro Orange (purchased from Bio-rad) was diluted 50 times with ultrapure water to obtain 100x fluorescent colorimetric reagent Sypro Orange. 50μl of 1mg / ml sample was measured, 10μl of 100x fluorescent colorimetric reagent Sypro Orange and 40μl of ultrapure water were added, mixed thoroughly, 30μl of the mixture was measured and added to a 96-well PCR plate, triplicate wells per sample, and the plate was placed in the PCR machine. The heating program was set as follows: keep constant at 25℃ for 5 minutes, heat to 99℃ at a rate of 0.5℃ / min. After the program was completed, the temperature value of the lowest point of the curve in the "melting curve" graph was read, which was taken as the Tm value of the sample. The specific results are shown in Table 5 below.

[0164] [Table 12]

[0165] The experimental results are shown in Table 5. All nine antibodies of the present application had some degree of thermal stability.

[0166] Example 5. Purified anti-c-Met candidates that bind to human / rhesus c-Met cells To confirm the binding ability of the antibodies of the present application to mammalian cells expressing c-Met, three existing antibodies, amivantamab, LY2875358 and onartuzumab, were selected as control antibodies for comparison.

[0167] Therein, the construction process of amivantamab and LY2875358 control antibodies was the same as that of the single-arm antibody of the present application, and both were constructed according to the steps of Example 2. Briefly, for the control antibody amivantamab, the insert fragments described in SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115 were inserted into a plasmid, respectively, and the plasmids were simultaneously transformed into Expi-CHO cells. For the control antibody LY2875358, the insert fragments described in SEQ ID NO:116, SEQ ID NO:117 and SEQ ID NO:118 were inserted into a plasmid, respectively, and the plasmids were simultaneously transformed into Expi-CHO cells. For the control antibody onartuzumab, the nucleotide sequences encoding SEQ ID NO:119, SEQ ID NO:120 and SEQ ID NO:115 were inserted into a plasmid, respectively, and the plasmids were simultaneously transformed into Expi-CHO cells.

[0168] HEK293T cells overexpressing human c-Met (HEK293-Hu c-Met) and rhesus c-Met (HEK293-Rhe c-Met) were generated by transfecting pCHO1.0 vector (purchased from Invitrogen) containing human or rhesus c-Met cDNA (purchased from Sino Biological) cloned into the MCS. The cell density of the expanded HEK293-Hu c-Met / HEK293-Rhe c-Met cells was 2 × 10 6 The purified c-Met antibody was diluted in PBS starting at 100 nM in 3-fold dilutions, totaling 10. The diluted samples were added to the 96-well flow cytometry plate at 100 μl / well with the cells, incubated at 4°C for 30 minutes, and washed twice with PBS. Goat F(ab') antibody diluted 100-fold in PBS was added to the 96-well flow cytometry plate starting at 100 nM. 2Anti-human IgG-Fc (PE) (purchased from Abcam) was added at 100 μl / well, incubated at 4° C. for 30 min, and washed twice with PBS. PBS was added at 100 μl / well, cells were resuspended, and cells were detected with a CytoFlex (Becman) flow cytometer and the corresponding MFI was calculated.

[0169] In the detection experiment of the above method, the experimental results are shown in Figure 2A and Figure 2B, and all of the anti-c-Met single-arm antibodies of the present invention had binding activity to HEK293-Hu c-Met cells. In particular, antibody 136 showed a strong binding activity similar to that of the control antibody amivantamab, and was superior to the control antibody LY2875358 and the control antibody onartuzumab. Figure 2C and Figure 2D showed that the binding activity of all of the anti-c-Met single-arm antibodies of the present invention to HEK293-Rhe c-Met cells was similar to that to human c-Met overexpressing cells. In particular, antibody 136 showed a strong binding activity similar to that of amivantamab, and was superior to other candidate antibodies and control antibodies. In addition, as shown in Tables 6 and 7, all of the anti-c-Met screening single-arm antibodies showed similar binding ability (EC50) to cells expressing human c-Met and rhesus c-Met.

