Monoclonal and bispecific antibodies against c-Met

Abstract

The present application relates to an antibody or antigen-binding fragment thereof capable of specifically binding to c-Met, as well as immunoconjugates, pharmaceutical compositions, and multispecific molecules comprising the same. The present application further relates to the use of an antibody or antigen-binding fragment thereof and multispecific molecules that specifically bind to c-Met. Compared to a control antibody, the bispecific antibody or defucosylated bispecific antibody of the present application can block HGF-dependent TKI resistance, block HGF-induced tumor cell proliferation and migration, induce ADCC effects, and inhibit tumor growth in vivo.

Description

[Technical field]

[0001] Technical Field The present application is in the field of biomedical technology. More specifically, the present application relates to an antibody or antigen-binding fragment thereof capable of specifically binding to c-Met, as well as immunoconjugates, pharmaceutical compositions and multispecific molecules comprising the antibody or antigen-binding fragment thereof. Furthermore, the present application relates to the use of the antibody or antigen-binding fragment thereof capable of specifically binding to c-Met and the multispecific molecules. [Background technology]

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

[0003] Studies have shown that c-Met may promote cancer progression and is highly expressed in various tumor tissues. Therefore, c-Met is a target for anticancer drug development. In addition to traditional RTK small molecule inhibitors, the development of antibody-targeting macromolecular drugs is gradually progressing and deepening in clinical studies. An example of this is the anti-c-Met monoclonal antibody MetMab. Another example is ADC molecules such as TR1801, which is conjugated with the antitubulin toxin Tecilin, and is effective in mouse tumor PDX models with low, intermediate and high MET expression. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, there is a need to develop novel anti-c-Met monoclonal antibodies and multispecific antibodies containing the same in order to increase the applicability in tumor treatment. [Means for solving the problem]

[0005] Contents of the invention In the present application, an antibody or an antigen-binding fragment thereof capable of specifically binding to c-Met is provided, from which a bispecific antibody capable of specifically binding to c-Met and EGFR is prepared.Furthermore, a non-fucosylated bispecific antibody is prepared, completing the present invention.

[0006] In a first aspect, the present application provides an antibody, or antigen-binding fragment thereof, capable of specifically binding to c-Met, comprising: (a) a heavy chain variable region (VH) having the following three complementarity determining regions (CDRs): (i) a VH CDR1 consisting of a sequence as set forth in SEQ ID NO: 27 or 33, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (ii) a VH CDR2 consisting of a sequence as set forth in SEQ ID NO: 28 or 34 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (iii) a VH CDR3 consisting of a sequence as set forth in any one of SEQ ID NOs: 29 or 35, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; and / or a heavy chain variable region (VH) comprising (b) a light chain variable region (VL) having the following three complementarity determining regions (CDRs): (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 30 or 36 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 31 or 37, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 32 or 38, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared thereto; A light chain variable region (VL) containing The present invention provides an antibody or antigen-binding fragment thereof comprising:

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

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

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

[0010] In certain embodiments, the above-described antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO:27, VH CDR2 set forth in SEQ ID NO:28, VH CDR3 set forth in SEQ ID NO:29, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO:30, VL CDR2 set forth in SEQ ID NO:31, VL CDR3 set forth in SEQ ID NO:32.

[0011] In certain embodiments, the above-described antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO: 33, VH CDR2 set forth in SEQ ID NO: 34, VH CDR3 set forth in SEQ ID NO: 35, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO: 36, VL CDR2 set forth in SEQ ID NO: 37, VL CDR3 set forth in SEQ ID NO: 38.

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a human immunoglobulin framework region.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof described above comprises: (a) a heavy chain variable region (VH), (i) a sequence as set forth in SEQ ID NO: 9 or 13; (ii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 9 or 13; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 9 or 13. and / or a heavy chain variable region (VH) comprising an amino acid sequence selected from (b) a light chain variable region (VL), (iv) a sequence as set forth in SEQ ID NO: 11 or 15; (v) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 11 or 15; or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 11 or 15. A light chain variable region (VL) comprising an amino acid sequence selected from Includes.

[0014] In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0015] In a particular embodiment, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:9 and a VL having the sequence set forth in SEQ ID NO:11.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:13 and a VL having the sequence set forth in SEQ ID NO:15.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof is as described above, further comprising a constant region derived from a human immunoglobulin.

[0018] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4).

[0019] In certain embodiments, the heavy chain constant region has the sequence set forth in SEQ ID NO: 19, 20, 39 or 40.

[0020] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda).

[0021] In certain embodiments, the light chain constant region has the sequence set forth in SEQ ID NO:21, 22 or 41.

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof has ADCC activity.

[0023] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a mutated or chemically modified Fc region.

[0024] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region having LALA mutations and / or knobs-into-hole modifications.

[0025] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain having the sequence shown in SEQ ID NO: 10 and a light chain having the sequence shown in SEQ ID NO: 12; (2) a heavy chain having the sequence shown in SEQ ID NO: 14 and a light chain having the sequence shown in SEQ ID NO: 16; or (3) a heavy chain having the sequence shown in SEQ ID NO: 23 and a light chain having the sequence shown in SEQ ID NO: 24 Includes.

[0026] Related "knob-into-hole" technology is described, for example, in U.S. Pat. Nos. 5,731,168 and 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001).

[0027] In a specific embodiment, the Fc region has the amino acid sequence set forth in SEQ ID NO:17 or 18.

[0028] In certain embodiments, the antibody or antigen-binding fragment thereof is hypofucosylated or non-fucosylated.

[0029] In certain embodiments, the antibodies or antigen-binding fragments thereof described above include 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.

[0030] On the other hand, the present application provides an isolated nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof.

[0031] On the other hand, the present application provides a vector comprising the above-described nucleic acid molecule. In certain embodiments, the vector is a cloning vector or an expression vector.

[0032] On the other hand, the present application provides a host cell comprising the above-described nucleic acid molecule or the above-described vector.

[0033] In certain embodiments, the host cell is a mammalian cell.

[0034] In certain embodiments, the host cell has low or no fucosylation activity, e.g., the host cell is selected from mammalian cells (e.g., CHO cells) that lack expression of genes encoding fucosyltransferases.

[0035] On the other hand, the present application provides a method for preparing the above-described antibody or antigen-binding fragment thereof, comprising culturing the above-described host cell under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cell.

[0036] In certain embodiments, the host cell has low or no fucosylation activity, e.g., the host cell is selected from mammalian cells (e.g., CHO cells) that lack expression of genes encoding fucosyltransferases.

[0037] On the other hand, the present application provides multispecific molecules comprising the above-described antibodies or antigen-binding fragments thereof.

[0038] In certain embodiments, the multispecific molecule specifically binds to c-Met and additionally specifically binds to one or more other targets.

[0039] In certain embodiments, the multispecific molecule is a bispecific molecule.

[0040] In certain embodiments, the bispecific molecule further comprises a molecule having a second binding specificity for a second target (eg, a second antibody).

[0041] In certain embodiments, the second target is epidermal growth factor receptor (EGFR) and the second antibody is an anti-EGFR antibody or an antigen-binding fragment thereof.

[0042] In certain embodiments, the bispecific molecule is modified by glycosylation to have a lower number of fucose than the same bispecific molecule that is not modified by glycosylation.

[0043] In certain embodiments, the anti-EGFR antibody, or antigen-binding fragment thereof, is hypofucosylated or non-fucosylated.

[0044] In certain embodiments, the antibodies or antigen-binding fragments thereof described above are hypofucosylated or non-fucosylated.

[0045] In a particular embodiment, the second antibody comprises the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO: 46, VH CDR2 set forth in SEQ ID NO: 47, VH CDR3 set forth in SEQ ID NO: 48, and the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO: 49, VL CDR2 set forth in SEQ ID NO: 50, VL CDR3 set forth in SEQ ID NO: 51.

[0046] In certain embodiments, the second antibody is (1) a VH having a sequence as set forth in SEQ ID NO: 1 and a VL having a sequence as set forth in SEQ ID NO: 3, or (2) VH having the sequence shown in SEQ ID NO: 52 and VL having the sequence shown in SEQ ID NO: 53 Includes.

[0047] In certain embodiments, the second antibody is (1) a heavy chain having the sequence shown in SEQ ID NO:2 and a light chain having the sequence shown in SEQ ID NO:4, or (2) a heavy chain having the sequence shown in SEQ ID NO: 25 and a light chain having the sequence shown in SEQ ID NO: 26 Includes.

[0048] In certain embodiments, the bispecific molecule comprises: (1) a first antibody comprising VH CDR1 to 3 shown in SEQ ID NOs: 27 to 29 and VL CDR1 to 3 shown in SEQ ID NOs: 30 to 32, and a second antibody comprising VH CDR1 to 3 shown in SEQ ID NOs: 46 to 48 and VL CDR1 to 3 shown in SEQ ID NOs: 49 to 51, or (2) A first antibody comprising VH CDR1 to 3 shown in SEQ ID NOs: 33 to 35 and VL CDR1 to 3 shown in SEQ ID NOs: 36 to 38, and a second antibody comprising VH CDR1 to 3 shown in SEQ ID NOs: 46 to 48 and VL CDR1 to 3 shown in SEQ ID NOs: 49 to 51. Includes.

[0049] In certain embodiments, the multispecific molecule comprises: (1) a first antibody comprising a VH having the sequence shown in SEQ ID NO:9 and a VL having the sequence shown in SEQ ID NO:11, and a second antibody comprising a VH having the sequence shown in SEQ ID NO:1 and a VL having the sequence shown in SEQ ID NO:3; (2) a first antibody comprising a VH having the sequence shown in SEQ ID NO: 9 and a VL having the sequence shown in SEQ ID NO: 11, and a second antibody comprising a VH having the sequence shown in SEQ ID NO: 52 and a VL having the sequence shown in SEQ ID NO: 53; (3) a first antibody comprising a VH having the sequence shown in SEQ ID NO: 13 and a VL having the sequence shown in SEQ ID NO: 15, and a second antibody comprising a VH having the sequence shown in SEQ ID NO: 1 and a VL having the sequence shown in SEQ ID NO: 3; or (4) A first antibody comprising a VH having the sequence shown in SEQ ID NO: 13 and a VL having the sequence shown in SEQ ID NO: 15, and a second antibody comprising a VH having the sequence shown in SEQ ID NO: 52 and a VL having the sequence shown in SEQ ID NO: 53. Includes.

[0050] In certain embodiments, the multispecific molecule comprises: (1) a first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 10 and a light chain having the sequence shown in SEQ ID NO: 12, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 25 and a light chain having the sequence shown in SEQ ID NO: 26; (2) a first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 10 and a light chain having the sequence shown in SEQ ID NO: 12, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 2 and a light chain having the sequence shown in SEQ ID NO: 4; (3) a first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 14 and a light chain having the sequence shown in SEQ ID NO: 16, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 25 and a light chain having the sequence shown in SEQ ID NO: 26; (4) a first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 14 and a light chain having the sequence shown in SEQ ID NO: 16, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 2 and a light chain having the sequence shown in SEQ ID NO: 4; or (5) A first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 23 and a light chain having the sequence shown in SEQ ID NO: 24, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 25 and a light chain having the sequence shown in SEQ ID NO: 26. Includes.

