multispecific antibodies targeting c-Met

Abstract

Multispecific antibodies targeting c-Met are provided. Specifically, multispecific antibodies, nucleic acids encoding them, vectors containing said nucleic acids, cells containing said vectors, and drug compositions containing multispecific antibodies are provided, along with their use in the treatment of related diseases.

Description

[Technical Field]

[0001] This disclosure relates to multispecific antibodies, and more particularly to multispecific antibodies targeting EGFR and c-Met. This disclosure also relates to methods for preparing said multispecific antibodies and their use. [Background technology]

[0002] The epidermal growth factor receptor (EGFR; sometimes called ErbB1 or HER1) is a 170 kDa type I transmembrane glycoprotein encoded by the proto-oncogene c-erbB1 and belongs to the receptor tyrosine kinase (RTK) family. EGFR is a member of the human epidermal growth factor receptor (HER) family, which includes HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Increased EGFR expression or kinase activity is associated with a range of human tumors, making EGFR an attractive target in tumor therapy.

[0003] The c-Mesenchymal-epithelial transition factor (c-Met) is a type of receptor tyrosine kinase and is an approximately 190 kDa heterodimer formed by the linkage of a 50 kDa extracellular chain (α chain) and a 145 kDa transmembrane chain (β chain). The transmembrane chain (β chain) contains a SEMA domain (sema homology region, SEMA), a PSI domain (plexin semaphorin-integrin, PSI), four immunoglobulin-like repeat domains (immunoglobulin-like regions in plexins and transcription factors, IPT), one transmembrane domain, one juxtamembrane domain (JM), a tyrosine kinase domain (tyrosine kinase, TK), and one carboxyl-terminal tail region (Carboxyl terminal, CT). c-Met is a receptor expressed on the cell surface, and its SEMA domain is one of the important elements for ligand binding and is considered to be the binding site of hepatocyte growth factor (HGF) as its ligand. HGF is synthesized by mesenchymal cells, fibroblasts, and smooth muscle cells, and the HGF / c-Met signal is activated by a paracrine mechanism to exert its biological function.

[0004] In various tumors, due to the overexpression of c-Met and HGF, the paracrine and autocrine positive feedback loops formed by HGF and c-Met, the HGF / c-Met signaling pathway is abnormally activated, promoting the proliferation, invasion, migration, and angiogenesis of tumor cells. Abnormal expression of the c-Met gene also exists in many cancer types, such as brain cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, liver cancer, etc. According to immunotherapeutic agents such as antibodies that bind to c-Met, the binding of HGF and c-Met can be inhibited.

[0005] In the IgG antibodies in camelids, in addition to the conventional IgG1 with a four-chain structure, heavy-chain-only antibodies (HcAb) IgG2 and IgG3 without light chains also naturally exist. The single variable domain (V H H) of the heavy-chain-only antibody can specifically bind to an antigen and has a relatively high affinity for the antigen. Due to its uniqueness, when the V H H domain is used as part of an antibody or antigen-binding fragment, it has significant advantages over conventional antibody fragments (such as scFv, Fab, etc.). For example, it only needs to specifically bind to an antigen with high affinity with a single domain, can be easily modified into multivalent and multispecific forms, the V H H domain is highly soluble and has no tendency to aggregate, the V H H molecule is small and thus has relatively high tissue permeability, and in V H H, there is no problem of light-chain and heavy-chain mismatch when constructing bispecific or multispecific antibodies, etc.

[0006] In all tumor patients resistant to EGFR tyrosine kinase inhibitors, about 60% of the patients have increased c-Met expression, c-Met amplification or increased HGF, indicating that when EGFR is inhibited, c-Met is activated as a compensatory pathway.

[0007] Therefore, there is a need to develop a multispecific antibody targeting EGFR and c-Met.

Summary of the Invention

[0008] The present disclosure provides a multispecific antibody targeting EGFR and c-Met. In addition, related nucleic acids, vectors, cells, pharmaceutical compositions, preparation methods and their uses capable of encoding the provided antibody are also provided.