[0170] [Table 13]

[0171] [Table 14]

[0172] Example 6: Blocking HGF-dependent TKI resistance with single-arm antibodies for anti-c-Met screening HCC827 cells are human non-small cell lung cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences, which could highly express epidermal growth factor receptor (EGFR) (exon 19 deletion) and c-Met receptor. Treatment with tyrosine kinase inhibitor (TKI) small molecule gefitinib (gefitinib, epidermal growth factor receptor tyrosine kinase inhibitor) could induce apoptosis in HCC827 cells. If HGF was added simultaneously under such conditions, the c-Met pathway would be activated, thereby inducing HCC827 to resist gefitinib and inhibiting apoptosis.

[0173] The cell density of cultured HCC827 cells was increased to 2 × 10 4 The cells were adjusted to 1000 nM per well and added to a 96-well cell culture plate at 100 μl per well and incubated overnight for later use. Using 1640 medium, the antibodies to be tested were diluted in 1640 medium to 1000 nM, HGF to 800 ng / ml, and gefitinib to 8 μM. 50 μl per well of diluted antibody, 25 μl per well of HGF, and 25 μl per well of gefitinib were added to a 96-well plate with cells according to the requirements of the experiment, and 1640 medium was replenished to reach a total volume of 200 μl per well. After incubation at 37°C and 5% carbon dioxide for 3 days, 100 μl of medium was removed, then Cell titer glo (purchased from Promega) was added at 100 μl per well, and the chemiluminescence signal was collected by a microplate reader.

[0174] The experimental results are shown in Figure 3. The single-arm antibodies for anti-c-Met screening of the present application inhibited the HGF-c-Met signaling pathway (e.g., antibodies 22, 23, 74, 85, 111, 136 and 187), thereby restoring the apoptosis of HCC827 induced by gefitinib. Among them, both antibodies 136 and 187 showed significant inhibitory activity, significantly better than other antibodies (e.g., antibodies 22, 23, 74.85, 111, 216, 221, 222, 223 and 225), and even better than some control antibodies.

[0175] Example 7. Blocking HGF-induced cell proliferation by single-arm antibodies for anti-c-Met screening H596 cells are human lung cancer cells, purchased from ATCC, and could express EGFR and c-Met receptors. Treatment with HGF could induce H596 cell proliferation. If the control antibody was added under such conditions, the HGF-c-Met signaling pathway would be inhibited, thereby inhibiting the HGF-induced proliferation of H596. The expanded H596 cells were cultured at 3 × 10 4 The cells were adjusted to reach a density of 100 μl / well of cells / ml and added to a 96-well cell culture plate at 100 μl / well and cultured overnight for later use. Antibodies to be tested were diluted to 400 nM and HGF was diluted to 200 ng / mL using 1640 medium. 50 μl / well of diluted antibodies and 50 μl / well of HGF were added to the 96-well plate with cells according to the requirements of the experiment, and 1640 medium was supplemented to reach a total volume of 200 μl / well. The cells were cultured at 37°C and 5% carbon dioxide for 5 days. After 5 days, 100 μl of medium was removed, then Cell titer glo (purchased from Promega) was added at 100 μl / well, and the chemiluminescence signal was collected by a microplate reader.

[0176] As shown in Figure 4, both single-arm antibodies 136 and 187 for anti-c-Met screening significantly inhibited HGF-induced proliferation of H596 cells. In addition, the inhibitory effect of antibody 136 was better than all control antibodies.

[0177] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings disclosed, and these modifications are within the scope of protection of the present invention. The whole of the present invention is indicated by the appended claims and any equivalents thereof.