[0051] On the other hand, there is provided a method for preparing a multispecific antibody as described above, comprising obtaining an antibody or antigen-binding fragment thereof as described above by a method as described above, contacting it with an anti-EGFR antibody or antigen-binding fragment thereof and optionally contacting it with a reducing agent (e.g. DTT).

[0052] On the other hand, there is provided an immunoconjugate comprising an antibody or antigen-binding fragment thereof as described above, or a multispecific molecule as described above, and a therapeutic agent linked to the antibody or antigen-binding fragment thereof, or the multispecific molecule.

[0053] In certain embodiments, the therapeutic agent is selected from a cytotoxic agent.

[0054] In certain embodiments, the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof.

[0055] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0056] In another aspect, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above or a multispecific molecule as described above and a pharma- ceutically acceptable carrier and / or excipient.

[0057] In certain embodiments, the pharmaceutical composition further comprises an additional pharma- ceutical active agent.

[0058] In certain embodiments, the pharmaceutical composition further comprises an EGFR inhibitor.

[0059] In certain embodiments, the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, osimertinib, or any combination thereof.

[0060] In certain embodiments, the EGFR inhibitor and the antibody or antigen-binding fragment thereof, or the multispecific molecule, each as an active ingredient, are contained in different preparations and are administered at the same time or at different times. In certain embodiments, the EGFR inhibitor is osimertinib.

[0061] In certain embodiments, the additional pharma- ceutically active agent is a drug with antitumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide drug, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0062] On the other hand, there is provided the use of an antibody or antigen-binding fragment thereof as described above, or a multispecific molecule as described above, in combination with an EGFR inhibitor in the manufacture of a medicament.

[0063] In certain embodiments, the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, osimertinib, or any combination thereof, and in certain embodiments, the EGFR inhibitor is osimertinib.

[0064] In certain embodiments, the pharmaceutical agent is (1) increasing immune cell activity in a subject in vitro or in vivo; (2) enhancing an immune response in a subject; (3) preventing and / or treating a tumor in a subject; or (4) Preventing and / or treating an infection in a subject. Used for.

[0065] In certain embodiments, the tumor expresses c-Met.

[0066] In certain embodiments, the tumor is associated with tumor cells that express c-Met, hi certain embodiments, c-Met is expressed on the surface of the tumor cells.

[0067] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, B-cell rich lymphoma of T-cell / histiocyte, EBV-positive and -negative PTLD and EBV-related diffuse large B-cell lymphoma. lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumor, and brain stem glioma.

[0068] In certain embodiments, the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.

[0069] In certain embodiments, the subject is a mammal, such as a human, a cynomolgus monkey, or a mouse.

[0070] In another aspect, a kit is provided that comprises an antibody or antigen-binding fragment thereof described above.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (eg, a chemiluminescent material), or biotin.

[0072] In some embodiments, the kit further comprises a second antibody that specifically recognizes the anti-EGFR antibody, or an antigen-binding fragment thereof.

[0073] In some embodiments, the second antibody further comprises a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorochrome, a luminescent material (eg, a chemiluminescent material), or biotin.

[0074] In some embodiments, the anti-EGFR antibody, or antigen-binding fragment thereof, is hypofucosylated or non-fucosylated.

[0075] In some embodiments, the antibodies or antigen-binding fragments thereof described above are hypofucosylated or non-fucosylated.

[0076] In another aspect, there is provided a chimeric antigen receptor comprising the antigen-binding domain of an antibody or antigen-binding fragment thereof described above.

[0077] In some embodiments, the antigen-binding domain comprises a heavy chain variable region and a light chain variable region of an antibody or antigen-binding fragment thereof described above.

[0078] In certain embodiments, the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell).

[0079] In another aspect, there is provided a method of inhibiting the proliferation of and / or killing a tumor cell expressing c-Met, comprising contacting said tumor cell with an antibody or antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or an immunoconjugate as described above, or a pharmaceutical composition as described above, or a chimeric antigen receptor as described above.

[0080] In another aspect, the use of an antibody or antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or an immunoconjugate as described above, or a pharmaceutical composition as described above, or a chimeric antigen receptor as described above, in the manufacture of a medicament, wherein the medicament comprises (1) increasing immune cell activity in a subject in vitro or in vivo; (2) enhancing an immune response in a subject; (3) preventing and / or treating a tumor in a subject; or (4) Preventing and / or treating an infection in a subject. For use, use is provided.

[0081] In certain embodiments, the tumor expresses c-Met.

[0082] In certain embodiments, the tumor is associated with tumor cells that express c-Met, hi certain embodiments, c-Met is expressed on the surface of the tumor cells.

[0083] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, Selected from the group consisting of T cell / histiocyte B cell rich lymphoma, EBV positive and negative PTLD and EBV associated diffuse large B cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T cell lymphoma, nasopharyngeal carcinoma and HHV8 associated primary effusion lymphoma, Hodgkin's lymphoma, central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumor, brain stem glioma.

[0084] In some embodiments, the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.

[0085] In some embodiments, the subject is a mammal, such as a human, a cynomolgus monkey, or a mouse.

[0086] In another embodiment, the use of an antibody or antigen-binding fragment thereof as described above in the manufacture of a kit, the kit being used to determine whether a tumor can be treated by an anti-tumor therapy that targets c-Met, (1) contacting a sample containing tumor cells with the antibody or antigen-binding fragment thereof described above; (2) detecting the formation of a complex containing the antibody or antigen-binding fragment thereof and c-Met; A use is provided in which

[0087] In some embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label.

[0088] In some embodiments, the c-Met is mammalian (eg, human, monkey) c-Met.

[0089] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, Selected from the group consisting of T cell / histiocyte B cell rich lymphoma, EBV positive and negative PTLD and EBV associated diffuse large B cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T cell lymphoma, nasopharyngeal carcinoma and HHV8 associated primary effusion lymphoma, Hodgkin's lymphoma, central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumor, brain stem glioma.

[0090] In one aspect, the present application provides a method of preventing and / or treating a tumor in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof as described above, or a bispecific or multispecific molecule as described above, or an immunoconjugate as described above, or a pharmaceutical composition as described above, or a chimeric antigen receptor as described above, or a host cell as described above.

[0091] In certain embodiments, the tumor expresses c-Met.

[0092] In certain embodiments, the tumor is associated with tumor cells that express c-Met, hi certain embodiments, c-Met is expressed on the surface of the tumor cells.

[0093] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, Selected from the group consisting of T cell / histiocyte B cell rich lymphoma, EBV positive and negative PTLD and EBV associated diffuse large B cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T cell lymphoma, nasopharyngeal carcinoma and HHV8 associated primary effusion lymphoma, Hodgkin's lymphoma, central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumor, brain stem glioma.

[0094] In certain embodiments, the subject is a mammal, for example a human.

[0095] In certain embodiments, the method further comprises administering an additional drug having anti-tumor activity (e.g., an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide drug, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus).

[0096] In certain embodiments, the method further comprises administering an additional anti-tumor therapy (eg, surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy, or palliative care).

[0097] On the other hand, the present application provides a method for determining whether a tumor can be treated by an antitumor therapy that targets c-Met, comprising: (1) contacting a sample containing tumor cells with the antibody or antigen-binding fragment thereof described above; (2) detecting the formation of a complex containing the antibody or antigen-binding fragment thereof and c-Met; The present invention provides a method comprising:

[0098] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label.

[0099] In certain embodiments, the c-Met is mammalian (eg, human, monkey) c-Met.

[0100] In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, Selected from the group consisting of T cell / histiocyte B cell rich lymphoma, EBV positive and negative PTLD and EBV associated diffuse large B cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T cell lymphoma, nasopharyngeal carcinoma and HHV8 associated primary effusion lymphoma, Hodgkin's lymphoma, central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumor, brain stem glioma.

[0101] On the other hand, the present application provides a method for detecting the presence or amount of c-Met in a sample, comprising: (1) contacting the sample with an antibody or antigen-binding fragment thereof described above; (2) detecting the formation of a complex containing the antibody or antigen-binding fragment thereof and c-Met, or detecting the amount of this complex. The present invention provides a method comprising:

[0102] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label.

[0103] In certain embodiments, the c-Met is mammalian (eg, human, monkey) c-Met.

[0104] Definition of Terms In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA manipulation procedures used herein are all conventional procedures widely used in the corresponding fields.In addition, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0105] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains, each having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as kappa light chains and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the antibody isotype can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and heavy chains also contain 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 consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions are not directly involved in binding the antibody to an antigen, but exhibit various effector functions (e.g., mediating the interaction of immunoglobulins with host tissues or factors, such as binding to various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q)). The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL), respectively, form an antigen-binding site. Assignment of amino acids to regions or domains may follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0106] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are involved in antigen binding. The heavy and light chain variable regions each contain three CDRs, designated CDR1, CDR2, and CDR3. The exact boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al. al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0107] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention may be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined according to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system.

[0108] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0109] The term "antibody" is not limited to any particular method for producing the antibody. For example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies are included. The antibody can be of various isotypes, such as IgG antibodies (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0110] As used herein, the terms "monoclonal antibody," "McAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or antibody fragment derived from a population of highly homologous antibody molecules (i.e., a population of antibody molecules that are identical except for natural mutations that may occur naturally). Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are referred to in comparison to monoclonal antibodies and usually contain at least two or more different antibodies that usually recognize different epitopes on an antigen. In addition, the modifier "monoclonal" merely indicates that the antibody is characterized as being obtained from a population of highly homologous antibodies and should not be construed as requiring that the antibody be prepared in any particular manner.

[0111] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma techniques (see, e.g., Kohler et al., Nature, 256:495, 1975), recombinant DNA techniques (see, e.g., U.S. Pat. No. 4,816,567), or phage antibody library techniques (see, e.g., Clackson et al. Nature 352: 624-628, 1991 or Marks et al. J. Mol. Biol. 222: 581-597, 1991).

[0112] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, which fragment 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 an antigen (the fragment is also referred to as an "antigen-binding portion"). See generally Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be obtained by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') and / or F(ab') fragments. 2 , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (Domantis technology), probodies, and other polypeptides that contain at least a portion of an antibody sufficient to confer specificity to a polypeptide with antigen-binding ability. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0113] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Among these, the "full-length heavy chain" refers to a polypeptide consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction, and optionally also includes a heavy chain constant region CH4 domain if the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. The "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. The two pairs of full-length antibody chains are linked to each other by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be derived from a single species, such as human, and may be a chimeric or humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by a VH pair and a VL pair, each of which specifically recognizes / binds the same antigen.