[0009] According to one aspect of the present disclosure, a multispecific antibody is provided, and such a multispecific antibody (i) a first antigen-binding module that binds to a first antigen, (ii) A second antigen-binding module that binds to the second antigen, and (iii) comprising a third antigen-binding module that binds to the first antigen,

[0010] The first antigen is c-Met, the second antigen is EGFR, and both the first antigen-binding module and the third antigen-binding module are single variable domains, each independently, (1) CDR1 containing the amino acid sequence represented by SEQ ID NO:1, CDR2 containing the amino acid sequence represented by SEQ ID NO:2, and CDR3 containing the amino acid sequence represented by SEQ ID NO:3, (2) CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, (4) CDR1 containing the amino acid sequence represented by SEQ ID NO:10, CDR2 containing the amino acid sequence represented by SEQ ID NO:11, and CDR3 containing the amino acid sequence represented by SEQ ID NO:12. (5) CDR1 containing the amino acid sequence represented by SEQ ID NO:13, CDR2 containing the amino acid sequence represented by SEQ ID NO:14, and CDR3 containing the amino acid sequence represented by SEQ ID NO:15. (6) CDR1 containing the amino acid sequence represented by SEQ ID NO:16, CDR2 containing the amino acid sequence represented by SEQ ID NO:17, and CDR3 containing the amino acid sequence represented by SEQ ID NO:18, (7) CDR1 containing the amino acid sequence represented by SEQ ID NO:19, CDR2 containing the amino acid sequence represented by SEQ ID NO:20, and CDR3 containing the amino acid sequence represented by SEQ ID NO:21, or (8) Containing one of the following: CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0011] In some embodiments, the first antigen-binding module and the third antigen-binding module are each independently, (2) CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, or (8) Containing one of the following: CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0012] In some embodiments, the first antigen-binding module and the third antigen-binding module are each independently, (2) CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6, or (3) It contains one of the following: CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9.

[0013] In some embodiments, the first antigen-binding module and the third antigen-binding module are each independently, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, or (8) Containing one of the following: CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0014] In some embodiments, the first antigen-binding module and the third antigen-binding module each independently include CDR1, CDR2, and CDR3, which are single variable domains whose amino acid sequences are represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39. In some embodiments, the first antigen-binding module and the third antigen-binding module each independently include CDR1, CDR2, and CDR3, which are single variable domains whose amino acid sequences are represented by SEQ ID NO: 35, 36, or 38.

[0015] In some embodiments, the first antigen-binding module and the third antigen-binding module each independently include CDR1, CDR2, and CDR3, which are single variable domains whose amino acid sequences are represented by SEQ ID NO: 35 or 36. In some other embodiments, the first antigen-binding module and the third antigen-binding module each independently include CDR1, CDR2, and CDR3, which are single variable domains whose amino acid sequences are represented by SEQ ID NO: 36 or 38.

[0016] In some specific embodiments, the first antigen-binding module comprises CDR1, CDR2, and CDR3, single variable domains whose amino acid sequence is represented by SEQ ID NO:36, and the third antigen-binding module comprises CDR1, CDR2, and CDR3, single variable domains whose amino acid sequence is represented by SEQ ID NO:35. In some other specific embodiments, the first antigen-binding module comprises CDR1, CDR2, and CDR3, single variable domains whose amino acid sequence is represented by SEQ ID NO:36, and the third antigen-binding module comprises CDR1, CDR2, and CDR3, single variable domains whose amino acid sequence is represented by SEQ ID NO:38.

[0017] In some embodiments, the first antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:7, CDR2 containing an amino acid sequence represented by SEQ ID NO:8, and CDR3 containing an amino acid sequence represented by SEQ ID NO:9, and the third antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:4, CDR2 containing an amino acid sequence represented by SEQ ID NO:5 (where X1 is S or T), and CDR3 containing an amino acid sequence represented by SEQ ID NO:6. Furthermore, in some embodiments, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:41, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:40. In some embodiments, the first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:35. In some specific embodiments, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:35.

[0018] In some other embodiments, the first antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:7, CDR2 containing an amino acid sequence represented by SEQ ID NO:8, and CDR3 containing an amino acid sequence represented by SEQ ID NO:9, and the third antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:22, CDR2 containing an amino acid sequence represented by SEQ ID NO:23, and CDR3 containing an amino acid sequence represented by SEQ ID NO:24. Furthermore, in some embodiments, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:41, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:42. In some embodiments, the first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:38. In some specific embodiments, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:38.

