TW202442686A - Antibodies against c-Met and their uses - Google Patents

Abstract

This application relates to an antibody that can specifically bind c-Met or an antigen-binding fragment thereof, as well as immunoconjugates, pharmaceutical compositions and kits containing containing such an antibody. This application also relates to the use of the antibody or the antigen-binding fragment in preparing kits or pharmaceuticals. The antibody has a relatively high affinity to cells that express or overexpress c-Met, and also has certain thermal stability. Moreover, the antibody can inhibit the HGF-c-Met signaling pathway, thereby causing cancer cell apoptosis and inhibiting proliferation of cancer cells, demonstrating significant potential in targeted tumor therapy.

Description

Antibodies against c-Met and their uses

The present application belongs to the field of biopharmaceutical technology. More specifically, the present application relates to an antibody or an antigen-binding fragment thereof that can specifically bind to c-Met, as well as an immunoconjugate, a drug composition and a reagent 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 reagent kit or a drug.

c-Met protein is a receptor tyrosine kinase. It is converted from a 170kDa leader protein into a 50kDa α subunit and a 145kDa β subunit after translational modification. It forms a transmembrane dimer after being linked by disulfide bonds. Currently, the main known ligand of c-Met is hepatocyte growth factor (HGF). After being stimulated by HGF, the tyrosine positions Y1234 and Y1235 of the intracellular end of c-Met are phosphorylated, and then the phosphorylation of Y1349 and Y1356 is transmitted, so that the intracellular end of c-Met binds to the framework factor. The signal activation pathways downstream of c-Met include PI3K/Akt, Rac1/Cdc42, and Erk/MAPK, which can significantly affect cell proliferation, migration, invasion, and vascular tissue formation. After the autophosphorylation of c-Met protein is activated, Cb1 ubiquitin ligase activates the ubiquitination of this protein and then enters the degradation process, negatively regulating the c-Met pathway.

Studies have shown that c-Met can promote cancer growth and is highly expressed in a variety of tumor tissues. This makes it a target for anticancer drug development. In addition to traditional RTK small molecule inhibitors, drug development of macromolecular target antibodies has gradually advanced and deepened into clinical research. For example, the anti-c-Met monoclonal antibody MetMab. There are also ADC molecules such as TR1801, which, with the help of the microtubule protein toxin tesirine, has an effect on mouse tumor PDX models with low, medium, and high MET abundance.

Therefore, it is necessary to develop a new anti-c-Met monoclonal antibody and its corresponding humanized antibody to improve affinity and facilitate production.

After a large number of experiments and repeated explorations, the inventors of the present application have provided an antibody or an antigen-binding fragment thereof that can specifically bind to c-Met, the antibody has a high affinity with cells expressing or over-expressing 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.

Therefore, in the first aspect, the present application provides an antibody or an antigen-binding fragment thereof capable of specifically binding to c-Met, the antibody or the antigen-binding fragment thereof comprising: (a) a heavy chain variable region (VH) comprising the following three complementary determining regions (CDRs): (i) VH CDR1, which is composed of a sequence shown in any one of SEQ ID NO:19, 21, 23, 25, 27, 29, 31, 33 or 35, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, (ii) VH CDR2, which is composed of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51 or 53, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and (iii) VH CDR3, which is composed of a sequence shown in any one of SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69 or 71, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and/or, (b) a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): (iv) VL CDR1, which is composed of the following sequence: SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34 or 36, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52 or 54, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 70 or 72, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

In certain embodiments, the substitutions described in any one of (i)-(vi) are conservative substitutions.

In certain embodiments, the CDRs described in any of (i)-(vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

In certain embodiments, the CDRs described in any of (i)-(vi) are defined according to the IMGT numbering system.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 19, VH CDR2 as shown in SEQ ID NO: 37, VH CDR3 as shown in SEQ ID NO: 55; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 20, VL CDR2 as shown in SEQ ID NO: 38, VL CDR3 as shown in SEQ ID NO: 56.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 21, VH CDR2 as shown in SEQ ID NO: 39, VH CDR3 as shown in SEQ ID NO: 57; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 22, VL CDR2 as shown in SEQ ID NO: 40, VL CDR3 as shown in SEQ ID NO: 58.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 23, VH CDR2 as shown in SEQ ID NO: 41, VH CDR3 as shown in SEQ ID NO: 59; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 24, VL CDR2 as shown in SEQ ID NO: 42, VL CDR3 as shown in SEQ ID NO: 60.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 25, VH CDR2 as shown in SEQ ID NO: 43, VH CDR3 as shown in SEQ ID NO: 61; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 26, VL CDR2 as shown in SEQ ID NO: 44, VL CDR3 as shown in SEQ ID NO: 62.

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

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 29, VH CDR2 as shown in SEQ ID NO: 47, VH CDR3 as shown in SEQ ID NO: 65; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 30, VL CDR2 as shown in SEQ ID NO: 48, VL CDR3 as shown in SEQ ID NO: 66.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 31, VH CDR2 as shown in SEQ ID NO: 49, VH CDR3 as shown in SEQ ID NO: 67; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 32, VL CDR2 as shown in SEQ ID NO: 50, VL CDR3 as shown in SEQ ID NO: 68.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 33, VH CDR2 as shown in SEQ ID NO: 51, VH CDR3 as shown in SEQ ID NO: 69; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 34, VL CDR2 as shown in SEQ ID NO: 52, VL CDR3 as shown in SEQ ID NO: 70.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 35, VH CDR2 as shown in SEQ ID NO: 53, VH CDR3 as shown in SEQ ID NO: 71; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 36, VL CDR2 as shown in SEQ ID NO: 54, VL CDR3 as shown in SEQ ID NO: 72.

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

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the following: (i) a sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to a sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17; or (iii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to a sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17 has a sequence with 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; and/or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the following: (iv) a sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18; (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to a sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18; or (vi) a sequence with ... NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18 has a sequence with 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.

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

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a VH having a sequence as shown in SEQ ID NO: 1 and a VL having a sequence as shown in SEQ ID NO: 2.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a VH having a sequence as shown in SEQ ID NO: 3 and a VL having a sequence as shown in SEQ ID NO: 4.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a VH having a sequence as shown in SEQ ID NO: 5 and a VL having a sequence as shown in SEQ ID NO: 6.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a VH having a sequence as shown in SEQ ID NO: 7 and a VL having a sequence as shown in SEQ ID NO: 8.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a VH having a sequence as shown in SEQ ID NO: 9 and a VL having a sequence as shown in SEQ ID NO: 10.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a VH having a sequence as shown in SEQ ID NO: 11 and a VL having a sequence as shown in SEQ ID NO: 12.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a VH having a sequence as shown in SEQ ID NO: 13 and a VL having a sequence as shown in SEQ ID NO: 14.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a VH having a sequence as shown in SEQ ID NO: 15 and a VL having a sequence as shown in SEQ ID NO: 16.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a VH having a sequence as shown in SEQ ID NO: 17 and a VL having a sequence as shown in SEQ ID NO: 18.

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, wherein the variant has one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids or any combination thereof); and/or (b) A human immunoglobulin light chain constant region (CL) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; for example, substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids or any combination thereof).

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.

In certain embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

In certain embodiments, the heavy chain constant region has a sequence as shown in SEQ ID NO: 121.

In certain embodiments, the light chain constant region has a sequence as shown in SEQ ID NO: 122.