[0114] As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains, the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)), the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains, and the term "F(ab') 2 The term "Fab' fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and the term "Fab' fragment" refers to an F(ab') fragment consisting of a complete light chain and an Fd fragment of a heavy chain (consisting of the VH and CH1 domains). 2 It refers to a fragment obtained by reducing the disulfide bond linking two heavy chain fragments in a fragment.

[0115] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. In general, six CDRs are considered to confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, but the affinity may be lower than that of the complete binding site.

[0116] As used herein, the term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of a first heavy chain and the second and third constant regions of a second heavy chain of an antibody. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.

[0117] As used herein, the term "LALA mutation" refers to an L to A mutation at amino acid 234 and an L to A mutation at amino acid 235 of the native Fc fragment.

[0118] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL domain and a VH domain, where the VL and VH are linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988) and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the following general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers may consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, although variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that may be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. Optionally, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the invention, scFvs may form di-scFvs, which refers to antibodies formed by linking two or more single scFvs in series. In certain embodiments of the invention, scFvs may form (scFv)2, which refers to antibodies formed by two or more single scFvs in parallel.

[0119] As used herein, the term "single domain antibody (sdAb)" has its meaning commonly understood by those skilled in the art and refers to an antibody fragment that is composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) and retains the ability to specifically bind to the same antigen as a full-length antibody. Single domain antibodies are also called nanobodies.

[0120] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as bound by the full-length antibody and / or competes with the full-length antibody for specific binding to antigen.

[0121] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of an antibody can be screened for specificity in the same way as an intact antibody.

[0122] As used herein, unless the context clearly indicates otherwise, reference to the term "antibody" includes not only intact antibodies but also antigen-binding fragments of such antibodies.

[0123] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light or / and heavy chain is derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass) and another portion of the light or / and heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), while still retaining binding activity for the target antigen in either case (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" may include an antibody in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody and the constant regions of the heavy and light chains of the antibody are derived from a second antibody.

[0124] As used herein, the term "identity" is used to refer to the sequence match between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at this position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of identity = number of identical overlapping positions / total number of positions x 100%). In certain embodiments, the two sequences are the same length.

[0125] Determining the percentage of identity between two sequences can also be achieved using a mathematical algorithm.A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified by Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877.Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0126] As used herein, in the context of a polypeptide (including a polypeptide), the term "variant" also refers to a polypeptide or peptide that contains an amino acid sequence that is altered by the introduction of substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that is modified (i.e., by covalently attaching any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide can be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. A derivatized polypeptide or peptide can be produced by chemical modification using techniques known to those skilled in the art (e.g., but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.). In addition, a variant has similar, identical, or improved functions as the polypeptide or peptide from which it is derived.

[0127] As used herein, the term "specifically bind" refers to a non-random binding reaction between two molecules (e.g., a reaction between an antibody and an antigen to which the antibody is directed). The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of this 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.

[0128] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex. Both the "association rate constant" (ka or k) and the "dissociation rate constant" (k or k) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of k / k is equal to the dissociation constant K (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). The values ​​of K, k and k can be measured by any valid method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured by bioluminescence interferometry or Kinexa.

[0129] As used herein, a detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are known in the art and 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 770 ... 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.

[0130] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transgenesis, and the genetic material elements carried by the vector are expressed in the host cell. Vectors are known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40). Vectors may contain various elements that control expression, including, but are not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may also contain an origin of replication.

[0131] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells, such as Escherichia coli or Bacillus subtilis, fungal cells, such as yeast cells or Aspergillus, insect cells, such as S2 Drosophila cells or Sf9, or animal cells, such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0132] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, such as one that is physically or functionally similar to the corresponding amino acid residue (e.g., one that has similar size, shape, charge, chemical properties such as the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. This family includes amino acids with basic side chains (e.g., lysine, arginine and histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0133] The twenty conventional amino acids referred to herein are written according to convention. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. In addition, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0134] As used herein, the term "pharmaceutical composition" refers to any product that contains one or more active ingredients (e.g., antibodies, small molecule drugs), optionally in a specific amount, and is made directly or indirectly by combining one or more active ingredients, optionally in a specific amount. Different active ingredients in a pharmaceutical composition may be administered independently in separate formulations, and these formulations may be administered simultaneously or at different times for a combined synergistic effect. In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0135] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient, which are known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, osmotic pressure maintaining agents, absorption retarding agents, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic surfactants, anionic surfactants, or nonionic surfactants (e.g., Tween-80). Ionic strength enhancing agents include, but are not limited to, sodium chloride. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and the like. Absorption retarding agents include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol) and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents (e.g., thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.). Stabilizers have the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the pharmaceutical product, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharma-ceutically acceptable carrier or excipient includes a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution).In certain exemplary embodiments, such a sterile injectable fluid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), a sodium chloride solution (e.g., 0.9% (w / v) NaCl), a glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer (e.g., phosphate buffer), Ringer's solution, and any combination thereof.

[0136] As used herein, the term "prevention" refers to a method carried out to prevent or delay the occurrence of a disease, disorder or condition in a subject. As used herein, the term "treatment" refers to a method carried out to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include (but are not limited to): alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., no longer worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief of symptoms (partial or complete alleviation), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to expected survival (if not receiving treatment).

[0137] As used herein, the term "subject" refers to a mammal, such as a human, a cynomolgus monkey, or a mouse. In certain embodiments, the subject (e.g., a human, a cynomolgus monkey, or a mouse) is suffering from a disease associated with c-Met or is at risk of suffering from the above-mentioned disease.

[0138] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired result. For example, an amount effective for disease prevention refers to an amount effective to prevent, stop or delay the onset of a disease, and an amount effective for disease treatment refers to an amount effective to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. It is well within the capabilities of a person skilled in the art to determine such an effective amount. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the subject's own immune system, the patient's overall condition, such as age, weight and sex, the mode of administration of the drug, and other treatments administered at the same time, etc.

[0139] As used herein, the term "single-arm antibody" refers to an antigen-binding fragment comprising a Fab segment and an Fc segment, where the Fab segment generally comprises a heavy chain (e.g., VH and CH1) and a light chain (e.g., VL and CL), and the Fc segment comprises a constant region (e.g., CH2 and CH3). The Fab segment and the Fc segment may or may not be connected by a linker. The single-arm antibody of the present invention may be prepared or synthesized in a variety of ways, for example, by constructing a sequence encoding a Fab heavy chain and a sequence encoding an Fc in the same vector, constructing a sequence encoding a Fab light chain in another vector, and transforming these two vectors separately into a host cell to obtain the single-arm antibody.

[0140] As used herein, the term "bispecific antibody" refers to a conjugate formed by a first antibody (or fragment thereof) and a second antibody (or fragment thereof) or antibody analogue via conjugation arms, the conjugation methods including, but not limited to, chemical reaction, gene fusion and enzymes. Bispecific antibodies can be linked or generated in a variety of ways, see for example the methods of Songsivilai et al. (Clin. Exp. Immunol., 79: 315-321 (1990)) and Kostelny et al. (J. Immunol., 148: 1547-1553 (1992)).

[0141] As used herein, the terms "fucosylated" or "modified by fucosylation" have the same meaning and refer to the presence of fucose residues in the oligosaccharides attached to the peptide backbone of an antibody. Similarly, the terms "non-fucosylated" or "defucosylation" or "modified by fucosylation" have the same meaning and refer to the removal of fucose residues in the oligosaccharides attached to the peptide backbone of an antibody.

[0142] Beneficial Effects of the Invention In the present application, an antibody or an antigen-binding fragment thereof capable of specifically binding to c-Met is obtained, and further, a bispecific antibody and a non-fucosylated bispecific antibody capable of specifically binding to c-Met and EGFR are prepared. Compared with the reference antibody, the bispecific antibody or the non-fucosylated bispecific antibody of the present application can block HGF-dependent TKI resistance, block tumor cell proliferation and migration induced by HGF, induce ADCC effect, and inhibit tumor growth in vivo, which is superior to the control antibody and the commercially available antibody Rybrevant, and shows synergistic tumor killing effect when combined with an EGFR inhibitor. Therefore, the bispecific antibody of the present application has outstanding clinical value.

[0143] Although the embodiments of the present invention will be described in detail below in conjunction with the drawings and examples, those skilled in the art will understand that the following drawings and examples are used only to illustrate the present invention, and not to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the drawings and the following detailed description of the preferred embodiments. [Brief description of the drawings]