Claims

**Claim 1**: Comprising a heavy chain variable region (VH) and a light chain variable region (VL), (1) said VH comprises VH CDR1 set forth in SEQ ID NO: 29, VH CDR2 set forth in SEQ ID NO: 47, and VH CDR3 set forth in SEQ ID NO: 65; and said VL comprises VL CDR1 set forth in SEQ ID NO: 30, VL CDR2 set forth in SEQ ID NO: 48, and VL CDR3 set forth in SEQ ID NO: 66; (2) said VH comprises VH CDR1 set forth in SEQ ID NO: 27, VH CDR2 set forth in SEQ ID NO: 45, and VH CDR3 set forth in SEQ ID NO: 63; and said VL comprises VL CDR1 set forth in SEQ ID NO: 28, VL CDR2 set forth in SEQ ID NO: 46, and VL CDR3 set forth in SEQ ID NO: 64; (3) said VH comprises VH CDR1 set forth in SEQ ID NO: 19, VH CDR2 set forth in SEQ ID NO: 37, and VH CDR3 set forth in SEQ ID NO: 55; and said VL comprises VL CDR1 set forth in SEQ ID NO: 20, VL CDR2 set forth in SEQ ID NO: 38, and VL CDR3 set forth in SEQ ID NO: 56; (4) said VH comprises VH CDR1 set forth in SEQ ID NO: 21, VH CDR2 set forth in SEQ ID NO: 39, and VH CDR3 set forth in SEQ ID NO: 57; and said VL comprises VL CDR1 set forth in SEQ ID NO: 22, VL CDR2 set forth in SEQ ID NO: 40, and VL CDR3 set forth in SEQ ID NO: 58; (5) said VH comprises VH CDR1 set forth in SEQ ID NO: 23, VH CDR2 set forth in SEQ ID NO: 41, and VH CDR3 set forth in SEQ ID NO: 59; and said VL comprises VL CDR1 set forth in SEQ ID NO: 24, VL CDR2 set forth in SEQ ID NO: 42, and VL CDR3 set forth in SEQ ID NO: 60; (6) said VH comprises VH CDR1 set forth in SEQ ID NO: 25, VH CDR2 set forth in SEQ ID NO: 43, and VH CDR3 set forth in SEQ ID NO: 61; and said VL comprises VL CDR1 set forth in SEQ ID NO: 26, VL CDR2 set forth in SEQ ID NO: 44, and VL CDR3 set forth in SEQ ID NO: 62; (7) The VH comprises a VH CDR1 set forth in SEQ ID NO: 31, a VH CDR2 set forth in SEQ ID NO: 49, and a VH CDR3 set forth in SEQ ID NO: 67; and the VL comprises a VL CDR1 set forth in SEQ ID NO: 32, a VL CDR2 set forth in SEQ ID NO: 50, and a VL CDR3 set forth in SEQ ID NO: 68; (8) The VH comprises a VH CDR1 set forth in SEQ ID NO: 33, a VH CDR2 set forth in SEQ ID NO: 51, and a VH CDR3 set forth in SEQ ID NO: 69; and the VL comprises a VL CDR1 set forth in SEQ ID NO: 34, a VL CDR2 set forth in SEQ ID NO: 52, and a VL CDR3 set forth in SEQ ID NO: 70; or (9) The VH comprises a VH CDR1 set forth in SEQ ID NO: 35, a VH CDR2 set forth in SEQ ID NO: 53, and a VH CDR3 set forth in SEQ ID NO: 71; and the VL comprises a VL CDR1 set forth in SEQ ID NO: 36, a VL CDR2 set forth in SEQ ID NO: 54, and a VL CDR3 set forth in SEQ ID NO: 72 An antibody or an antigen-binding fragment thereof that can specifically bind to c-Met.