In certain embodiments, the antibody or antigen-binding fragment thereof as described above, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab') ² , Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb).

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

In certain embodiments, the antigen binding fragment is Fab.

In certain embodiments, the antigen-binding fragment further comprises an Fc fragment (e.g., an Fc fragment of human IgG1) or a mutant thereof.

In certain embodiments, the Fc fragment has a LALA mutation and a knob mutation; alternatively, the Fc fragment has a LALA mutation and a hole mutation.

In certain embodiments, the Fc fragment has a sequence as shown in SEQ ID NO: 111 or 112.

In another aspect, the present application provides an isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described above.

On the other hand, the present application provides a vector comprising a nucleic acid molecule as described above. In certain embodiments, the vector is a clone vector or an expression vector.

On the other hand, the present application provides a host cell comprising a nucleic acid molecule as described above or a vector as described above. In certain embodiments, the host cell is a mammalian cell.

In another aspect, the present application provides a method for preparing the antibody or antigen-binding fragment thereof as described above, which comprises culturing the host cell as described above under conditions that allow the expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

In another aspect, the present application provides a multispecific molecule comprising the antibody or antigen-binding fragment thereof as described above.

In certain embodiments, the multispecific molecule specifically binds c-Met and additionally specifically binds one or more other targets.

In certain embodiments, the multispecific molecule is a bispecific molecule.

In certain embodiments, the bispecific molecule further comprises a molecule having a second binding specificity for a second target (e.g., a second antibody).

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

In certain embodiments, the therapeutic agent is selected from cytotoxic agents.

In certain embodiments, the therapeutic agent is selected from an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an anti-metabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, and any combination thereof.

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

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

In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an anti-metabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

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

In another aspect, the present application provides a kit containing the antibody or antigen-binding fragment thereof as described above.

In certain embodiments, the antibody or antigen-binding fragment thereof is detectably labeled, for example, with an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

In certain embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof as described above.

In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

In another aspect, the present application provides a chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof as described above.

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 as described above.

In certain embodiments, the antigen binding domain is a scFv.

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

On the other hand, the present application provides a method for inhibiting the growth of tumor cells expressing c-Met and/or killing the tumor cells, which comprises 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.

On the other hand, the present application provides a 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 drug composition as described above, or a chimeric antigen receptor as described above, in the preparation of a medicament for preventing and/or treating tumors in a subject (e.g., a human).

In certain embodiments, the medicament further comprises an additional pharmaceutically active agent.

In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an anti-metabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

In certain embodiments, the tumor expresses c-Met.

In certain embodiments, the tumor involves a tumor cell that expresses c-Met. In certain embodiments, the c-Met is expressed on the surface of the tumor cell.

In certain embodiments, the tumor is selected from 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 ... fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary diaphragmatic large B-cell lymphoma, T-cell/tissue cell-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal tumors, brain stem neuroglioma.

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

Use of the antibody or antigen-binding fragment thereof as described above in preparing a kit for detecting whether a tumor can be treated by an anti-tumor therapy targeting c-Met. (1) contacting a sample containing the tumor cells with the antibody or antigen-binding fragment thereof as described above; (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and c-Met.

In certain embodiments, the antibody or antigen-binding fragment thereof is detectably labeled.

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

In certain embodiments, the tumor is selected from 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 ... fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary diaphragmatic large B-cell lymphoma, T-cell/tissue cell-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal tumors, brain stem neuroglioma.

On the other hand, the present application provides a method for preventing and/or treating tumors in a subject, the method comprising administering to a subject in need thereof an effective amount of an antibody or an 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.

In certain embodiments, the tumor expresses c-Met.

In certain embodiments, the tumor involves tumor cells expressing c-Met. In certain embodiments, the c-Met is expressed on the surface of the tumor cells.

In certain embodiments, the tumor is selected from 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, glioma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary diaphragmatic large B-cell lymphoma, T-cell/tissue cell-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal tumors, brain stem neuroglioma.

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

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

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 therapy.

On the other hand, the present application provides a method for detecting whether a tumor can be treated by an anti-tumor therapy targeting c-Met, comprising the following steps: (1) contacting a sample containing the tumor cells with the antibody or its antigen-binding fragment as described above; (2) detecting the formation of a complex between the antibody or its antigen-binding fragment and c-Met.

In certain embodiments, the antibody or antigen-binding fragment thereof is detectably labeled.

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

In certain embodiments, the tumor is selected from 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, glioma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary diaphragmatic large B-cell lymphoma, T-cell/tissue cell-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal tumors, brain stem neuroglioma.

On the other hand, the present application provides a method for detecting the presence or amount of c-Met in a sample, comprising the following steps: (1) contacting the sample with the antibody or antigen-binding fragment thereof as described above; (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and c-Met or detecting the amount of the complex.

In certain embodiments, the antibody or antigen-binding fragment thereof is detectably labeled.

In certain embodiments, the c-Met is mammalian (e.g., human, monkey) c-Met. Term Definitions

In the present invention, unless otherwise specified, the 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 steps used herein are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally 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, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists 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 domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability, called complementation determining regions (CDRs), with more conserved regions called framework regions (FRs) interspersed between them. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form antigen binding sites, respectively. The distribution of amino acids in each region or domain can follow the definition 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.

As used herein, the term "complementary determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the heavy and light chain variable regions, named CDR1, CDR2, and CDR3. The precise 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., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person 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 skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

In the present invention, the CDR contained in the antibody of the present invention or its antigen-binding fragment can be determined according to various numbering systems known in the art. In certain embodiments, the CDR contained in the antibody of the present invention or its antigen-binding fragment is preferably determined by the Kabat, Chothia or IMGT numbering system.

As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable regions of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

As used herein, the terms "monoclonal antibody", "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably, referring to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which generally contain at least two or more different antibodies, and these different antibodies generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.

The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al. Nature 352: 624-628, 1991, or Marks et al. J. Mol. Biol. 222: 581-597, 1991).

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 specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementary determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are summarized in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that is composed 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 the direction from N-terminus to C-terminus; and, when the full-length antibody is an IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. Preferably, "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N-terminus to C-terminus. "Full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. Two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between HR of two full-length heavy chains. The full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention comprises two antigen binding sites formed by VH and VL pairs, respectively, and these two antigen binding sites specifically recognize/bind to the same antigen.

As used herein, the term "Fd" means an antibody fragment consisting of a VH and CH1 domain; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CH1 domain; the term "F(ab') ² fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region; the term "Fab'fragment" means a fragment obtained by reducing the disulfide bonds linking two heavy chain fragments in the F(ab') ² fragment, consisting of an intact light chain and a heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains 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.

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

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein 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, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can 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. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the present invention, scFv can form di-scFv, which refers to two or more single scFvs connected in series to form an antibody. In certain embodiments of the present invention, scFv can form (scFv)2, which refers to two or more single scFvs connected in parallel to form an antibody.

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

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

Antibody antigen-binding fragments (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain and/or the heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and/or the heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (U.S.P 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 antibodies in which the heavy chain and light chain variable regions of the antibody are derived from a first antibody and the heavy chain and light chain constant regions of the antibody are derived from a second antibody.

As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (for example, a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions/total number of positions × 100%). In certain embodiments, the two sequences are of the same length.