[0144] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] FIG. 1 shows a schematic diagram of the structure of a single-arm antibody for anti-c-Met screening in the present application. [Figure 1B] FIG. 1 shows a schematic diagram of the structure of the anti-EGFRxc-Met bispecific antibody of the present application. [Diagram 2] 1 shows the inhibitory effects of the anti-c-Met antibodies of the present application (136 single-arm antibody, 187 single-arm antibody) and a control antibody on the proliferation of HCC827 cells. [Diagram 3] 1 shows the results of blocking EGF-EGFR interaction by anti-EGFRxc-MET bispecific antibodies of the present application (EGFRxc-Met-136, EGFRxc-Met-187) and control antibodies. [Figure 4] 1 shows the inhibitory effects of the anti-EGFRxc-Met bispecific antibodies of the present application (EGFRxc-Met-136, EGFRxc-Met-187) and a control antibody on the proliferation of HCC827 cells. [Diagram 5] 1 shows the inhibitory effects of the anti-EGFRxc-Met bispecific antibodies of the present application (EGFRxc-Met-136, EGFRxc-Met-187) and a control antibody on the proliferation of H596 cells. [Figure 6] 1 shows the blocking effect of the anti-EGFRxc-Met bispecific antibodies of the present application (EGFRxc-Met-136, EGFRxc-Met-187) and a control antibody on HGF-induced migration of HepG2 cells. [Figure 7A] FIG. 1 shows the quality check results of the stable cell line product and demonstrates the purity of the bispecific antibody obtained by affinity purification. [Figure 7B] FIG. 1 shows the quality check results of the stable cell line product, showing the percentage of correctly paired product of purified bispecific antibodies. [Figure 8A] 1 shows the cell binding activity results of nonfucosylated bispecific antibodies (EGFRxc-Met-136 Afu) and (EGFRxc-Met-187 Afu) and a control antibody against A375 cells. [Figure 8B] 1 shows the cell binding activity results of nonfucosylated bispecific antibodies (EGFRxc-Met-136 Afu) and (EGFRxc-Met-187 Afu) and a control antibody against H292 cells. [Figure 8C] 1 shows the cell binding activity results of nonfucosylated bispecific antibodies (EGFRxc-Met-136 Afu) and (EGFRxc-Met-187 Afu) and a control antibody against HCC827 cells. [Figure 9A] 1 shows the ADCC effects induced by the anti-EGFRxc-Met bispecific antibodies of the present application (EGFRxc-Met-136, EGFRxc-Met-187), a non-fucosylated bispecific antibody (EGFRxc-Met-136 Afu), and a control antibody against A375 cells. [Figure 9B] 1 shows the ADCC effects induced by the anti-EGFRxc-Met bispecific antibodies of the present application (EGFRxc-Met-136, EGFRxc-Met-187), a non-fucosylated bispecific antibody (EGFRxc-Met-136 Afu), and a control antibody against H1975 cells. [Figure 9C] 1 shows the ADCC effects induced by the anti-EGFRxc-Met bispecific antibodies of the present application (EGFRxc-Met-136, EGFRxc-Met-187), a non-fucosylated bispecific antibody (EGFRxc-Met-136 Afu), and a control antibody against HCC827 cells. [Figure 10]1 shows the killing effect of PBMCs on A375 cells induced by anti-EGFRxc-Met bispecific antibodies of the present application (EGFRxc-Met-136, EGFRxc-Met-187), a non-fucosylated bispecific antibody (EGFRxc-Met-136 Afu), and a control antibody. [Figure 11] 1 shows the killing effect of PBMCs on HCC827 cells induced by a combination of the nonfucosylated anti-EGFRxc-Met bispecific antibody of the present application (EGFRxc-Met-136 Afu) and an EGFR inhibitor. [Figure 12] 1 shows the inhibitory effects of the nonfucosylated anti-EGFRxc-Met bispecific antibodies of the present application (EGFRxc-Met-136 Afu, EGFRxc-Met-187 Afu) and a control antibody on the proliferation of H292 human lung cancer cells. [Figure 13] 1 shows the inhibitory effect of various doses of the nonfucosylated anti-EGFRxc-Met bispecific antibody of the present application (EGFRxc-Met-136 Afu) on the proliferation of H1975 human lung cancer cells. [Figure 14] 1 shows the inhibitory effect of various doses of the nonfucosylated anti-EGFRxc-Met bispecific antibody of the present application (EGFRxc-Met-136 Afu) on the proliferation of H292 human lung cancer cells. [Figure 15] 1 shows the inhibitory effect of a single dose of the nonfucosylated anti-EGFRxc-Met bispecific antibody of the present application (EGFRxc-Met-136 Afu) and the control commercial antibody Rybrevant on the proliferation of H292 human lung cancer cells. [Figure 16] 1 shows the inhibitory effect of a combination of a nonfucosylated anti-EGFRxc-Met bispecific antibody of the present application (EGFRxc-Met-136 Afu) and a small molecule inhibitor (osimertinib) on the proliferation of H1975-HGF human lung cancer cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0145] Sequence information Some sequence information relevant to the present invention is shown in Table 1 below.

[0146] [Table 1]

[0147] [Table 2]

[0148] [Table 3]

[0149] [Table 4]

[0150] [Table 5]

[0151] [Table 6]

[0152] Specific Model for Implementing the Invention The invention will now be described with reference to the following examples which are intended to illustrate, but not limit, the invention.

[0153] Unless otherwise specified, the experiments and methods described in the examples are essentially carried out according to conventional methods known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in: Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL METHOD. APPROACH) (MJ MacPherson, BD Hames and GR Taylor, ed. (1995)) and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0154] In addition, if no specific conditions are specified in the examples, they were carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or equipment used without manufacturer indication were all conventional products that could be obtained commercially. It is known to those skilled in the art that the examples are illustrative of the present invention and are not intended to limit the scope of protection of the present invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety. EXAMPLES

[0155] Example 1: Antibody construction and protein expression and purification Monoclonal antibody screening Mice were immunized with cell mesenchymal epithelial transition factor (c-Met) antigen (purchased from AcroBiosystems), total RNA was extracted and reverse transcribed, and the yeast display library constructed by PCR amplification was screened to obtain the sequences of anti-c-MET antibodies 136 and 187 used in this application. The sequences of antibodies 136 and 187 were obtained by sequencing, and the CDR sequences of these antibodies were determined by the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). Specifically, a fully humanized anti-c-MET antibody library (having a diversity of 3×10^7) was first constructed from five immunized mice, and human c-MET protein was labeled according to the product instructions of a biotin labeling kit (purchased from Thermo). Yeast cells capable of specifically binding to c-MET were enriched by MACS, and finally, yeast cells capable of specifically binding to biotin-labeled c-MET protein with high affinity were obtained after multiple rounds of flow cytometry sorting. The final selected yeast templates were used to extract antibody heavy and light chain sequences, and these sequences were then constructed into expression vectors to prepare antibodies.

[0156] After preparing the antibodies, ForteBio affinity assay was performed according to the reported method (Estep, P et al., Determination of antibody-antigen affinity and epitope binding based on high-throughput methods. MAbs, 2013.5(2):p.270-8). The results showed that anti-c-MET antibodies 136 and 187 had good binding activity with human c-MET protein and monkey c-MET protein (Table 2, Table 3).

[0157] [Table 7]

[0158] [Table 8]

[0159] The construction of bispecific antibodies targeting cMet and EGFR was carried out according to the method described in the patent application (patent application no. 201611016435.0), where nucleotide sequences encoding the variable regions of anti-EGFR and anti-cMet antibodies were synthesized and ligated to constant regions capable of spontaneously forming heterodimers (sequences were derived from patent application PCT / CN2017 / 111310). Among these, three different anti-cMet antibodies were selected. The first anti-cMet antibody was the antibody disclosed in patent application WO 2011 / 110642A2 (whose sequence is shown in Table 1), which was used as a control antibody, and the second and third anti-cMet antibodies were antibodies 136 and 187 obtained from the above screening, whose sequences are shown in Table 1.

[0160] A nucleotide sequence encoding an anti-EGFR antibody heavy chain variable region (the amino acid sequence of which is set forth in SEQ ID NO: 1 and disclosed in patent application WO 02 / 100348A2) was synthesized by conventional methods, ligated to a nucleotide sequence encoding a heavy chain constant region sequence 1 (SEQ ID NO: 39) capable of spontaneously forming a heterodimer, and then EcoRI and XhoI restriction enzyme sites were added to both ends, followed by cloning into vector pCDNA3.1 (Genwiss) to obtain plasmid EGFR-HC-pCDNA3.1 (EGFR-HC-pCDNA3.1). The nucleotide sequence encoding the light chain variable region of an anti-EGFR antibody (whose amino acid sequence is set forth in SEQ ID NO: 3 and disclosed in patent application WO 02 / 100348 A2) was synthesized by conventional methods and ligated to a nucleotide sequence encoding a light chain constant region sequence (SEQ ID NO: 41). EcoRI and XhoI restriction enzyme sites were then added to both ends, and the plasmid was cloned into vector pCDNA3.1 to construct plasmid EGFR-LC-pCDNA3.1 (the amino acid sequence of EGFR-LC is set forth in SEQ ID NO: 4).

[0161] A nucleotide sequence encoding the anti-cMet control antibody heavy chain variable region (SEQ ID NO: 5, Patent Application WO 2011 / 110642A2) was synthesized by a conventional method, ligated to a nucleotide sequence encoding a heavy chain constant region sequence 2 (SEQ ID NO: 40) capable of spontaneously forming a heterodimer, and then EcoRI and XhoI restriction enzyme sites were added to both ends. The plasmid was cloned into the vector pCDNA3.1 to obtain the plasmid cMetGen-HC-pCDNA3.1 (cMet-2 The amino acid sequence of Fc is set forth in SEQ ID NO:6) was constructed, and a nucleotide sequence encoding an anti-cMet control antibody light chain variable region (SEQ ID NO:7, Patent Application WO 2011 / 110642A2) was synthesized by conventional methods and ligated to a nucleotide sequence encoding a light chain constant region sequence (SEQ ID NO:41), followed by adding EcoRI and XhoI restriction enzyme sites to both ends and cloning into the vector pCDNA3.1 to construct the plasmid cMetGen-LC-pCDNA3.1 (the amino acid sequence of cMet LC is set forth in SEQ ID NO:8).

[0162] A nucleotide sequence encoding an anti-cMet antibody heavy chain variable region (SEQ ID NO: 9) was synthesized by a conventional method and ligated to a nucleotide sequence encoding a heavy chain constant region sequence 2 (SEQ ID NO: 40) capable of spontaneously forming a heterodimer, followed by the addition of EcoRI and XhoI restriction enzyme sites to both ends and cloning into the vector pCDNA3.1 to construct the plasmid cMet136-HC-pCDNA3.1 (the amino acid sequence of 136-2 Fc is set forth in SEQ ID NO: 10). A nucleotide sequence encoding an anti-cMet antibody light chain variable region (SEQ ID NO: 11) was synthesized by a conventional method and ligated to a nucleotide sequence encoding a light chain constant region sequence (SEQ ID NO: 41), followed by the addition of EcoRI and XhoI restriction enzyme sites to both ends and cloning into the vector pCDNA3.1 to construct the plasmid cMet136-LC-pCDNA3.1 (the amino acid sequence of 136 LC is set forth in SEQ ID NO: 12).

[0163] A nucleotide sequence encoding an anti-cMet antibody heavy chain variable region (SEQ ID NO: 13) was synthesized by a conventional method and ligated to a nucleotide sequence encoding a heavy chain constant region sequence 2 (SEQ ID NO: 40) capable of spontaneously forming a heterodimer, followed by the addition of EcoRI and XhoI restriction enzyme sites to both ends and cloning into the vector pCDNA3.1 to construct the plasmid cMet187-HC-pCDNA3.1 (the amino acid sequence of 187-2Fc is set forth in SEQ ID NO: 14). A nucleotide sequence encoding an anti-cMet antibody light chain variable region (SEQ ID NO: 15) was synthesized by a conventional method and ligated to a nucleotide sequence encoding a light chain constant region sequence (SEQ ID NO: 41) to construct the plasmid cMet187-LC-pCDNA3.1 (the amino acid sequence of 187 LC is set forth in SEQ ID NO: 16).

[0164] The products of homologous recombination were transformed into Top10 competent cells, which were plated onto ampicillin-resistant plates and grown overnight at 37°C, and single colonies were picked and sequenced.

[0165] Cellular transfection and protein expression A normal host cell line (ExpiCHO-S cell F1) (Yida) and a non-fucosylated cell line by gene knockout (CHOS-ADP Fut8 KO) (self-constructed by BIOTHEUS; Fut8 knockout CHOS cells were obtained by knocking out the FUT8 allele in CHO cells) were used in a transient expression scheme by electroporation for gene transfer and expression of normal and non-fucosylated antibodies. The specific method was referred to the product manual. The supernatant was collected after 13 days of cell culture.

[0166] Protein purification The target protein was purified using a Protein A affinity chromatography column (MabSelect™ PrismA, GE Healthcare). The purification column was equilibrated with 5-10 column volumes of equilibration buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) until the conductivity and pH of the effluent did not change, and then loading was performed. After loading, the column was continuously rinsed with equilibration buffer until the UV value of the effluent no longer decreased. The sample was eluted using elution buffer (20 mM glycine-HCl, pH 2.7) and the effluent was collected. The eluate was neutralized with alkaline buffer (1 M Tris-HCl, pH 8.0).