[0019] In some embodiments, the first antigen-binding module and the third antigen-binding module bind to different epitopes of c-Met.

[0020] The second antigen-binding module provides the ability to bind to EGFR, and in some embodiments, the second antigen-binding module includes a heavy chain variable region and a light chain variable region, the heavy chain variable region including HCDR1 containing an amino acid sequence represented by SEQ ID NO:63, HCDR2 containing an amino acid sequence represented by SEQ ID NO:64, and HCDR3 containing an amino acid sequence represented by SEQ ID NO:65, and the light chain variable region including LCDR1 containing an amino acid sequence represented by SEQ ID NO:66, LCDR2 containing an amino acid sequence represented by SEQ ID NO:67, and LCDR3 containing an amino acid sequence represented by SEQ ID NO:68. Furthermore, in some embodiments, the second antigen-binding module includes a heavy chain variable region of an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:69, and a light chain variable region of an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:70. In some specific embodiments, the second antigen-binding module includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes an amino acid sequence represented by SEQ ID NO:69 and the light chain variable region includes an amino acid sequence represented by SEQ ID NO:70.

[0021] In some embodiments, the multispecific antibody further comprises an Fc region composed of two Fc polypeptides.

[0022] In some specific embodiments, in a multispecific antibody, the first antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:7, CDR2 containing an amino acid sequence represented by SEQ ID NO:8, and CDR3 containing an amino acid sequence represented by SEQ ID NO:9; the third antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:4, CDR2 containing an amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 containing an amino acid sequence represented by SEQ ID NO:6; furthermore, the second antigen-binding module includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes HCDR1 containing an amino acid sequence represented by SEQ ID NO:63, HCDR2 containing an amino acid sequence represented by SEQ ID NO:64, and HCDR3 containing an amino acid sequence represented by SEQ ID NO:65; the light chain variable region includes LCDR1 containing an amino acid sequence represented by SEQ ID NO:66, LCDR2 containing an amino acid sequence represented by SEQ ID NO:67, and SEQ ID The LCDR3 includes an amino acid sequence represented by NO:68. Furthermore, in a specific embodiment, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:41, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:40, and the heavy chain variable region of the second antigen-binding module includes an amino acid sequence represented by SEQ ID NO:69, and its light chain variable region includes an amino acid sequence represented by SEQ ID NO:70. Furthermore, in a specific embodiment, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:35, and the heavy chain variable region of the second antigen-binding module includes an amino acid sequence represented by SEQ ID NO:69, and its light chain variable region includes an amino acid sequence represented by SEQ ID NO:70.

[0023] In several other specific embodiments, in a multispecific antibody, the first antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:7, CDR2 containing an amino acid sequence represented by SEQ ID NO:8, and CDR3 containing an amino acid sequence represented by SEQ ID NO:9; the third antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:22, CDR2 containing an amino acid sequence represented by SEQ ID NO:23, and CDR3 containing an amino acid sequence represented by SEQ ID NO:24; furthermore, the second antigen-binding module includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes HCDR1 containing an amino acid sequence represented by SEQ ID NO:63, HCDR2 containing an amino acid sequence represented by SEQ ID NO:64, and HCDR3 containing an amino acid sequence represented by SEQ ID NO:65; and the light chain variable region includes LCDR1 containing an amino acid sequence represented by SEQ ID NO:66, LCDR2 containing an amino acid sequence represented by SEQ ID NO:67, and LCDR3 containing an amino acid sequence represented by SEQ ID NO:68. Furthermore, in a specific embodiment, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:41, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:42, the heavy chain variable region of the second antigen-binding module includes an amino acid sequence represented by SEQ ID NO:69, and its light chain variable region includes an amino acid sequence represented by SEQ ID NO:70. Furthermore, in a specific embodiment, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:38, the heavy chain variable region of the second antigen-binding module includes an amino acid sequence represented by SEQ ID NO:69, and its light chain variable region includes an amino acid sequence represented by SEQ ID NO:70.