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

As used herein, the term "variant", in the context of a polypeptide (including a polypeptide), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by the introduction of amino acid residue substitutions, deletions or additions. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limiting, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection/blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, 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 of chlamydial, etc. Furthermore, the variant has similar, identical or improved function as the polypeptide or peptide from which it is derived.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be represented 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.

The specific binding properties between two molecules can be measured using methods known in the art. One method involves measuring the speed of antigen binding site/antigen complex formation and dissociation. Both "binding rate constant" (ka or kon) and "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values can be measured by any effective method. In some embodiments, surface plasmon resonance (SPR) can be used to measure the dissociation constant in Biacore. In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

As used herein, the 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, but are not limited to, 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 dye derivatives (e.g., Cy7, Alexa Fluor, 5-Chloro-1-Y-D-Y-S, 5-Chloro-1-Y-S ... 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., Dynabeads®), calorimetric 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.

As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express 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 genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomas (such as SV40). A vector 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, the vector may also contain a replication initiation site.

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 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising an 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 substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions carried out by residues similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.) physically or functionally. Families of amino acid residues with 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, glutamine), 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well 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).

The writing of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. 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 can be used interchangeably. And in the present invention, amino acids are usually represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

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

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

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) suffers from a disease associated with c-Met, or is at risk of suffering from the above disease.

As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease refers to an amount sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

As used herein, the term "single-armed antibody" refers to an antigen-binding fragment comprising a Fab segment and an Fc segment, wherein 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 segment and the Fc segment may be connected by a linker or not. The single-armed antibody of the present invention may be prepared or synthesized by a variety of methods, for example, constructing a sequence encoding the Fab heavy chain and a sequence encoding the Fc into the same vector, and constructing a sequence encoding the Fab light chain into another vector, and transforming the two vectors into host cells separately to obtain the single-armed antibody.

As used herein, the term "bispecific antibody" refers to a conjugate formed by a first antibody (or a fragment thereof) and a second antibody (or a fragment thereof) or an antibody analog via a coupling arm, and the coupling method includes but is not limited to chemical reaction, gene fusion and enzymatic reaction. Bispecific antibodies can be linked or produced by various methods, for example, see the method of Songsivilai et al. (Clin. Exp. Immunol., 79: 315-321 (1990)), and the method of Kostelny et al. (J. Immunol., 148: 1547-1553 (1992)). Beneficial effects of the invention

Compared with the prior art, the antibody prepared in the present application can specifically bind to the antibody or antigen-binding fragment of c-Met, and then specifically recognize or bind to cells expressing c-Met. The antibody has a high affinity with cells expressing or over-expressing c-Met, and also 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 application potential in targeted therapy of tumors.

The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but those skilled in the art will understand that the following drawings and embodiments are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art.