2. The antibody or an antigen-binding fragment thereof is (1) a VH having a sequence set forth in SEQ ID NO: 11 or a sequence having at least 80% sequence identity thereto, and a VL having a sequence set forth in SEQ ID NO: 12 or a sequence having at least 80% sequence identity thereto; (2) a VH having a sequence set forth in SEQ ID NO: 9 or a sequence having at least 80% sequence identity thereto, and a VL having a sequence set forth in SEQ ID NO: 10 or a sequence having at least 80% sequence identity thereto; (3) a VH having a sequence set forth in SEQ ID NO: 1 or a sequence having at least 80% sequence identity thereto, and a VL having a sequence set forth in SEQ ID NO: 2 or a sequence having at least 80% sequence identity thereto; (4) a VH having a sequence set forth in SEQ ID NO: 3 or a sequence having at least 80% sequence identity thereto, and a VL having a sequence set forth in SEQ ID NO: 4 or a sequence having at least 80% sequence identity thereto; VH having the sequence set forth in SEQ ID NO: 5 or a sequence having at least 80% sequence identity thereto, and VL having the sequence set forth in SEQ ID NO: 6 or a sequence having at least 80% sequence identity thereto; VH having the sequence set forth in SEQ ID NO: 7 or a sequence having at least 80% sequence identity thereto, and VL having the sequence set forth in SEQ ID NO: 8 or a sequence having at least 80% sequence identity thereto; VH having the sequence set forth in SEQ ID NO: 13 or a sequence having at least 80% sequence identity thereto, and VL having the sequence set forth in SEQ ID NO: 14 or a sequence having at least 80% sequence identity thereto; VH having the sequence set forth in SEQ ID NO: 15 or a sequence having at least 80% sequence identity thereto, and VL having the sequence set forth in SEQ ID NO: 16 or a sequence having at least 80% sequence identity thereto; or VH having the sequence set forth in SEQ ID NO: 17 or a sequence having at least 80% sequence identity thereto, and VL having the sequence set forth in SEQ ID NO: 18 or a sequence having at least 80% sequence identity thereto; The antibody or antigen-binding fragment thereof according to claim 1, comprising the same.

3. The antibody or antigen-binding fragment thereof is a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, wherein the variant has 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived, a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, and / or b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof, wherein the variant has 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived, a light chain constant region (CL) of a human immunoglobulin or a variant thereof The antibody or antigen-binding fragment thereof according to claim 1, comprising the same.

4. The following: i) The heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, etc.; ii) The light chain constant region is a κ light chain constant region or a λ light chain constant region; (iii) The heavy chain constant region has the sequence set forth in SEQ ID NO: 121; (iv) The light chain constant region has the sequence set forth in SEQ ID NO: 122; and (v) The antibody or antigen-binding fragment thereof further comprises a framework region of human immunoglobulin The antibody or antigen-binding fragment thereof according to claim 3, characterized by one or more of the above.

5. The antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') ２ , Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb), and / or the antibody is a mouse antibody, chimeric antibody, humanized antibody, or multispecific antibody. The antibody or antigen-binding fragment thereof according to claim 1.

6. (i) The antigen-binding fragment further comprises an Fc fragment of human IgG1 or a variant thereof; (ii) The antigen-binding fragment further comprises an Fc fragment having a LALA mutation and a knob mutation or an Fc fragment having a LALA mutation and a hole mutation; or (iii) The antigen-binding fragment further comprises an Fc fragment having the sequence set forth in SEQ ID NO: 111 or 112 The antibody or antigen-binding fragment thereof according to claim 5.

7. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to claim 1.

8. A vector comprising the nucleic acid molecule according to claim 7.

9. A host cell comprising the nucleic acid molecule according to claim 7 or the vector comprising the isolated nucleic acid molecule.

10. A method for preparing the antibody or antigen-binding fragment thereof according to claim 1, comprising culturing a host cell comprising an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof under conditions enabling the expression of the antibody or antigen-binding fragment thereof and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cell.

11. A multispecific molecule comprising the antibody or antigen-binding fragment thereof according to claim 1.

12. The following: (i) The multispecific molecule can specifically bind to c-Met and, in addition, can specifically bind to one or more other targets; (ii) The multispecific molecule is a bispecific molecule; and (iii) The multispecific molecule further comprises a molecule having a second binding specificity for a second target The multispecific molecule according to claim 11, characterized by one or more of the above.

13. The immune complex comprising the antibody or antigen-binding fragment thereof according to claim 1, or a bispecific or multispecific molecule comprising said antibody or antigen-binding fragment thereof, and a therapeutic agent linked to said antibody or antigen-binding fragment thereof or said multispecific molecule.

14. The following: (i) The therapeutic agent is selected from cytotoxic agents; (ii) The therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, anticancer antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof; and (iii) The immune complex is an antibody-drug conjugate (ADC) The immune complex according to claim 13, characterized by one or more of the above.