[0167] After obtaining the monoclonal antibodies, they were concentrated and exchanged into PBS buffer to a final protein concentration of 5-10 mg / mL. Anti-cMet and anti-EGFR antibodies were mixed in a 1:1 molar ratio, and reduction was performed at 4 °C for 4 h by adding the reducing agent DTT. The solution was then changed to 20 mM sodium phosphate buffer (pH 6.0) to remove DTT. Fragments and aggregates were removed using a cation exchange method. Components with higher purity were collected and pooled, and the proteins were completely oxidized in air. The antibody Fc segments formed heterodimers, which resulted in five anti-EGFRxcMet bispecific antibody proteins, the structures of which are shown in Figure 1B. The antibodies obtained in normal host cells were named amivantamab analogs (control antibodies), EGFRxMET-136, and EGFRxMET-187. The antibodies obtained from the gene knockout nonfucosylated cell line were named EGFRxMET-187 Afu and amivantamab analog Afu.

[0168] Construction and preparation of single-arm control antibody The nucleotide sequence encoding the anti-EGFR antibody heavy chain variable region (SEQ ID NO: 1) and the nucleotide sequences of the three anti-cMet antibody heavy chain variable regions (SEQ ID NO: 5, 9, 13) were linked to the nucleotide sequence encoding the human IgG1-CH1-Fc (LALA mutation, knob mutation) segment (SEQ ID NO: 17) to obtain the nucleotide sequences of the anti-EGFR antibody heavy chain (SEQ ID NO: 42) and the three anti-cMet antibody heavy chains (SEQ ID NO: 43, 44, 45), which were assembled into EcoR I / Not I double-digested linearized pCDNA3.1 vector using homologous recombination (purchased from Vazyme). The nucleotide sequences encoding the four antibody light chains (SEQ ID NO: 4, 8, 12, 16) were assembled into EcoR I / Xhol I double-digested linearized pCDNA3.1 vector, and the process was carried out according to the product instructions. The homologous recombination product was introduced into Top10 competent cells, plated on ampicillin-resistant plates, and grown overnight at 37°C, and single clones were picked and sequenced, and the plasmid was extracted. The nucleotide sequence encoding Fc-LALA-hole (SEQ ID NO: 18) was constructed into EcoR I / Xhol I double-digested linearized pCDNA3.1 vector.

[0169] The three extracted plasmids of heavy chain (Fc-LALA-knob), light chain and Fc-LALA-hole were co-transfected into Expi-CHO cells to form a single Fab antibody structure (Figure 1A) using the ExpiCHO™ Expression System Kit (purchased from Thermo). The transfection method was performed according to the product instructions. After 5 days of cell culture, the supernatant was collected and the target protein was purified by Protein A magnetic bead (purchased from GenScript) selection. The magnetic beads were resuspended in an appropriate amount of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1-4 times the volume of the magnetic beads) and added to the sample to be purified, and incubated at room temperature for 1 hour with gentle shaking. 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 (0.1 M sodium citrate, pH 3.2) was added at 3-5 times the volume of magnetic beads, and the mixture was shaken at room temperature for 5-10 minutes. The mixture was returned to the magnetic stand, and the elution buffer was collected and transferred to a collection tube containing neutralization buffer (1 M Tris, pH 8.54) and mixed thoroughly. Four target proteins were obtained, which were named anti-EGFR single-arm antibody, anti-MET single-arm antibody, 136 single-arm antibody, and 187 single-arm antibody.

[0170] Blocking of HGF-c-MET signaling pathway by c-MET antibodies 136 and 187 HCC827 cells are human non-small cell lung cancer cells (purchased from Cell Bank of the Chinese Academy of Sciences), which highly express epidermal growth factor receptor EGFR (exon 19 deletion) and c-Met receptor. Treatment with the small molecule tyrosine kinase inhibitor (TKI) gefitinib (gefitinib, epidermal growth factor receptor tyrosine kinase inhibitor) induces apoptosis in HCC827 cells. Under such conditions, when HGF is added simultaneously, the c-Met pathway is activated, causing HCC827 to become resistant to gefitinib, thereby inhibiting apoptosis.

[0171] The cell density of HCC827 cells after expansion culture was 2 × 10 4 The diluted antibodies were adjusted to 1000 nM, HGF to 800 ng / mL, and gefitinib to 8 μM in 1640 medium. According to the experimental requirements, 50 μl / well of diluted antibodies, 25 μl / well of HGF, and 25 μl / well of gefitinib were added to the 96-well plate containing HCC827 cells, and supplemented with 1640 medium to make the total volume 200 μl / well. After 3 days of incubation at 37°C and 5% carbon dioxide, 100 μl of medium was removed, and then Cell titer glo (purchased from Promega) was added at 100 μl / well, and the chemiluminescence signal was collected by a microplate reader.

[0172] The results of this experiment are shown in Figure 2. The anti-c-MET antibodies 136 and 187 were able to restore the growth inhibitory effect of gefitinib on HCC827 cells by blocking the HGF-c-MET signaling pathway.

[0173] Example 2: Anti-EGFRxc-Met bispecific antibodies that block EGF-EGFR interaction H292 cells, human non-small cell lung cancer cells purchased from Procell, were capable of expressing EGFR and c-Met receptors. H292 cells were grown to an appropriate density, digested, detached from cell culture flasks, and cultured at 1 × 10 6 The cells were resuspended to 100 cells / ml and placed in a 96-well flow plate at 100 μL per well and centrifuged for later use. Antibodies to be tested were diluted 3-fold in PBS starting at 200 nM and the diluted samples were added to the 96-well flow plate with cells at 100 μL / well and incubated at 25° C. for 60 minutes. Biotin-h.EGF was diluted to 100 nM in PBS and added to the 96-well flow plate with cells at 100 μL / well and incubated at 25° C. for 60 minutes and then washed twice with PBS. Streptavidin-FITC (purchased from Jackson) was diluted 100-fold in PBS and added to the plate at 100 μL / well, incubated at 4° C. for 30 minutes and washed twice with PBS. PBS was added at 100 μL / well to resuspend the cells, and then the cells were detected with a CytoFlex (Beckman) flow cytometer and the corresponding MFI was calculated.

[0174] As shown in Figure 3, the candidate bispecific antibody molecules of the present application, EGFRxMET-136 and EGFRxMET-187, both blocked the interaction of EGF with EGFR expressed on H292 cells, with IC50 values ​​of 3.15 nM and 2.90 nM, respectively. As negative controls, anti-c-Met 136 single-arm antibody and anti-c-Met single-arm molecule constructed based on the control molecule amivantamab were unable to block the interaction of EGF with EGFR.

[0175] Example 3: Anti-EGFRxc-Met bispecific antibodies that block HGF-dependent TKI resistance In this experiment, the strength of the anti-EGFRxc-Met bispecific antibody of the present application in blocking HGF signaling was detected. Proliferated HCC827 cells (HCC827 cells are human non-small cell lung cancer cells purchased from the Cell Bank of the Chinese Academy of Sciences, and highly express epidermal growth factor receptor EGFR (exon 19 deletion) and c-Met receptor. Treatment with tyrosine kinase inhibitor TKI small molecule gefitinib (gefitinib, epidermal growth factor receptor tyrosine kinase inhibitor) will induce apoptosis in HCC827 cells. Under such conditions, if HGF is added simultaneously, the c-Met pathway will be activated, which will cause HCC827 to develop resistance to gefitinib and inhibit apoptosis) were cultured at a cell density of 2×10 4 The cells were adjusted to 100 μl / well and added to a 96-well cell culture plate at 100 μl / well and incubated overnight for later use. The antibodies to be tested were diluted to 300 nM in 1640 medium, then serially diluted 3-fold, HGF was diluted to 300 ng / mL, and gefitinib was diluted to 0.6 μM. According to the requirements of the experiment, 50 μl / well of the diluted antibodies, 25 μl / well of HGF, and 25 μl / well of gefitinib were added to the 96-well plate with cells, supplemented with 1640 medium to a total volume of 150 μl / well, and incubated at 37° C. and 5% carbon dioxide for 5 days. After 5 days, 100 μl of medium was removed, then Cell titer glo (purchased from Promega) was added at 100 μl / well, and the chemiluminescence signal was collected by a microplate reader.

[0176] As shown in Figure 4, the blocking potency of the EGFRxc-Met bispecific antibody molecules (EGFRxMET-136 and EGFRxMET-187) was significantly better than that of the anti-c-Met single-arm molecule, indicating that the blocking effect of the bispecific antibody molecules on HGF-c-Met signaling could be improved by binding to EGFR.

[0177] Example 4: Anti-EGFRxc-Met bispecific antibodies block HGF-induced cell proliferation In this experiment, we detected the blocking effect of anti-EGFRxc-Met bispecific antibody on cell proliferation induced by HGF. H596 cells were grown at 3 × 10 4 The cells were adjusted to a density of 100 cells / ml and added to a 96-well cell culture plate at 100 μl / well and cultured overnight for later use. The antibodies to be tested were diluted to 1200 nM in 1640 culture medium, then diluted 3-fold, and HGF was diluted to 200 ng / mL. According to the requirements of the experiment, 50 μl / well of the diluted antibodies and 50 μl / well of HGF were added to the 96-well plate containing cells, supplemented with 1640 culture medium to a total volume of 200 μl / well, and cultured at 37°C and 5% carbon dioxide for 5 days. After 5 days, 100 μl of culture medium was removed, then Cell titer glo (purchased from Promega) was added at 100 μl / well, and the chemiluminescence signal was collected by a microplate reader.

[0178] As shown in Figure 5, the blocking potency of the EGFRxc-Met bispecific antibody molecules EGFRxMET-136 and EGFRxMET-187 was significantly better than that of the anti-c-Met single-arm molecule, again indicating that binding to EGFR could improve the blocking effect of bispecific antibody molecules on HGF-c-Met signaling.

[0179] Example 5: Anti-EGFRxc-Met bispecific antibodies block HGF-induced cell migration HepG2 cells were human liver cancer cells purchased from ATCC and could express EGFR and c-Met receptors. When HGF was added to the culture medium in the lower chamber, the HepG2 cells in the upper chamber would be induced to migrate to the lower chamber through the filter membrane. The specific experimental method was as follows: HepG2 cells were grown to an appropriate density, and the cells were digested and detached from the cell culture flask, and 1 × 10 6The cells were resuspended to 100 cells / ml and added to the top chamber of a 24-well cell migration plate (purchased from Corning) at 100 μL per well for later use. Bispecific antibodies were diluted in MEM to 200 nM and added to the top chamber of a 24-well cell migration plate at 100 μL per well and incubated at 25°C for 60 min. h.HGF (purchased from R&D) was diluted in MEM to 50 ng / mL and added to the bottom chamber of a cell migration plate at 500 μL / well and cultured at 37°C and 5% carbon dioxide for 3 days. Non-migrated cells on the top chamber membrane were gently wiped off with a cotton swab, and cells that had migrated to the bottom chamber membrane were lysed with Cell titer glo (purchased from Promega), and chemiluminescence signals were collected with a microplate plate reader.