Sequence information

The information of some sequences involved in the present invention is provided in Table 1 below. Table 1: Description of sequences SEQ ID NO: describe sequence 1 22-VH QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYGISWVRQAPGQGLEWMGWISAYNGHTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDRRTGTSFFDYWGQGTLVTVSS 2 22-VL DIVMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSKFSGSGSGTEFTLTISSLQPEDFATYFCQQAYGFPLTFGGGTKVEIK 3 23-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMHSLRAEDTAVYYCAKVITFERGRTFDIWGQGTMVTVSS 4 23-VL DIVMTQSPSSSLSASVGDRVTITCRASQSFSDYLNWYRQKPGKAPKLLLFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSPPYTFGQGTKLEIK 5 74-VH QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQSPGKGLEWIGSIYYSGNTYYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCVRQVYDFWRDWGQGTLVTVSS 6 74-VL DIVMTQSPSSVSASVGDRVTITCRASQGINSWLAWYQQKPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFSLTISSLHPEDFATYYCQQAKGFPLTFGGGTKVEIK 7 111-VH QVQLVQSGAEVKKPGASMKVSCKASGNTFISYGINWVRQAPGQGLEWMGWISAYNGNTNYAQKFQGRVTMTTDTSTSSAYMELRNLRSDDTAVYYCATGDTTSSGYYNYYMDVWGKGTTVTVSS 8 111-VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGQGTKVDIK 9 136-VH QVQLQESGPGLVKPSETLSLTCTVSGGSVTSVNYYWKWIRQPPGKGLEWIGYISYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVRAPYYYMDVWSKGTTVTVSS 10 136-VL DIVMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAVSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPLTFGGGTKVEIK 11 187-VH QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQSPGKGLEWIGSRYYSGNTYYNPSLKSRVTMSVDTSKNQFSLKLRSVTAADTAVYYCARQVYDYWRDWGQGALVTVSS 12 187-VL DIVMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPNLLIYAASNLPSGVPSRFSGSGSGTVFTLTISSLQPEDFATYYCQQSNSFPLTFGGGTKVEIK 13 216-VH EVQLVESGGGLVQPGGSLRLFCGASGFTFSSYAMSWVRQAPGKGLEWVSAITGSGGSTYYADSVKGRFTISRDNSKNTLFLQLNSLRAEDTAIYYCAKIVTVEAGRWLDPWGQGTMVTVSS 14 216-VL DIRMTQSPSSSLSASVGDRVTITCRASQSISNYLNWFQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPYTFGQGTKVEIK 15 221-VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGLEWVSVISGSGGSTYYEDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVITFERGRTFDIWGQGTMVTVSS 16 221-VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGPSSRATGIPDRFSGSGSGTDFTLTISSLQPADFASYYCQQSYITPYTFGQGTKVEIK 17 223-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSAISGSGVGTYYADSVKGRFTISRDNSKNTLFLQMNSLRVEDTAVYYCVRVMTVEFSRWLDPWGQGTLVTVSS 18 223-VL DIQLTQSPSSSLSASVGDRVTITCRASQSIITYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLNISSLQPEDFATYYCQQANSLPYTFGQGTKVEIK 19 22-VH CDR1 GYTFTNYG 20 22-VL CDR1 QGISSW twenty one 23-VH CDR1 GFTFSSYA twenty two 23-VL CDR1 QS twenty three 74-VH CDR1 GGSISSSSYY twenty four 74-VL CDR1 QGINSW 25 111-VH CDR1 GNTFISYG 26 111-VL CDR1 QSVSSSY 27 136-VH CDR1 GGSVTSVNYY 28 136-VL CDR1 QGISSW 29 187-VH CDR1 GGSISSSSYY 30 187-VL CDR1 QGISSW 31 216-VH CDR1 GFTFSSYA 32 216-VL CDR1 QSISNY 33 221-VH CDR1 GFTFSSYA 34 221-VL CDR1 QSVSSSY 35 223-VH CDR1 GFTFRNYA 36 223-VL CDR1 QSIITY 37 22-VH CDR2 ISAYNGHT 38 22-VL CDR2 AAS 39 23-VH CDR2 ISGSGGTT 40 23-VL CDR2 AAS 41 74-VH CDR2 YYYSGNT 42 74-VL CDR2 AAS 43 111-VH CDR2 ISAYNGNT 44 111-VL CDR2 GAF 45 136-VH CDR2 ISYSGNT 46 136-VL CDR2 AVS 47 187-VH CDR2 RYYSGNT 48 187-VL CDR2 AAS 49 216-VH CDR2 ITGSGGST 50 216-VL CDR2 ATS 51 221-VH CDR2 ISGSGGST 52 221-VL CDR2 GPS 53 223-VH CDR2 ISGSGVGT 54 223-VL CDR2 AAS 55 22-VH CDR3 ARDRRTGTSFFDY 56 22-VL CDR3 QQAYGFPLT 57 23-VH CDR3 AKVITFERGRTFDI 58 23-VL CDR3 QQSYSPPYT 59 74-VH CDR3 VRQVYDFW 60 74-VL CDR3 QQAKGFPLT 61 111-VH CDR3 ATGDTTSSGYYNYYMDV 62 111-VL CDR3 QQYGSSPRT 63 136-VH CDR3 VRAPYYYMDV 64 136-VL CDR3 QQANSFPLT 65 187-VH CDR3 ARQVYDYWRD 66 187-VL CDR3 QQSNSFPLT 67 216-VH CDR3 AKIVTVEAGRWLDP 68 216-VL CDR3 QQSYSIPYT 69 221-VH CDR3 AKVITFERGRTFDI 70 221-VL CDR3 QQSYITPYT 71 223-VH CDR3 VRVMTVEFSRWLDP 72 223-VL CDR3 QQANSLPYT 73 Rechain Upstream Primer 1 ACAGGTGCCCACTCCCAGGTGCAG 74 Rechain Upstream Primer 2 AAGGTGTCCAGTGTGARGTGCAG 75 Rechain Upstream Primer 3 CCCAGATGGGTCCTGTCCCAGGTGCAG 76 Rechain Upstream Primer 4 CAAGGAGTCTGTTCCGAGGTGCAG 77 Reconstitute downstream primer 1 TGAGCAGCAGAACCTGGAGCCAAGGGATAGACAGATGGGGCTGTTGT 78 Rechain downstream primer 2 TGAGCAGCAGAACCTGGAGCCAGTGGATAGACAGATGGGGCTGTTGT 79 Light chain upstream primer 1 ATGAGGSTCCCYGCTCAGCTGCTGG 80 Light chain upstream primer 2 CTCTTCCTCCTGCTACTCTGGCTCCCAG 81 Light chain upstream primer 3 ATTTCTCTGTTGCTCTGGATCTCTG 82 Light chain downstream primer 1 TGTTCAGAAGATGGTGGGAAGATAGATACAGTTGGTGCAGCATCAGC 83 Rechain Upstream Primer 5 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGACAGGTGCAGCTGGTGCAG 84 Rechain Upstream Primer 6 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGAGAGGTGCAGCTGGTGCAG 85 Reconstitute upstream primer 7 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGAGAGGTGCAGCTGGTGGAG 86 Rechain upstream primer 8 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGAGAGGTGCAGCTGTTGGAG 87 Rechain upstream primer 9 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGACAGGTGCAGCTGCAGGAG 88 Reconstitute upstream primer 10 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGACAGGTGCAGCTACAGCAGTG 89 Rechain upstream primer 11 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGACAGGTTCAGCTGGTGCAG 90 Reconstitute upstream primer 12 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGACAGGTCCAGCTGGTACAG 91 Reconstitute upstream primer 13 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGACAGGTGCAGCTGGTGGAG 92 Reconstitute upstream primer 14 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGAGAAGTGCAGCTGGTGGAG 93 Reconstitute upstream primer 15 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGACAGCTGCAGCTGCAGGAG 94 Reconstitute upstream primer 16 GAGAAGCTGTTGCTGAAGCTTCTTTGGACAAGAGACAGGTACAGCTGCAGCAG 95 Reconstitute downstream primer 3 GCCAGGGGGAAGACCGATGGGCCCTTGGTGCTAGCTGAGGAGACGGTGACCAG 96 Rechain downstream primer 4 GCCAGGGGGAAGACCGATGGGCCCTTGGTGCTAGCTGAAGAGACGGTGACCATTG 97 Re-chain downstream primer 5 GCCAGGGGGAAGACCGATGGGCCCTTGGTGCTAGCTGAGGAGACGGTGACCGTG 98 Light chain upstream primer 4 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGACATCCAGATGACCCAGTC 99 Light chain upstream primer 5 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGACATCCAGTTGACCCAGTCT 100 Light chain upstream primer 6 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGATATCCGGATGACCCAGTC 101 Light chain upstream primer 7 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGATATTGTGATGACCCAGAC 102 Light chain upstream primer 8 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGATATTGTGATGACTCAGTC 103 Light chain upstream primer 9 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGATGTTGTGATGACTCAGTC 104 Light chain upstream primer 10 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGAAATTGTGTTGACACAGTC 105 Light chain upstream primer 11 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGAAATAGTGATGACGCAGTC 106 Light chain upstream primer 12 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGAAATTGTGTTGACGCAGTCT 107 Light chain upstream primer 13 CCTTCTTTTTCTCTTTAGCTCGGCTTATTCCGACATCGTGATGACCCAGTC 108 Light chain downstream primer 2 GGAAGATGAAGACAGATGGTGCAGCCACCGTACGTTTGATYTCCACCTTGGTC 109 Light chain downstream primer 3 GGAAGATGAAGACAGATGGTGCAGCCACCGTACGTTTGATCTCCAGCTTGGTC 110 Light chain downstream primer 4 GGAAGATGAAGACAGATGGTGCAGCCACCGTACGTTTGATATCCACTTTGGTC 111 Human IgG1-Fc (LALA mutation, knob mutation) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 112 Fc-LALA-hole DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 113 Knob rechain of the single-arm antibody Amivantamab QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISWVRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDLRGTNYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 114 The light chain of the one-armed antibody Amivantamab DIQMTQSPSSSVSASVGDRVTITCRASQGISNWLAWFQHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 115 Hole rechain of the single-armed antibody Amivantamab and the antibody onartuzumab DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 116 Single-arm antibody LY2875358Konb rechain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGRVNPNRRGTTYNQKFEGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARANWLDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 117 Light chain of the one-armed antibody LY2875358 DIQMTQSPSSSLSASVGDRVTITCSVSSSVSSIYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQVYSGYPLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 118 Single-arm antibody LY2875358Hole rechain DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 119 Knob rechain of the antibody onartuzumab EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 120 The light chain of the antibody onartuzumab DIQMTQSPSSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 121 CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 122 CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The present invention will now be described with reference to the following embodiments which are intended to illustrate the present invention (but not to limit the present invention).

Unless otherwise specified, the experiments and methods described in the examples were performed essentially 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 ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al., ed., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL METHOD. APPROACH) (M.J. MacPherson, B.D. Hames, and G.R. Taylor, eds. (1995)), and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

In addition, if no specific conditions are specified in the embodiments, the experiments are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are conventional products that can be purchased commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed by the present invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

Example 1. Anti-c-Met antibody screening 1.1. Construction of antibody library Animal immunization

1 mg of cellular mesenchymal epithelial transition factor (c-Met) antigen (purchased from AcroBiosystems) was mixed with an equal volume of Freund's adjuvant and immunized 5 fully humanized mice (purchased from Alloy) once a week for a total of 4 times to stimulate B cells to express antigen-specific antibodies. After 4 immunizations, the spleen of the mice was taken and total RNA was extracted using RNA extraction reagent Trizol (purchased from Invitrogen). The total cDNA of fully humanized mice was reverse transcribed using a cDNA synthesis kit (purchased from Invitrogen). Antibody gene amplification

First round of PCR: Using heavy chain upstream primers 1-4 (SEQ ID NO: 73 to SEQ ID NO: 76) and heavy chain downstream primers 1-2 (SEQ ID NO: 77 and SEQ ID NO: 78) as shown in Table 1, and light chain upstream primers 1-3 (SEQ ID NO: 79 to SEQ ID NO: 81) and light chain downstream primer 1 (SEQ ID NO: 82), the fully humanized antibody heavy and light chain variable region sequences were amplified from cDNA.