[0024] In these embodiments, as one selectable configuration, the first antigen-binding module and the third antigen-binding module are both single variable domains, the second antigen-binding module is a Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc polypeptides in the Fc region, the second antigen-binding module is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region at the C-terminus of its Fab heavy chain or Fab light chain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. In this embodiment, in a more specific embodiment, the multispecific antibody is composed of three polypeptide chains, the first polypeptide chain comprising the first antigen-binding module, the third antigen-binding module and one of the Fc polypeptides in the Fc region, the second polypeptide chain comprising the Fab heavy chain of the second antigen-binding module and the other Fc polypeptide in the Fc region, and the third polypeptide chain being the Fab light chain of the second antigen-binding module.

[0025] In some embodiments, the multispecific antibody is trivalent.

[0026] One aspect of this disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a multispecific antibody described herein.

[0027] In one aspect of this disclosure, a vector containing the nucleic acid described herein is provided.

[0028] One aspect of this disclosure provides a host cell containing the nucleic acid or vector described in this disclosure.

[0029] Another aspect of the present disclosure provides a method for preparing a multispecific antibody as described in the present disclosure, which includes culturing the host cells such that the multispecific antibody is expressed, and isolating and purifying the multispecific antibody in a system.

[0030] In another aspect of the present disclosure, a drug composition is provided comprising the multispecific antibody and a pharmaceutically acceptable auxiliary material.

[0031] Another aspect of this disclosure provides the use of the multispecific antibody or the drug composition in the preparation of drugs for the treatment of c-Met and / or EGFR expression disorders.

[0032] Another aspect of the present disclosure provides a method for treating c-Met and / or EGFR-expressing tumors, which comprises administering the multispecific antibody or the drug composition to a subject in a therapeutically effective dose.

[0033] In yet another aspect of this disclosure, a c-Met-conjugated antibody comprising a single variable domain is provided, the single variable domain is (1) CDR1 containing the amino acid sequence represented by SEQ ID NO:1, CDR2 containing the amino acid sequence represented by SEQ ID NO:2, and CDR3 containing the amino acid sequence represented by SEQ ID NO:3, (2) CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, (4) CDR1 containing the amino acid sequence represented by SEQ ID NO:10, CDR2 containing the amino acid sequence represented by SEQ ID NO:11, and CDR3 containing the amino acid sequence represented by SEQ ID NO:12. (5) CDR1 containing the amino acid sequence represented by SEQ ID NO:13, CDR2 containing the amino acid sequence represented by SEQ ID NO:14, and CDR3 containing the amino acid sequence represented by SEQ ID NO:15. (6) CDR1 containing the amino acid sequence represented by SEQ ID NO:16, CDR2 containing the amino acid sequence represented by SEQ ID NO:17, and CDR3 containing the amino acid sequence represented by SEQ ID NO:18, (7) CDR1 containing the amino acid sequence represented by SEQ ID NO:19, CDR2 containing the amino acid sequence represented by SEQ ID NO:20, and CDR3 containing the amino acid sequence represented by SEQ ID NO:21, or (8) Includes CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0034] In some embodiments, the single variable domain includes an amino acid sequence represented by SEQ ID NO: 40, 41, or 42, but does not include an amino acid sequence represented by SEQ ID NO: 29, 30, or 31.

[0035] The EGFR and c-Met-targeted multispecific antibodies provided in this disclosure exhibit favorable antitumor efficacy and / or safety. [Brief explanation of the drawing]