[0180] As shown in Figure 6, the EGFRxc-Met bispecific antibody molecules EGFRxMET-136 and EGFRxMET-187 almost completely blocked the migration of HepG2 cells induced by HGF. As a negative control, an anti-EGFR single-arm antibody prepared based on the control antibody amivantamab was unable to block the signal induced by HGF.

[0181] Example 6: Cell line screening In this example, the EGFRxMET-136 Afu molecule was constructed using CH1 / CL preferential mutation (patent application WO 2021 / 067404A2) and knob-in-hole technology, the heavy chain variable regions of the anti-cMet antibody and the anti-EGFR antibody were constructed into the CH1 mutant heavy chain constant region CH SET1 (sequence number 19) and CH SET2 (sequence number 20), respectively, and the light chain variable regions of the anti-cMet antibody and the anti-EGFR antibody were constructed into the CL mutant light chain constant region CL SET1 (sequence number 21) and CL SET2 (sequence number 22), respectively. A 1+1 asymmetric anti-EGFRxcMet bispecific antibody cell line was constructed.

[0182] Cell line construction Vector pCHO2.0-GS-Puro-H1-L1 containing nucleotide sequences encoding the heavy chain (SEQ ID NO:23) and light chain (SEQ ID NO:24) of anti-c-Met antibody and vector pCHO2.0-GS-Puro-H2-L2 containing nucleotide sequences encoding the heavy chain (SEQ ID NO:25) and light chain (SEQ ID NO:26) of anti-EGFR antibody were co-transfected into host cells CHOS-ADP Fut8 KO (a non-fucosylated cell line by gene knockout) by electroporation. The cells were screened by puromycin and MSX screening pressure to obtain high-yield minipools, and then rounds of limiting dilution and monoclonal identification were performed to obtain high-yield stable clonal cell lines. Clonal cell line 6F5 was finally determined to be a recombinant engineered cell line by comparing the growth conditions, protein expression, and physicochemical properties of the expressed proteins.

[0183] cell culture Dynamis AGT Medium was used as the base medium, and cells were cultured at (1.0±0.2)×10 6 The cells were cultured at an inoculation density of 1000 cells / ml. On the 3rd, 5th, 7th, 9th and 11th days of the culture, 5.0±0.5% (w / w) of 7a supplement medium based on the initial culture weight was added in a fed-batch manner, and 0.5±0.05% (w / w) of 7b supplement medium based on the initial culture weight was added in a fed-batch manner. The dissolved oxygen was set at 40%, and the initial culture temperature was 36.5°C, which was cooled to 33.0°C on the 4th day. According to the detection results of glucose concentration, 300 g / kg of glucose concentrate was supplemented every day, except for the harvest day, to make the glucose concentration in the cell solution 6.0 g / L. The culture was terminated on the 14th day or when the cell viability was less than 80%. During this culture process, the cell density and viability were detected using Vicell (Beckman). Starting from the 7th day, antibody production was detected every day using Cedex (Roche). The results are shown in Table 4. The expression of the bispecific antibody reached 2.432 g / L.

[0184] [Table 9]

[0185] Quality confirmation of stable cell line products The one-step purification method was the same as that of the single-arm antibody in Example 1. The quality of the obtained protein was detected by HPLC. The HPLC method was as follows: Mobile phase: 150 mM Na 2 HPO 4 12H 2 0, pH 7.0, chromatographic conditions: detection wavelength: 280 nm, column temperature: 25°C, flow rate: 0.5 ml / min, detection time: 30 min, TSKgel G3000SWXL column. The SEC results are shown in Figure 7A. The purity of the bispecific antibody obtained by one-step affinity purification was 98.18%.

[0186] The pairing of the heavy and light chains of the obtained protein was detected by high performance liquid chromatography mass spectrometry. The equipment used was a liquid phase system Vanquish UHPLC (Thermo), a mass spectrometer Q Exactive (Thermo) and a chromatography column Waters ACQUITY UPLC BEH C4, 2.1 mm x 100 mm. 50 μg of sample was taken and diluted to 25 μl by adding ultrapure water, centrifuged, then 20 μl of sample was taken and placed in an injection bottle, 5 μl of sample was injected and the complete molecular weight was analyzed using LC-MS. Chromatography conditions: column temperature: 80 °C; UV detection wavelength: 280 nm; flow rate: 0.3 mL / min; mobile phase A: aqueous solution (containing 0.1% formic acid); mobile phase B: acetonitrile solution (containing 0.1% formic acid). Mass spectrometry parameters: ESI ion source; ion transport tube temperature 320 °C, voltage 3.8 kV, gas flow rate 36 L / min; mode: positive ion full MS; resolution: 17500; scan range: 600-4000 m / z. The results are shown in Figure 7B, and the percentage of correctly paired products of the purified bispecific antibody was more than 98%.

[0187] In this example, a nonfucosylated bispecific antibody Final EGFRxMET-136 (designated EGFRxMET-136 Afu) was prepared, and this antibody molecule was used as the final molecule for subsequent in vivo and in vitro activity detection experiments.

[0188] Example 7: Binding activity of anti-EGFRxc-Met bispecific antibodies to EGFR and c-Met positive tumor cells A375 cells were human malignant melanoma cells purchased from Addexbio, capable of low expression of EGFR and c-Met receptor. H292 cells were human lung adenocarcinoma cells purchased from procell, capable of high expression of EGFR and c-Met receptor. HCC827 cells were human non-small cell lung cancer cells purchased from Cell Bank of the Chinese Academy of Sciences, capable of high expression of EGFR and c-Met receptor. The grown cells were adjusted to an appropriate cell density and added to a 96-well flow plate. After centrifugation, the samples to be tested were added in serial dilutions and incubated at 4°C for 1 hour. The cells were washed twice with PBS, then fluorescent secondary antibodies diluted to appropriate concentrations were added, incubated at 4°C for 30 minutes, and washed twice with PBS. The cells were resuspended in PBS and detected with a CytoFlex flow cytometer, and the corresponding MFI was calculated.

[0189] The results are shown in Figures 8A, 8B and 8C. For tumor cells with different expression levels of EGFR and MET, both EGFRxc-Met bispecific antibody candidate molecules, EGFRxMET-136 Afu and EGFRxMET-187 Afu, showed relatively high cell binding activity. For A375 cells, H292 cells and HCC827 cells, the binding activity EC50 values ​​of EGFRxMET-136 Afu were 1.35nM, 2.34nM and 3.67nM, respectively, and the binding activity EC50 values ​​of EGFRxMET-187 Afu were 1.05nM, 1.46nM and 2.89nM, respectively.

[0190] The EC50 values ​​of antibody binding strength are shown in Table 5.

[0191] [Table 10]

[0192] Example 8: Effect of anti-EGFRxc-Met bispecific antibodies in inducing ADCC A375 cells were human malignant melanoma cells purchased from Addexbio, which could express EGFR and c-Met receptors. H1975 cells were human lung adenocarcinoma cells purchased from Addexbio, which could highly express EGFR and c-Met receptors. HCC827 cells were human non-small cell lung carcinoma cells purchased from Cell Bank of the Chinese Academy of Sciences, which could highly express EGFR and c-Met receptors. The propagated A375 / H1975 / HCC827 cells were cultured at 1.2 × 10 6 The CD16a-NFAT-Luc Jurkat reporter gene cells (self-produced by BIOTHEUS; CD16a and NFAT-Luc gene sequences were constructed in pCDNA3.1 vector and then transfected into Jurkat cells, and CD16a-NFAT-Luc Jurkat reporter gene cells were obtained by antibiotic resistance pressure screening) were resuspended in 1640 medium at a cell density of 6 × 10 cells / ml. 6 100 cells / ml, the antibody was diluted to 300 nM in 1640 medium, then serially diluted 4-fold, 25 ul of the serially diluted antibody as described above was added to each well of a 96-well cell culture plate, 25 ul of the target cells as described above were added to each well, mixed, and incubated at room temperature for 30 minutes, then 25 ul of the effector cells as described above were added to each well, mixed, and incubated at 37° C., 5% CO for 6 hours. 2 Then 75ul of Bio-turbo reagent (purchased from Ruian) was added to each well, mixed and the chemiluminescence signal was collected by a microplate reader.

[0193] The results are shown in Figures 9A, 9B and 9C. The candidate EGFRxc-Met bispecific antibody molecules EGFRxMET-136 and EGFRxMET-187 and the control molecule amivantamab analogue all induced ADCC effects and showed a dependency on the antigen expression level (HCC827>H1975>A375). The non-fucosylated bispecific antibodies (EGFRxMET-136 Afu, amivantamab analogue Afu) showed a stronger ADCC effect compared to the wild-type antibodies containing fucose (EGFRxMET-136, amivantamab analogue), and this enhancement was especially evident in cells with relatively low antigen expression (A375 / H1975).

[0194] A luciferase stable transfected cell line (A375-luc) was constructed by transfecting A375 cells with a plasmid encoding luciferase cDNA (the sequence was from Uniprot, P08659, constructed by General Biol in pLVX-neu vector) using lentiviral transfection method. Target cells and PBMC cells were resuspended in 1640 medium and each was diluted at 2 × 10 per well. 4 cells and 2 x 10 5 Cells were seeded in a 96-well culture plate, and the antibodies were diluted to 300 nM in 1640 medium, then serially diluted 3-fold, 50 μl of which was taken and added to the 96-well plate, and finally supplemented with 1640 medium to a total volume of 150 μl / well. The cells were incubated at 37°C, 5% CO for 48 hours. 2 and 100 μl of Bio-turbo reagent (purchased from Ruian) was added to each well and mixed thoroughly, and then the chemiluminescence signal was collected using a microplate reader.

[0195] All candidate EGFRxc-Met bispecific antibody molecules induced PBMCs to kill target cells, as shown in Figure 10. Similarly, the non-fucosylated bispecific antibody showed a stronger ability to induce PBMCs to kill tumor cells compared to the fucose-containing wild-type antibody.