The above PCR products were subjected to agarose gel electrophoresis to determine the band size and uniqueness. The target fragment was at 400 bp~500 bp.

Second round of PCR: Using heavy chain upstream primers 5-16 (SEQ ID NO: 83 to SEQ ID NO: 94) and heavy chain downstream primers 3-5 (SEQ ID NO: 95 to SEQ ID NO: 97), as well as light chain upstream primers 4-13 (SEQ ID NO: 98 to SEQ ID NO: 107) and light chain downstream primers 2-4 (SEQ ID NO: 108 to SEQ ID NO: 110) as shown in Table 1, a second round of sequence amplification was performed using the first round of PCR products as a template to add homology arms to the heavy and light chain genes.

Use PCR purification kit (purchased from QIAGEN) to recover the target fragment. Library construction

The linearized yeast display vector and the second round of PCR products were mixed and electroporated into brewing yeast (purchased from ATCC) to construct a fully humanized anti-c-Met antibody library from five animals. The library capacity was determined to be 3×10 ⁷ . 1.2. Screening of c-Met antibodies Biotinylation of c-Met protein

Take an appropriate volume of double distilled water to dissolve human c-Met protein (purchased from AcroBiosystems). According to the instructions of the biotin labeling kit (purchased from Thermo), dissolve the biotin and mix it with the protein solution, and incubate at 4°C for 2 hours. Use a desalting column (purchased from Thermo) to remove excess biotin. The desalting column pretreatment and sample collection operations are all carried out according to the steps in the product instructions. Magnetic bead sorting (MACS) amplifies yeast that can specifically bind to human c-Met

The antibody library constructed in Example 1.1 was inoculated into SD-CAA expansion medium (1L SD-CAA expansion medium contains 6.7g YNB, 5g tyrosine, 13.62g Na ₂ HPO ₄ ·12H ₂ O, 7.44g NaH ₂ PO ₄ and 2% glucose) and cultured at 30°C, 225rpm overnight. An appropriate amount of yeast cells was taken, centrifuged at 3000rpm×5min (the following centrifugation operations are the same), the medium was removed, and the yeast cells were resuspended in SD-CAA induction medium and induced overnight. The concentration of the library after induction was measured, and an appropriate amount of yeast cells was taken, centrifuged and the medium was removed. Resuspend the yeast cells in 50 ml PBS and remove the supernatant by centrifugation. Resuspend the yeast cells in 10 ml PBS.

Add biotin-labeled human c-Met protein (final concentration 100nM), incubate at room temperature for 30min, collect yeast cells by centrifugation, and wash the yeast 3 times with 50ml PBS. Resuspend the yeast cells with 5ml washing solution, add 200μl SA magnetic beads (purchased from Miltenyi Biotec), and incubate upside down for 10min. Wash the yeast and magnetic bead mixture 3 times with PBS, and add the mixture to the LS purification column (purchased from Miltenyi Biotec). Place the LS purification column on the magnetic stand and wash with PBS to remove non-specifically bound yeast cells. Remove the purification column from the magnetic stand and add PBS to elute the yeast. After centrifugation, the eluted yeast is transferred to SD-CAA expansion medium for expansion. Flow cytometry (FACS) to obtain high affinity yeast cells

Inoculate the yeast cells amplified by MACS into SD-CAA expansion medium. Incubate overnight at 30°C, 225 rpm. Resuspend the yeast cells in SD-CAA induction medium and induce overnight. Add anti-c-Myc mouse antibody (purchased from Thermo) and 100 nM biotinylated c-Met antigen and incubate for 10 min. Wash the yeast three times with PBS, add goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (purchased from Invitrogen) and streptavidin APC conjugate fluorescent antibody (purchased from Invitrogen) and incubate for 15 min. Add PBS to resuspend the cells, and use the BD AriaIII instrument to sort out yeast with high binding ability to c-Met antigen. Retrieval of c-Met antibody candidate molecule antibody gene

The yeast suspension with high binding ability to human c-Met antigen obtained by MACS and FACS amplification was spread on SD-CAA solid culture plates, and then the monoclonal strains were picked and cultured in SD-CAA expansion medium at 30°C and 225rpm overnight. The expanded monoclonal strains were treated with 0.1% SDS, centrifuged and the supernatant was used as a template for PCR amplification. The PCR products were sent for sequencing to obtain gene sequences. A total of 9 antibodies were obtained, which were named antibodies 22, 23, 74, 111, 136, 187, 216, 221 and 223, respectively. The specific sequences are shown in Table 1, where the CDR sequences of the antibodies are all based on the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). Example 2. Construction and expression purification of one-armed antibodies 2.1. Constructing antibody genes into pCDNA3.1 expression vector

The nucleotide sequence encoding the heavy chain variable region (as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17) was linked to the nucleotide sequence encoding the heavy chain constant region 1 (as shown in SEQ ID NO: 121) and the nucleotide sequence encoding the human IgG1-Fc (LALA mutation, knob mutation) segment (SEQ ID NO: 111), and constructed into the EcoR I/ Not I double enzyme linearized pCDNA3.1 vector using homologous recombinase (purchased from Vazyme); the nucleotide sequence encoding the light chain variable region (as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18) and the nucleotide sequence encoding the light chain constant region (as shown in SEQ ID NO: 122) were constructed into the EcoR I/ Xhol I double enzyme linearization pCDNA3.1 vector; the nucleotide sequence encoding Fc-LALA-hole (SEQ ID NO: 112) was constructed into EcoR I/ Xhol I double enzyme linearization pCDNA3.1 vector. The process is in accordance with the product manual. The homologous recombination product was transformed into Top10 competent cells, coated with benzyl resistant plates, cultured at 37℃ overnight, single colonies were picked for sequencing, and plasmids were extracted. 2.2. Cell transfection and protein purification

The ExpiCHO™ Expression System Kit (purchased from Thermo) was used to co-transfect the heavy chain (Fc-LALA-knob), light chain and Fc-LALA-hole (SEQ ID NO: 112) extracted above into Expi-CHO cells to form a single Fab antibody structure (Figure 1). The transfection method was in accordance with the product instructions. After 5 days of cell culture, the supernatant was collected and the target protein was purified using protein A magnetic beads (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 magnetic beads) and added to the sample to be purified. Incubate at room temperature for 1 hour with gentle shaking during the period. 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. Elution buffer (0.1M sodium citrate, pH 3.2) was added according to 3-5 times the volume of the magnetic beads, and the beads were shaken at room temperature for 5-10 minutes, and then placed back on the magnetic stand. The elution buffer was collected and transferred to a collection tube with neutralization buffer (1M Tris, pH 8.54) added to mix and obtain the target protein. Example 3. Determination of c-Met antibody affinity