[0036] [Figure 1-1] Figures 1A to 1G are curves showing the binding of different anti-human c-Met VHH-Fc chimeric antibodies to target cells at different c-Met expression levels, as measured by flow cytometry. [Figure 1-2] Figures 1A to 1G are curves showing the binding of different anti-human c-Met VHH-Fc chimeric antibodies to target cells at different c-Met expression levels, as measured by flow cytometry. [Figure 2] Figure 2 shows the structure of an exemplary multispecific antibody according to this disclosure. [Figure 3]Figures 3A to 3C show curves illustrating the binding of anti-EGFR / anti-c-Met multispecific antibodies to EGFR and c-Met expressing cells, as measured by flow cytometry. [Figure 4] Figure 4 shows the inhibition of c-Met phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies as measured by Western blotting. In the figure, the leftmost lane is the marker, BM is amivantamab, negative is hIgG1, blank indicates the absence of the antibody, HGF "-" or "+" indicates no HGF added or HGF added, respectively, and p-cMet, p-Akt, p-Erk, and p-EGFR represent phosphorylated c-Met, phosphorylated Akt, phosphorylated Erk, and phosphorylated EGFR, respectively. [Figure 5] Figure 5 shows the inhibition of EGFR phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies as measured by Western blotting. In the figure, the leftmost lane is the marker, BM is amivantamab, negative is hIgG1, blank indicates the absence of the antibody, EGF "-" or "+" indicates no EGF or EGF added, respectively, and p-cMet, p-Akt, p-Erk, and p-EGFR represent phosphorylated c-Met, phosphorylated Akt, phosphorylated Erk, and phosphorylated EGFR, respectively. [Figure 6] Figures 6A to 6C show curves illustrating how anti-EGFR / anti-c-Met multispecific antibodies, measured by ELISA, compete with HGF ligands at different concentrations to bind to c-Met. [Figure 7] Figures 7A and 7B show the inhibition of EGFR and c-Met expression tumor cell proliferation by anti-EGFR / anti-c-Met multispecific antibodies. [Figure 8] Figures 8A and 8B illustrate the toxic effects of anti-EGFR / anti-c-Met multispecific antibodies on EGFR and c-Met expressing tumor cells. [Figure 9]Figures 9A to 9C show the endocytosis activity of anti-EGFR / anti-c-Met multispecific antibodies against EGFR and c-Met expressing cells, as measured by flow cytometry. [Modes for carrying out the invention]

[0037] Definition and Description As used in this disclosure, the following terms have the meanings set forth below unless otherwise specified. Any particular term, unless specifically defined, should not be considered uncertain or ambiguous, but should be understood in accordance with its general meaning in the art. Where a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0038] The term "antibody" is used in its broadest sense, encompassing both natural and artificial antibodies with various structures. It includes, but is not limited to, various antibody structures such as monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific and triplicate antibodies), as well as single-chain antibodies, as long as they exhibit the desired antigen-binding activity.

[0039] The term "multispecificity" refers to an antibody that can specifically bind to various different antigenic determinants, for example, to two or more different antigenic determinants. In this specification, antigenic determinants are synonymous with antigenic epitopes. Typically, bispecific antibodies have two antigen-binding sites, each specific to a different antigenic determinant. Different antigenic determinants can be expressed on the same or different cells. The different antigenic determinants may differ depending on the type of antigen (e.g., binding to EGFR or c-Met), or they may be present on the same antigen. An antigenic determinant is a specific chemical group on the surface or other site of an antigenic substance molecule that has a particular composition and structure and can specifically bind to the corresponding antibody or sensitized lymphocyte. As an example of an antigenic determinant, c-Met, as an antigenic substance, has multiple antigenic determinants, some of which are structurally identified or unspecified. In this specification, an antibody that can bind to two different antigenic determinants on an antigen is called a bispecific antibody. Specific examples of bispecific antibodies include those that can bind to EGFR and c-Met.

[0040] The term "N-valent antibody" refers to an antibody that has N antigen-binding sites. For example, "bivalent antibody" or "the antibody is bivalent" means that the antibody has two antigen-binding sites, and "trivalent antibody" or "the antibody is trivalent" means that the antibody has three antigen-binding sites. Natural human immunoglobulin molecules usually have two antigen-binding sites, Fab usually has one antigen-binding site and a single variable domain, and scFv usually has one antigen-binding site.

[0041] The term "antigen-binding module" refers to a polypeptide molecule that specifically binds to an antigenic determinant. Specific examples of antigen-binding modules include Fab, scFv, or single variable domains.

[0042] In this disclosure, the terms “first,” “second,” or “third” used with respect to antigen-binding modules, antigens, Fc polypeptides, peptide linkers, polypeptide chains, etc., are merely used to facilitate distinction when one or more parts of each type are present. The use of these terms is not intended to imply any specific order or direction unless explicitly stated.

[0043] The term "fusion" refers to the direct linking of components (e.g., antigen-binding modules, Fc polypeptides, etc.) or linking via peptide bonds through one or more peptide linkers. For example, some peptide linkers consist of 1 to 50 amino acids linked by peptide bonds, the amino acids being selected from 20 naturally occurring amino acids. In a more preferred embodiment, the 1 to 50 amino acids are selected from glycine, alanine, proline, serine, asparagine, glutamine, and lysine.