[0196] Example 9: Cell killing of anti-EGFRxc-Met bispecific antibodies combined with EGFR small molecule inhibitors Using lentiviral transfection, cDNAs encoding luciferase (the sequence was from Uniprot, P08659) and human HGF (hepatocyte growth factor) (the sequence was from UniProtKB P14210) were constructed on pLVX-neu vector by General Biol, and this plasmid was transfected into HCC827 cells to construct a cell line (HCC827-HGF luc) stably co-expressing luciferase and HGF. Since overexpression of HGF makes HCC827 cells resistant to first generation EGFR small molecule inhibitors, an experiment was designed to combine antibodies with first generation small molecules. The grown luciferase stable transfected cell line (HCC827-HGF Luc) was digested, centrifuged, counted, and resuspended in working medium (10% FBS+RPMI 1640). Cells were cultured at 3×10 4 The cells were adjusted to a density of 1.8 × 10 cells / ml and added to the culture plate at 100 μL per well. PBMC cells were resuscitated from liquid nitrogen, centrifuged, then resuspended in working medium and added to culture flasks for overnight adherent culture to remove mononuclear cells. After overnight culture, the concentration of suspended immune cells was adjusted to 1.8 × 10 cells / ml for later use. 5 The antibody was adjusted to 800 nM in working medium, and then serially diluted 3-fold to prepare a solution having a small molecule gefitinib at a concentration of 0.8 μM and a small molecule erlotinib at a concentration of 8 μM using working medium. 50 μL of the PBMC cell suspension, 25 μL of the serially diluted antibody and 25 μL of the gefitinib / erlotinib solution were added to the corresponding experimental wells on the culture plate containing HCC827-HGF luc cells according to the requirements of the experiment. Among these, the experimental wells to which only a part of the components were added were filled with working medium to a final volume of 200 μL per well, and after thorough mixing, the plate was incubated at 37 °C for 48 hours in CO 2After culturing in an incubator, the cell culture plate was taken out, 100 μL of culture medium supernatant was removed, and then 100 μL of Bio-turbo reagent (purchased from Ruian) was added to each well, and the fluorescence signal value was read using a SpectraMAX microplate reader.

[0197] In this example, EGFRxMET-136 Afu molecule was used in combination with small molecule EGFR inhibitors (gefitinib on the left and erlotinib on the right) and PBMCs were added simultaneously to simulate tumor cell killing by immune cells. The results are shown in Figure 11. Without the addition of PBMCs, EGFRxMET-136 Afu molecule blocked the HGF-MET signaling pathway, alleviated the resistance of HGF-dependent tumor cells to small molecule inhibitors, and promoted tumor killing by small molecule inhibitors. More importantly, after the addition of PBMCs, these combinations showed synergistic effects and could kill tumor cells more effectively compared with the use of EGFRxMET-136 Afu or small molecule inhibitors alone.

[0198] Example 10: Anti-EGFRxc-Met Bispecific Antibody Inhibits Tumor Growth In Vivo To study the in vivo efficacy of EGFRxMET-136 Afu and EGFRxMET-187 Afu bispecific molecules, in this example, five different animal experimental models were designed for validation.

[0199] Experimental model 1 In this experiment, CB-17 SCID mice were subcutaneously inoculated with H292 human lung cancer tumor cells to establish a tumor-bearing model and determine the antitumor effect of anti-EGFRxc-Met bispecific antibodies. Sufficient H292 cells were cultured and expanded in vitro, and the cells were harvested after trypsin digestion. After washing three times with PBS, the cells were counted and 3 × 10 6The mice were subcutaneously inoculated with 10 ...

[0200] [Table 11]

[0201] The tumor volume and body weight of the mice were measured 2 to 3 times a week. The body weight and tumor volume of the mice were measured until the 29th day after tumor cell inoculation, and then the mice were euthanized.

[0202] The results are shown in Figure 12. Compared with the PBS group, all three bispecific antibody groups showed significant inhibitory effects on tumor growth, and among these, the EGFRxMET-136 Afu bispecific antibody group was significantly more effective than the amivantamab analog Afu. The tumor (volume) inhibition rate (TGI) corresponding to each group was calculated, and among these, the TGI of the EGFRxMET-136 Afu molecule group was 97.3%, the TGI of the EGFRxMET-187 Afu molecule group was 81.0%, and the TGI of the amivantamab analog Afu molecule group was 74.5% (the TGI of EGFRxMET-136 Afu was significantly different from the TGI of the amivantamab analog Afu).

[0203] Experimental model 2 In this experiment, CB-17 SCID mice were subcutaneously inoculated with H1975 human lung cancer tumor cells to establish a tumor-bearing model and determine the antitumor effect of anti-EGFRxc-Met bispecific antibodies. Sufficient H1975 cells were cultured and expanded in vitro, and the cells were harvested after trypsin digestion. After washing three times with PBS, the cells were counted and found to be 3 × 10 6 Cells / mouse were subcutaneously inoculated into the right flank of female 8-week-old CB-17 SCID mice (purchased from Beijing Weitong Lihua). Subcutaneous tumor formation of tumor cells in mice was observed daily. The maximum width axis W and maximum length axis L of the subcutaneous tumor in the right flank of each animal were measured with a vernier caliper, and the body weight of each mouse was weighed with an electronic balance. The subcutaneous tumor volume in the right flank of each mouse was calculated according to the following formula: tumor volume T = 1 / 2 × W × W × L. Mice with excessively large and small tumor volumes were excluded, and the remaining mice were divided into 4 groups according to the average tumor volume (6 mice per group). Mice were divided into groups according to the grouping and administration scheme in Table 7, and the corresponding doses of antibodies were injected.

[0204] [Table 12]

[0205] The tumor volume and body weight of the mice were measured 2 to 3 times a week. The body weight and tumor volume of the mice were measured until the 24th day after tumor cell inoculation, and then the mice were euthanized.

[0206] The results are shown in Figure 13. Compared with the PBS group, the three bispecific antibody groups with different doses (2 mg / kg, 8 mg / kg, 32 mg / kg) all showed significant inhibitory effects on tumor growth. Among these, the TGI of the 2 mg / kg treatment group was 97.4%, the TGI of the 8 mg / kg treatment group was 99.3%, and the TGI of the 32 mg / kg treatment group was 99.4%. It was found that the bispecific antibody of the present application almost completely inhibited tumor growth, with the tumor inhibition rate reaching more than 99%.

[0207] Experimental model 3 In this experiment, CB-17 SCID mice were subcutaneously inoculated with H292 human lung cancer tumor cells to establish a tumor-bearing model and compare the anti-tumor effects of different doses of anti-EGFRxc-Met bispecific antibodies. The establishment process of the mouse model was the same as that of experimental model 1. The groups were divided according to the grouping and administration scheme in Table 8, and the corresponding doses of antibodies were injected.

[0208] [Table 13]

[0209] The tumor volume and body weight of the mice were measured 2 to 3 times a week. The body weight and tumor volume of the mice were measured until day 30 after tumor cell inoculation, and then the mice were euthanized.

[0210] The results are shown in Figure 14. Compared with the PBS group, the EGFRxMET-136 Afu molecule showed a significant effect of inhibiting tumor growth. Compared with the 4mg / kg group, the 16mg / kg group had a stronger tumor inhibition effect, among which the EGFRxMET-136 Afu molecule 4mg / kg group showed TGI=40.3%, and the EGFRxMET-136 Afu molecule 16mg / kg group showed TGI=90.5%.

[0211] Experimental model 4 In this experiment, CB-17 SCID mice were subcutaneously inoculated with H292 human lung cancer tumor cells to establish a tumor-bearing model, and the anti-EGFRxc-Met bispecific antibody was compared with the commercial EGFRxc-Met bispecific antibody Rybrevant (purchased from Johnson & Johnson, batch number: LHS3G02) (this antibody has been presented at various oncology conferences and was also known as JNJ-6372) in terms of anti-tumor effect. The establishment process of the mouse model was the same as that of experimental model 1. The groups were divided according to the group division and administration scheme in Table 9, and the corresponding doses of the antibodies were injected.

[0212] [Table 14]

[0213] The tumor volume and body weight of the mice were measured 2 to 3 times a week. The body weight and tumor volume of the mice were measured until the 27th day after tumor cell inoculation, and then the mice were euthanized.

[0214] The results are shown in Figure 15. Compared with the PBS group, the EGFRxMET-136 Afu group had a significantly stronger tumor inhibitory effect than that of the Rybrevant group. Among these, the TGI of the EGFRxMET-136 Afu molecule group was 79.9%, and the TGI of the Rybrevant molecule group was 67.9% (these two TGI results were significantly different).

[0215] Experimental model 5 In this experiment, the in vivo efficacy of the bispecific antibody of the present application combined with a small molecule EGFR inhibitor (e.g., osimertinib) was detected. Using lentiviral gene transfer method, cDNA encoding human HGF (hepatocyte growth factor) (the sequence was derived from UniProtKB P14210) was constructed on pLVX-neu vector by General Biol, and this plasmid was transfected into H1975 cells to construct a cell line (H975-HGF) stably expressing HGF. In this experiment, CB-17 SCID mice were subcutaneously inoculated with H1975-HGF human lung cancer cells to establish a tumor-bearing model and determine the anti-tumor effect of anti-EGFRxc-Met bispecific antibody. The construction process of this model was the same as that of experimental model 2. Mice were divided into groups according to the grouping and administration scheme in Table 10, and the corresponding doses of antibodies were injected.

[0216] [Table 15]

[0217] The tumor volume and body weight of the mice were measured 2 to 3 times a week. The body weight and tumor volume of the mice were measured until the 28th day after tumor cell inoculation, and then the mice were euthanized.

[0218] The results are shown in Figure 16. Compared with the PBS group, each experimental group showed a significant antitumor effect, and EGFRxMET-136 Afu combined with a small molecule inhibitor had a stronger antitumor activity than the bispecific antibody alone or small molecule alone group, and was significantly stronger than other treatment groups. At the same time, the EGFRxMET-136 Afu group combined with a small molecule had a stronger tumor inhibitory effect than the Rybrevant group combined with a small molecule. Among these, the osimertinib treatment group showed TGI = 38.9%, the Rybrevant treatment group showed TGI = 44.1%, the EGFRxMET-136 Afu treatment group showed TGI = 72.3%, the Rybrevant group combined with a small molecule showed TGI = 78.7%, and the EGFRxMET-136 Afu group combined with a small molecule showed TGI = 89.8%.

[0219] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that, based on all the teachings disclosed, various modifications and changes can be made to these details, and these modifications are within the scope of protection of the present invention. The whole of the present invention is indicated by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to c-Met, comprising a heavy chain variable region (VH) and a light chain variable region (VL), (1) The VH comprises a VH CDR1 set forth in SEQ ID NO: 27, a VH CDR2 set forth in SEQ ID NO: 28, and a VH CDR3 set forth in SEQ ID NO: 29, and the VL comprises a VL CDR1 set forth in SEQ ID NO: 30, a VL CDR2 set forth in SEQ ID NO: 31, and a VL CDR3 set forth in SEQ ID NO: 32; or (2) The antibody or antigen-binding fragment thereof, wherein the VH comprises a VH CDR1 shown in SEQ ID NO: 33, a VH CDR2 shown in SEQ ID NO: 34, and a VH CDR3 shown in SEQ ID NO: 35, and the VL comprises a VL CDR1 shown in SEQ ID NO: 36, a VL CDR2 shown in SEQ ID NO: 37, and a VL CDR3 shown in SEQ ID NO:

38.