ForteBio affinity determination was performed according to existing methods (Estep, P et al., Antibody-antigen affinity and epitope binding determination based on high-throughput methods. MAbs, 2013.5(2):p.270-8). Briefly, the sensor was equilibrated offline in the analytical buffer for 30 minutes, then detected 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 100nM human c-Met antigen for 5 minutes, and then the sensor was transferred to PBS for dissociation for 5 minutes. Kinetic analysis was performed using a 1:1 binding model. Table 2. Affinity of candidate molecules to human c-Met No. KD(M) Kon(1/Ms) Koff(1/s) twenty two 4.29E-09 1.28E+05 5.48E-04 twenty three 5.96E-10 1.60E+05 9.50E-05 74 6.01E-09 1.79E+05 1.08E-03 111 2.34E-09 1.60E+05 3.74E-04 136 2.93E-11 2.29E+05 6.71E-06 187 1.68E-09 2.20E+05 3.69E-04 216 6.54E-10 2.19E+05 1.43E-04 221 2.01E-09 1.84E+05 3.70E-04 223 9.76E-09 1.67E+05 1.63E-03

The candidate molecules were also tested for cross-reactivity with proteins from different species, including proteins from cynomolgus monkeys (purchased from AcroBiosystems). Table 3. Affinity of candidate molecules for monkey c-Met No. KD(M) Kon(1/Ms) Koff(1/s) twenty two 1.90E-08 1.36E+05 2.59E-03 twenty three 2.65E-08 5.69E+04 1.51E-03 74 9.70E-09 2.69E+05 2.60E-03 111 1.15E-08 1.02E+05 1.17E-03 136 1.16E-09 3.69E+05 4.26E-04 187 2.92E-09 3.24E+05 9.47E-04 216 3.84E-09 1.28E+05 4.93E-04 221 6.38E-08 3.01E+04 1.92E-03 223 1.62E-08 6.73E+04 1.09E-03

The experimental results are shown in Tables 2 and 3. The 9 antibodies of the present application all have a certain affinity with the c-Met protein of mammals (eg, humans, monkeys).

The protein construction and expression purification method was the same as in Example 2.2, and the purity of the protein was determined by HPLC. The HPLC method was as follows: mobile phase: 150 mM Na ₂ HPO ₄ •12H ₂ O, 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 chromatographic column (SEPAX 4.6×300mm, 3μm). Table 4. Purity test results of Anti-c-Met antibody No. Monomer ratio (%) twenty two 97.20% twenty three 98.50% 74 98.00% 111 97.20% 136 94.80% 187 97.70% 216 97.30% 221 94.00% 223 96.80%

The experimental results are shown in Table 4. The purity of the 9 antibodies in this application is relatively high, all at 94% and above. Example 4. Thermal stability of anti-c-Met samples

DSC (Differential scanning calorimetry) was used to detect the thermal stability of different antibodies. The sample was concentrated and diluted to 1 mg/ml with PBS; 5000× fluorescent colorimetric agent Cypro Orange (purchased from Bio-Rad) was diluted 50 times with ultrapure water to obtain 100× fluorescent colorimetric agent Sypro Orange. Take 50μl of 1mg/ml sample, add 10μl 100× fluorescent colorimetric agent Sypro Orange and 40μl ultrapure water, mix well, take 30μl and add it to a 96-well PCR plate, make 3 replicates for each sample, put it into the PCR instrument, and set the heating program as follows: keep constant at 25℃ for 5min, and heat to 99℃ at a rate of 0.5℃/min. After the program is finished, read the temperature value of the lowest point of the curve in the "Melt Curve" graph, which is the Tm value of the sample. The specific results are shown in Table 8 below: Table 5. Tm value of anti-c-Met antibody No. Tm（℃） twenty two 67.34 twenty three 67.95 74 67.42 111 64.26 136 67.86 187 67.42 216 67.42 221 67.42 223 67.42

The experimental results are shown in Table 5. All nine antibodies in this application have a certain degree of thermal stability. Example 5. Anti-c-Met purified samples and cell binding of human/Gangetic monkey c-Met

In order to verify the binding ability of the antibody of the present application to mammalian cells expressing c-Met, three existing antibodies, Amivantamab, LY2875358 and onartuzumab, were selected as control antibodies for comparison.

Among them, the construction procedures of Amivantamab and LY2875358 control antibodies are the same as those of the one-armed antibody of the present application, and are all carried out according to the steps of Example 2. Briefly, the control antibody Amivantamab is that the insert fragments shown in SEQ ID NO: 113, SEQ ID NO: 114 and SEQ ID NO: 115 are respectively integrated into the plasmid, and the plasmid is co-transformed into Expi-CHO cells; the control antibody LY2875358 is that the insert fragments shown in SEQ ID NO: 116, SEQ ID NO: 117 and SEQ ID NO: 118 are respectively integrated into the plasmid, and the plasmid is co-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 plasmids respectively, and the plasmids were co-transfected into Expi-CHO cells.

HEK293T cells expressing human c-Met (HEK293-Hu c-Met) and HEK293T cells expressing rhesus macaque c-Met (HEK293-Rhe c-Met) were generated by transfecting pCHO1.0 vector (purchased from Invitrogen) with human or rhesus macaque c-Met cDNA (purchased from Sino Biological) cloned into MCS. The cell density of the expanded HEK293-Hu c-Met/HEK293-Rhe c-Met cells was adjusted to 2×10 ⁶ cells/ml, 100 μl/well was added to a 96-well flow cytometry plate, and centrifuged for use. The purified c-Met antibody was diluted with PBS, starting from 100nM and diluted 3 times for a total of 10 points. 100μl/well of the diluted sample was added to the 96-well flow plate with cells, incubated at 4℃ for 30min, and washed twice with PBS. 100μl/well of Goat F(ab') ² Anti-Human IgG - Fc (PE) (purchased from Abcam) diluted 100 times with PBS was added, incubated at 4℃ for 30min, and washed twice with PBS. 100μl/well of PBS was added to resuspend the cells, and the detection was performed on the CytoFlex (Bechman) flow cytometer and the corresponding MFI was calculated.

In the assay experiment of the above method, the experimental results are shown in Figures 2A and 2B. All anti-c-Met one-armed antibodies of the present invention have binding activity to HEK293-Hu c-Met cells. In particular, antibody 136 showed strong binding similar to that of the control antibody Amivantamab, and was better than the control antibody LY2875358 and the control antibody onartuzumab. Figures 2C and 2D show that the binding of all anti-c-Met one-armed antibodies of the present invention to HEK293-Rhe c-Met cells is similar to that to human c-Met. In particular, antibody 136 showed strong binding similar to that of Amivantamab, which was better than other candidate antibodies and control antibodies. As shown in Tables 6 and 7, all anti-c-Met screening single-arm antibodies showed similar binding abilities (EC50) to cells expressing human c-Met and macaque c-Met. Table 6. Binding assay of anti-c-Met single-arm antibodies and HEK293-Hu c-Met Anti-clonal strain EC50 (nM) EC50 (nM) twenty two 0.947 / twenty three 0.8211 / 74 1.901 / 85 2.092 / 111 25.72 / 136 0.4515 / 187 / 0.7322 216 / 0.744 221 / 1.731 222 / 0.78 223 / 0.7649 225 / 2.352 LY2875358 single-arm antibody 1.245 1.391 Amivantamab single-arm antibody 0.3227 0.3957 Onartuzumab 1.032 0.5398 Table 7. Anti-c-Met one-armed antibody and HEK293-Rhe c-Met binding assay Anti-clonal strain EC50 (nM) EC50 (nM) twenty two 0.7258 / twenty three 0.7464 / 74 1.461 / 85 0.8092 / 111 26.17 / 136 0.3062 / 187 / 0.5922 216 / 0.6006 221 / 1.478 222 / 0.4646 223 / 1.064 225 / 7.536 LY2875358 One-armed antibody 0.8682 0.7487 Amivantamab One-armed Antibody 0.2414 0.2001 Onartuzumab 0.9699 1.241 Example 6. Screening with anti-c-Met single-armed antibodies to block HGF-dependent TKI resistance