15. A pharmaceutically acceptable carrier and / or excipient, and the following: The antibody or antigen-binding fragment thereof according to claim 1, or A bispecific or multispecific molecule comprising said antibody or antigen-binding fragment thereof, or An immune complex comprising said antibody or antigen-binding fragment thereof, and a therapeutic agent linked to said antibody or antigen-binding fragment thereof A pharmaceutical composition comprising one of the above.

16. The following: (i) The pharmaceutical composition further comprises an additional pharmaceutically active agent; (ii) The pharmaceutical composition further comprises an alkylating agent, mitotic inhibitor, anticancer antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, anti-angiogenic agent, cytokine, molecular target drug, immune checkpoint inhibitor or oncolytic virus; and (iii) The antibody or antigen-binding fragment thereof, the multispecific molecule or the immune complex and the additional pharmaceutically active agent are provided as separate components or as components of the same composition The pharmaceutical composition according to claim 15, characterized by one or more of the above.

17. A kit comprising the antibody or antigen-binding fragment thereof according to claim 1.

18. The following: (i) The antibody or antigen-binding fragment thereof comprises an enzyme, radionuclide, fluorescent dye, luminescent substance or biotin; (ii) The antibody or antigen-binding fragment thereof comprises a detectable label; and (iii) The kit further comprises a second antibody capable of specifically recognizing the antibody or antigen-binding fragment thereof according to claim 1 The kit according to claim 17, characterized by one or more thereof.

19. A chimeric antigen receptor comprising an antigen-binding domain of the antibody according to claim 1 or an antigen-binding fragment thereof.

20. The following: (i) The antigen-binding domain comprises a heavy-chain variable region and a light-chain variable region of the antibody according to claim 1 or an antigen-binding fragment thereof; (ii) The antigen-binding domain is a scFv; and (iii) The chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell) The chimeric antigen receptor according to claim 19, characterized by one or more thereof.

21. A method for inhibiting the growth of tumor cells expressing c-Met and / or killing said tumor cells, comprising contacting said tumor cells with an effective amount of the following: The antibody according to claim 1 or an antigen-binding fragment thereof, or A multispecific molecule comprising said antibody or an antigen-binding fragment thereof, or An immune complex comprising said antibody or an antigen-binding fragment thereof and a therapeutic agent linked to said antibody or an antigen-binding fragment thereof, or A pharmaceutical composition comprising said antibody or an antigen-binding fragment thereof, said bispecific or multispecific molecule, or said immune complex A method comprising contacting with any one of the above.

22. For use in the prevention and / or treatment of tumors in a subject, the antibody according to claim 1 or an antigen-binding fragment thereof, a bispecific or multispecific molecule comprising said antibody or an antigen-binding fragment thereof, or an immune complex comprising said antibody or an antigen-binding fragment thereof and a therapeutic agent linked to said antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising said antibody or an antigen-binding fragment thereof, said bispecific or multispecific molecule, or said immune complex, or a chimeric antigen receptor comprising an antigen-binding domain of said antibody or an antigen-binding fragment thereof.

23. The following: (i) The tumor is accompanied by tumor cells expressing c-Met; (ii) The tumor is accompanied by tumor cells having c-Met expressed on the cell surface; (iii) the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, multiple myeloma, fungating polypoid tumor, Merkel cell carcinoma, and other malignant blood diseases; and (iv) the subject is human The use according to claim 22, characterized by one or more of the above.

24. A method for determining whether a tumor can be treated by an anti-tumor therapy targeting c-Met, comprising the following steps: (1) contacting a sample containing tumor cells with the antibody or antigen-binding fragment thereof according to claim 1; (2) detecting the formation of a complex comprising the antibody or antigen-binding fragment thereof and c-Met A method comprising the above.

25. The following: (i) the antibody or antigen-binding fragment thereof comprises a detectable label; (ii) the c-Met is human c-Met; and (iii) the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, multiple myeloma, fungating polypoid tumor, Merkel cell carcinoma, and other malignant blood tumors The method according to claim 24, characterized by one or more of the above.

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