2. The antibody or antigen-binding fragment thereof (1) VH having a sequence shown in SEQ ID NO: 9 or a sequence having at least 80% sequence identity thereto, and VL having a sequence shown in SEQ ID NO: 11 or a sequence having at least 80% sequence identity thereto; (2) VH having the sequence shown in SEQ ID NO: 13 or a sequence having at least 80% sequence identity thereto, and VL having the sequence shown in SEQ ID NO: 15 or a sequence having at least 80% sequence identity thereto. The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. The following: (i) the antibody or antigen-binding fragment thereof further comprises a human immunoglobulin framework region; (ii) the heavy chain of the antibody or antigen-binding fragment thereof comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region derived from a human immunoglobulin; (iii) the heavy chain constant region has the sequence set forth in SEQ ID NO: 19, 20, 39, or 40; (iii) the light chain of the antibody or antigen-binding fragment thereof comprises a kappa or lambda light chain constant region derived from a human immunoglobulin; and (iii) the light chain constant region has the sequence set forth in SEQ ID NO: 21, 22, or 41.

2. The antibody or antigen-binding fragment thereof of claim 1, characterized by one or more of the following:

4. The following: (i) the antibody or antigen-binding fragment thereof comprises a mutated or chemically modified Fc region; (ii) the antibody or antigen-binding fragment thereof comprises an Fc region with a LALA mutation and / or a knob-into-hole modification; (iii) the Fc region has the amino acid sequence set forth in SEQ ID NO: 17 or 18; (iii) the antibody or antigen-binding fragment thereof is hypofucosylated or nonfucosylated; and (iiiiii) the antibody or antigen-binding fragment thereof (1) a heavy chain having the sequence shown in SEQ ID NO: 10 and a light chain having the sequence shown in SEQ ID NO: 12; (2) a heavy chain having the sequence shown in SEQ ID NO: 14 and a light chain having the sequence shown in SEQ ID NO: 16; or (3) A heavy chain having the sequence shown in SEQ ID NO: 23 and a light chain having the sequence shown in SEQ ID NO:

24.

2. The antibody or antigen-binding fragment thereof of claim 1, characterized by one or more of the following:

5. The antigen-binding fragment may be Fab, Fab', (Fab') ２ , Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb), and / or said antibody is a murine antibody, a chimeric antibody, a humanized antibody or a multispecific antibody. The antibody or antigen-binding fragment thereof of claim 1.

6. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of claim 1.

7. A vector comprising the nucleic acid molecule of claim 6.

8. 10. A host cell comprising the nucleic acid molecule of claim 6 or a vector comprising said isolated nucleic acid molecule, Optionally, a host cell that has low or no fucosylation activity.

9. 2. A method for preparing the antibody or antigen-binding fragment thereof described in claim 1, comprising culturing a host cell containing an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cell.

10. A multispecific molecule comprising the antibody or antigen-binding fragment thereof of claim 1, Preferably, a multispecific molecule that specifically binds to c-Met and additionally specifically binds to one or more other targets.

11. A bispecific molecule, Preferably, the bispecific molecule further comprises a second antibody having a second binding specificity for a second target; 11. The multispecific molecule of claim 10, wherein the second target is epidermal growth factor receptor (EGFR) and the second antibody is an anti-EGFR antibody or an antigen-binding fragment thereof.

12. The following: (i) the bispecific molecule has been modified by glycosylation such that it has a lower number of fucose cocoates than the same bispecific molecule that has not been modified by glycosylation; (ii) the anti-EGFR antibody or antigen-binding fragment thereof is hypofucosylated or non-fucosylated; (iii) the antibody or antigen-binding fragment thereof is hypofucosylated or nonfucosylated; (iii) the second antibody comprises the following three heavy chain CDRs: a VH CDR1 set forth in SEQ ID NO: 46, a VH CDR2 set forth in SEQ ID NO: 47, and a VH CDR3 set forth in SEQ ID NO: 48, and the following three light chain CDRs: a VL CDR1 set forth in SEQ ID NO: 49, a VL CDR2 set forth in SEQ ID NO: 50, and a VL CDR3 set forth in SEQ ID NO: 51; (iii) the second antibody is (1) A VH having the sequence shown in SEQ ID NO: 1 and a VL having the sequence shown in SEQ ID NO: 3, or (2) VH having the sequence set forth in SEQ ID NO: 52 and VL having the sequence set forth in SEQ ID NO: 53; and (iiiiii) the second antibody is (1) A heavy chain having the sequence shown in SEQ ID NO: 2 and a nucleic acid sequence shown in SEQ ID NO: 4 light chain, or (2) A heavy chain having the sequence shown in SEQ ID NO: 25 and a light chain having the sequence shown in SEQ ID NO:

26.

12. The multispecific molecule of claim 11, characterized by one or more of the following:

13. The bispecific molecule comprising: (1) A first antibody comprising VH CDR1-3 shown in SEQ ID NOs: 27-29 and VL CDR1-3 shown in SEQ ID NOs: 30-32, and a second antibody comprising VH CDR1-3 shown in SEQ ID NOs: 46-48 and VL CDR1-3 shown in SEQ ID NOs: 49-51, or (2) A first antibody comprising VH CDR1-3 shown in SEQ ID NOs: 33-35 and VL CDR1-3 shown in SEQ ID NOs: 36-38, and a second antibody comprising VH CDR1-3 shown in SEQ ID NOs: 46-48 and VL CDR1-3 shown in SEQ ID NOs: 49-51. Including, Preferably, the multispecific molecule comprises: (1) A first antibody comprising a VH having the sequence shown in SEQ ID NO: 9 and a VL having the sequence shown in SEQ ID NO: 11, and a second antibody comprising a VH having the sequence shown in SEQ ID NO: 1 and a VL having the sequence shown in SEQ ID NO: 3; (2) A first antibody comprising a VH having the sequence shown in SEQ ID NO: 9 and a VL having the sequence shown in SEQ ID NO: 11, and a second antibody comprising a VH having the sequence shown in SEQ ID NO: 52 and a VL having the sequence shown in SEQ ID NO: 53; (3) A first antibody comprising a VH having the sequence shown in SEQ ID NO: 13 and a VL having the sequence shown in SEQ ID NO: 15, and a second antibody comprising a VH having the sequence shown in SEQ ID NO: 1 and a VL having the sequence shown in SEQ ID NO: 3; or (4) A first antibody comprising a VH having the sequence shown in SEQ ID NO: 13 and a VL having the sequence shown in SEQ ID NO: 15, and a second antibody comprising a VH having the sequence shown in SEQ ID NO: 52 and a VL having the sequence shown in SEQ ID NO:

53. Including, Preferably, the multispecific molecule comprises: (1) A first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 10 and a light chain having the sequence shown in SEQ ID NO: 12, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 25 and a light chain having the sequence shown in SEQ ID NO: 26; (2) A first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 10 and a light chain having the sequence shown in SEQ ID NO: 12, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 2 and a light chain having the sequence shown in SEQ ID NO: 4; (3) A first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 14 and a light chain having the sequence shown in SEQ ID NO: 16, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 25 and a light chain having the sequence shown in SEQ ID NO: 26; (4) A first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 14 and a light chain having the sequence shown in SEQ ID NO: 16, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 2 and a light chain having the sequence shown in SEQ ID NO: 4, or (5) A first antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 23 and a light chain having the sequence shown in SEQ ID NO: 24, and a second antibody comprising a heavy chain having the sequence shown in SEQ ID NO: 25 and a light chain having the sequence shown in SEQ ID NO:

26.

12. The multispecific molecule of claim 11 , comprising:

14. 10. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of claim 1, or a bispecific or multispecific molecule comprising said antibody or antigen-binding fragment thereof, and a therapeutic agent linked to said antibody or antigen-binding fragment thereof or said multispecific molecule.

15. The following: (i) the therapeutic agent is selected from a cytotoxic agent; (ii) the therapeutic agent is selected from the group consisting of alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof; and (iii) the immunoconjugate is an antibody-drug conjugate (ADC).

15. The immunoconjugate of claim 14, characterized by one or more of:

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim 1 or a bispecific or multispecific molecule comprising said antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and / or excipient.

17. The following: (i) further comprising an additional pharmaceutically active agent; (ii) further comprising an EGFR inhibitor; Preferably, the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, osimertinib, or any combination thereof, and preferably, the EGFR inhibitor and the antibody or antigen-binding fragment thereof, or the multispecific molecule are contained as active ingredients in different preparations and are administered simultaneously or at different times; and (iii) The pharmaceutical composition further comprises a drug having antitumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide drug, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

17. The pharmaceutical composition of claim 16, characterized by one or more of:

18. A kit comprising the antibody or antigen-binding fragment thereof of claim 1.

19. The following: (i) the antibody or antigen-binding fragment thereof comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material), or biotin; (ii) the kit further comprises a second antibody that specifically recognizes the anti-EGFR antibody or antigen-binding fragment thereof; (iii) the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material), or biotin; and (iii) the antibody or antigen-binding fragment thereof is hypofucosylated or nonfucosylated.

19. The kit of claim 18, characterized by one or more of the following:

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

21. The following: (i) the antigen-binding domain comprises a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment thereof; and (ii) the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell).

21. The chimeric antigen receptor of claim 20, characterized by one or more of:

22. 1. A method of inhibiting the growth of and / or killing tumor cells that express c-Met, comprising treating said tumor cells with an effective amount of: The antibody or antigen-binding fragment thereof according to claim 1, or a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof; or an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a therapeutic agent linked to the antibody or antigen-binding fragment thereof; or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, said bispecific or multispecific molecule, or said immunoconjugate; or A chimeric antigen receptor comprising the antigen-binding domain of the antibody or its antigen-binding fragment. The method comprises contacting the subject with any of the following: (1) Increasing immune cell activity in a subject in vitro or in vivo; (2) enhancing an immune response in a subject; (3) preventing and / or treating tumors in a subject; or (4) Preventing and / or treating infection in a subject 10. The antibody or antigen-binding fragment thereof of claim 1, or a bispecific or multispecific molecule comprising said antibody or antigen-binding fragment thereof, or an immunoconjugate comprising said antibody or antigen-binding fragment thereof and a therapeutic agent linked to said antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, said bispecific or multispecific molecule, or said immunoconjugate, or a chimeric antigen receptor comprising the antigen-binding domain of said antibody or antigen-binding fragment thereof, for use in

24. The following: (i) the tumor expresses c-Met; (ii) the tumor is associated with tumor cells that have c-Met expressed on the cell surface; (iii) the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies; (iii) the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection; and (iii) the subject is a mammal, e.g., a human, a cynomolgus monkey, or a mouse.

24. The use according to claim 23, characterized by one or more of the following:

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

26. The following: (i) the antibody or antigen-binding fragment thereof comprises a detectable label; (ii) the c-Met is human c-Met; and (iii) the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies.

26. The method of claim 25, characterized by one or more of:

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