HCC827 cells are human non-small cell lung cancer cells purchased from the Chinese Academy of Sciences Cell Bank. They highly express the epidermal growth factor receptor EGFR (exon 19 loss) and c-Met receptor. Treatment with the tyrosine kinase inhibitor TKI small molecule Gefitinib (gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor) induces apoptosis of HCC827 cells. If HGF is added at the same time under such conditions, the c-Met pathway will be activated, thereby inducing HCC827 to produce resistance to Gefitinib and inhibiting apoptosis.

The cell density of the expanded cultured HCC827 cells was adjusted to 2×10 ⁴ cells/ml, and 100 μl/well was added to a 96-well cell culture plate and cultured overnight for use. The antibody to be tested was diluted to 1000 nM, HGF was diluted to 800 ng/mL, and Gefitinib was diluted to 8 μM with 1640 medium. 50 μl/well of the diluted antibody, 25 μl/well of HGF, and 25 μl/well of Gefitinib were added to the 96-well plate with cells according to experimental needs. The total volume was supplemented with 1640 medium to 200 μl/well. After culturing at 37°C and 5% carbon dioxide for 3 days, 100 μl of the medium was removed, and then Cell titer glo (purchased from Promega) was added at 100 μl/well. The chemiluminescent signal was collected using an enzyme labeler.

The experimental results are shown in Figure 3. The anti-c-Met screening single-armed antibodies of the present application inhibited the HGF-c-Met signaling pathway (e.g., antibodies 22, 23, 74, 85, 111, 136 and 187), thereby restoring Gefitinib-induced HCC827 apoptosis. Among them, antibodies 136 and 187 both showed significant inhibitory activity, which was 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. Example 7. Anti-c-Met screening single-armed antibodies block HGF-induced cell proliferation

H596 cells are human lung cancer cells purchased from ATCC that express EGFR and c-Met receptors. HGF treatment induces H596 cell proliferation. If control antibodies are added under such conditions, the HGF-c-Met signaling pathway will be inhibited, thereby inhibiting HGF-induced proliferation of H596. The density of the expanded cultured H596 cells was adjusted to 3×10 ⁴ cells/ml, and 100 μl/well was added to a 96-well cell culture plate and cultured overnight for use. Dilute the antibody to be tested to 400 nM and HGF to 200 ng/mL with 1640 medium. Add 50 μl/well of the diluted antibody and 50 μl/well of HGF to the 96-well plate with cells as required. Add 1640 medium to a total volume of 200 μl/well and culture at 37°C with 5% carbon dioxide for 5 days. After 5 days, remove 100 μl of the medium and then add 100 μl/well of Cell titer glo (purchased from Promega) to collect the chemiluminescent signal using an enzyme labeler.

The results are shown in Figure 4. The anti-c-Met screening single-arm antibodies 136 and 187 both significantly inhibited HGF-induced H596 cell proliferation. Moreover, the inhibitory effect of antibody 136 was superior to that of all control antibodies.

Although the specific implementation of the present invention has been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the details based on all the teachings published, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached patent application scope and any equivalent thereof.

without

Figure 1 shows a schematic diagram of the structure of the antibody constructed in Example 2 of the present application. Figure 2 shows the binding activity of the anti-c-Met antibody of the present application and cells overexpressing c-Met; wherein, Figures 2A and 2B show the binding activity of the antibodies of the present application (antibodies 22, 23, 74, 111, 136, 187, 216, 221, 223) and control antibodies to HEK293-Hu c-Met cells; Figures 2C and 2D show the binding activity of the antibodies of the present application (antibodies 22, 23, 74, 111, 136, 187, 216, 221, 223) and control antibodies to HEK293-Rhec-Met cells. Figure 3 shows the inhibition results of the anti-c-Met antibodies (antibodies 22, 23, 74, 111, 136, 187, 216, 221, 223) and control antibodies of this application on HCC827 cells. Figure 4 shows the inhibition results of the anti-c-Met antibodies (antibodies 136, 187) and control antibodies of this application on H596 cells

TW202442686A_112115165_SEQL.xml

Claims (17)

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) comprising the following three complementary determining regions (CDRs): (i) VH CDR1, which is composed of a sequence shown in any one of SEQ ID NO:19, 21, 23, 25, 27, 29, 31, 33 or 35, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, (ii) VH CDR2, which is composed of a sequence shown in any one of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51 or 53, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and (iii) VH CDR3, which is composed of a sequence shown in any one of SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69 or 71, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; and/or, (b) a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): (iv) VL CDR1, which is composed of the following sequence: SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34 or 36, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52 or 54, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 70 or 72, or a sequence having a substitution, deletion or addition of one or more amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the CDR described in any one of (i)-(vi) is defined according to the Kabat, IMGT or Chothia numbering system; Preferably, the CDR described in any one of (i)-(vi) is defined according to the IMGT numbering system. The antibody or antigen-binding fragment thereof as described in claim 1, comprising: (1) the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 19, VH CDR2 as shown in SEQ ID NO: 37, VH CDR3 as shown in SEQ ID NO: 55; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 20, VL CDR2 as shown in SEQ ID NO: 38, VL CDR3 as shown in SEQ ID NO: 56; (2) the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 21, VH CDR2 as shown in SEQ ID NO: 39, VH CDR3 as shown in SEQ ID NO: 57; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 22, VL CDR2 as shown in SEQ ID NO: 40, VL CDR3 as shown in SEQ ID NO: ID NO: 58; (3) the following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 23, VH CDR2 as shown in SEQ ID NO: 41, VH CDR3 as shown in SEQ ID NO: 59; and/or, the following 3 light chain CDRs: VL CDR1 as shown in SEQ ID NO: 24, VL CDR2 as shown in SEQ ID NO: 42, VL CDR3 as shown in SEQ ID NO: 60; (4) the following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 25, VH CDR2 as shown in SEQ ID NO: 43, VH CDR3 as shown in SEQ ID NO: 61; and/or, the following 3 light chain CDRs: VL CDR1 as shown in SEQ ID NO: 26, VL CDR2 as shown in SEQ ID NO: 44, VL CDR3 as shown in SEQ ID NO: 62; (5) the following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 27, VH CDR2 as shown in SEQ ID NO: 45, VH CDR3 as shown in SEQ ID NO: 63; and/or, the following 3 light chain CDRs: VL CDR1 as shown in SEQ ID NO: 28, VL CDR2 as shown in SEQ ID NO: 46, VL CDR3 as shown in SEQ ID NO: 64; (6) the following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 29, VH CDR2 as shown in SEQ ID NO: 47, VH CDR3 as shown in SEQ ID NO: 65; and/or, the following 3 light chain CDRs: VL CDR1 as shown in SEQ ID NO: 30, VL CDR2 as shown in SEQ ID NO: 48, VL CDR3 as shown in SEQ ID NO: 66; (7) the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 31, VH CDR2 as shown in SEQ ID NO: 49, VH CDR3 as shown in SEQ ID NO: 67; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 32, VL CDR2 as shown in SEQ ID NO: 50, VL CDR3 as shown in SEQ ID NO: 68; (8) the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 33, VH CDR2 as shown in SEQ ID NO: 51, VH CDR3 as shown in SEQ ID NO: 69; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 34, VL CDR2 as shown in SEQ ID NO: 52, VL CDR3 as shown in SEQ ID NO: 70; or (9) the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 35, VH CDR2 as shown in SEQ ID NO: 53, VH CDR3 as shown in SEQ ID NO: 71; and/or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 36, VL CDR2 as shown in SEQ ID NO: 54, VL CDR3 as shown in SEQ ID NO: 72; Preferably, the antibody or antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin. An antibody or antigen-binding fragment thereof as described in claim 1 or claim 2, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the following: (i) a sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to a sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17; or (iii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to a sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17 has a sequence with 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; and/or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the following: (iv) a sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18; (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to a sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18; or (vi) a sequence with ... NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18 has a sequence identity of 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%; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: (1) a VH having a sequence as shown in SEQ ID NO: 1 and a VL having a sequence as shown in SEQ ID NO: 2; (2) a VH having a sequence as shown in SEQ ID NO: 3 and a VL having a sequence as shown in SEQ ID NO: 4; (3) a VH having a sequence as shown in SEQ ID NO: 5 and a VL having a sequence as shown in SEQ ID NO: 6; (4) a VH having a sequence as shown in SEQ ID NO: 7 and a VH having a sequence as shown in SEQ ID NO: 8; (5) a VH having a sequence as shown in SEQ ID NO: 9 and a VL having a sequence as shown in SEQ ID NO: 10; (6) a VH having a sequence as shown in SEQ ID NO: 11 and a VL having a sequence as shown in SEQ ID NO: 12; (7) a VH having a sequence as shown in SEQ ID NO: 13 and a VL having a sequence as shown in SEQ ID NO: 14; (8) a VH having a sequence as shown in SEQ ID NO: 15 and a VL having a sequence as shown in SEQ ID NO: 16; or (9) a VH having a sequence as shown in SEQ ID NO: 17 and a VL having a sequence as shown in SEQ ID NO: 18. An antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; for example, substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids or any combination thereof); and/or (b) A light chain constant region (CL) of a human immunoglobulin or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids or any combination thereof); Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; Preferably, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region; Preferably, the heavy chain constant region has a sequence such as SEQ ID NO: 121; Preferably, the light chain constant region has a sequence as shown in SEQ ID NO: 122. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab') ² , Fv, disulfide-linked Fv, scFv, diabody and single-domain antibody (sdAb); and/or the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a multispecific antibody; Preferably, the antigen-binding fragment is Fab; Preferably, the antigen-binding fragment also comprises an Fc fragment (e.g., an Fc fragment of human IgG1) or a mutant thereof; Preferably, the Fc fragment has a LALA mutation and a knob mutation; or, the Fc fragment has a LALA mutation and a hole mutation; Preferably, the Fc fragment has a sequence as shown in SEQ ID NO: 111 or 112. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5. A vector comprising the nucleic acid molecule as described in claim 6; preferably, the vector is a clone vector or an expression vector. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7; preferably, the host cell is a mammalian cell. A method for preparing the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, comprising culturing the host cell as described in claim 8 under conditions that allow the expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture. A multispecific molecule comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5; Preferably, the multispecific molecule specifically binds to c-Met, and additionally specifically binds to one or more other targets; Preferably, the multispecific molecule is a bispecific molecule; Preferably, the bispecific molecule further comprises a molecule having a second binding specificity for a second target (e.g., a second antibody). An immunoconjugate comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5 or a multispecific molecule as described in claim 10, and a therapeutic agent linked to the antibody or antigen-binding fragment thereof or the multispecific molecule; Preferably, the therapeutic agent is selected from cytotoxic agents; Preferably, the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, anti-metabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof; Preferably, the immunoconjugate is an antibody-drug conjugate (ADC). A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, or the multispecific molecule as described in claim 10, or the immunoconjugate as described in claim 11, and a pharmaceutically acceptable carrier and/or formulation; Preferably, the pharmaceutical composition further comprises another pharmaceutically active agent; Preferably, the other pharmaceutically active agent is a drug having anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an anti-metabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecular target drug, an immune checkpoint inhibitor, or an oncolytic virus; Preferably, the antibody or antigen-binding fragment thereof, multispecific molecule or immunoconjugate is provided as separate components or as components of the same composition as the additional pharmaceutically active agent. A reagent kit, comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5; Preferably, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or biotin; Preferably, the reagent kit also includes a second antibody, which specifically identifies the antibody or antigen-binding fragment thereof as described in any one of claims 1-5; Preferably, the second antibody also includes a detectable label, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or biotin. A chimeric antigen receptor comprising an antigen binding domain of an antibody or an antigen binding fragment thereof as described in any one of claims 1 to 5; Preferably, the antigen binding domain comprises a heavy chain variable region and a light chain variable region of an antibody or an antigen binding fragment thereof as described in any one of claims 1 to 5; Preferably, the antigen binding domain is scFv; Preferably, the chimeric antigen receptor is expressed by immune effector cells (e.g., T cells). A method for inhibiting the growth of tumor cells expressing c-Met and/or killing the tumor cells, comprising contacting the tumor cells with an effective amount of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, or the multispecific molecule as described in claim 10, or the immunoconjugate as described in claim 11, or the pharmaceutical composition as described in claim 12, or the chimeric antigen receptor as described in claim 14. Use of an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5, or a multispecific molecule as described in claim 10, or an immunoconjugate as described in claim 11, or a drug composition as described in claim 12, or a chimeric antigen receptor as described in claim 14 in the preparation of a drug for preventing and/or treating tumors in a subject (e.g., a human); Preferably, the drug also contains another pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an anti-metabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecular target drug, an immune checkpoint inhibitor or an oncolytic virus; Preferably, the tumor expresses c-Met; Preferably, the tumor involves a tumor cell expressing c-Met; Preferably, the c-Met is expressed on the surface of the tumor cell; Preferably, the tumor is selected from 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, glioma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, fungoids), Merkel cell carcinoma and other malignant blood diseases, such as classical Hodgkin lymphoma (CHL), primary diaphragmatic large B-cell lymphoma, T-cell/tissue cell-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary exudative lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal tumors, brain stem neuroglioma. Preferably, the subject is a mammal, such as a human. Use of an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5 in preparing a kit for detecting whether a tumor can be treated by an anti-tumor therapy targeting c-Met; (1) contacting a sample containing the tumor cells with the antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5; (2) detecting the formation of a complex between the antibody or an antigen-binding fragment thereof and c-Met; Preferably, the antibody or an antigen-binding fragment thereof carries a detectable label; Preferably, the c-Met is c-Met of a mammal (e.g., human, monkey); Preferably, the tumor is selected from 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, glioma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary diaphragmatic large B-cell lymphoma, T-cell/tissue cell-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary exudative lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal tumors, brain stem neuroglioma.