[0044] The term "variable domain" or "variable region" refers to the domain in an antibody that is involved in the binding of the antibody to an antigen. For example, natural quadruple antibodies (e.g., those derived from humans, mice, etc.) have a heavy chain variable region (sometimes called a heavy chain variable domain, VH, or VH domain) and a light chain variable region (sometimes called a light chain variable domain, VL, or VL domain), while heavy chain-only antibodies derived from animals such as camelids and sharks have a single variable domain. In most cases, each variable domain of a natural antibody basically consists of four "framework regions (FRs)" and three "complementary determination regions (CDRs)". The four framework regions are called framework region 1 (or FR1), framework region 2 (or FR2), framework region 3 (or FR3), and framework region 4 (or FR4), respectively, and these framework regions are separated by three complementary determination regions, which are called complementary determination region 1 (or CDR1), complementary determination region 2 (or CDR2), and complementary determination region 3 (or CDR3), respectively, in this art and the following description. Therefore, the general structure of the variable domain can be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Because the variable domain has an antigen-binding site, it gives the antibody specificity to the antigen.

[0045] The term "single variable domain" refers to a variable domain that can specifically bind to an antigen epitope without pairing with other variable domains. A single variable domain typically contains three CDRs (CDR1, CDR2, CDR3) present on a single domain. In some cases, a single variable domain can be a heavy chain variable domain (e.g., VH) if it can form a single antigen-binding unit (i.e., a functional antigen-binding unit composed essentially of a single variable domain, thus allowing the single antigen-binding domain to form a functional antigen-binding unit without interacting with other variable domains). Another example of a single variable domain is the "V" in camelids. H H domain ("V H It is sometimes abbreviated as "H" or "VHH".

[0046] The term “V HThe "H domain" distinguishes these variable domains from the heavy chain variable domains present in normal four-chain antibodies and the light chain variable domains present in normal four-chain antibodies. V H The H domain specifically binds to an epitope without the need for other antigen-binding domains (this is different from the VH or VL domains in normal four-chain antibodies, where the epitope is recognized by both the VL domain and the VH domain). V H The H domain is a small, stable, and efficient antigen recognition unit composed of a single domain.

[0047] The term "complementary determining region" (CDR) may also be referred to as "hypervariable region" (HVR). A natural four-chain antibody usually consists of six CDRs, with three in the heavy chain variable region, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and three in the light chain variable region, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3). An antibody of only the heavy chain or a single variable domain usually consists of three CDRs (CDR1, CDR2, CDR3).

[0048] Currently, there are many methods for classifying and defining CDRs. Among them, the Kabat definition, which classifies CDRs based on sequence variability, is the most commonly used (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), while the Chothia definition is based on the position of structural loops (Cyrus Chothia et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901-917 (1987)). The AbM definition is intermediate between the Kabat and Chothia definitions and is used in Oxford Molecular's AbM antibody modeling software. The "contact" definition of CDRs is based on the analysis of the crystal structure of available complexes. There is also a CCG definition. However, it should be noted that the boundaries of CDRs in the same antibody variable region, as classified and defined in different ways, may differ, meaning that the CDR sequences in the same antibody variable region, as classified and defined in different ways, may differ. Therefore, when an antibody is limited by a specific CDR sequence, the range of the antibody also covers antibodies limited by CDR sequences according to other arbitrary definitions (e.g., one or a combination of several selected from definitions such as Kabat, IMGT, Chothia, Contact, AbM, CCG, etc.).

[0049] The term "Fab" refers to a protein consisting of the VH and CH1 heavy chains and the VL and CL light chains of an immunoglobulin. In this specification, Fab means a native or modified Fab molecule, i.e., a Fab heavy chain (VH-CH1, from the N-terminus to the C-terminus) consisting of the variable heavy chain region VH and the constant region CH1, and a Fab light chain (VL-CL, from the N-terminus to the C-terminus) consisting of the variable light chain region and the constant region CL. An example of a modified Fab is a Fab in which amino acid substitutions have been introduced into the CH1 / CL domain and / or the VH / VL domain. Specifically, a modified Fab may be a Fab in which amino acid substitutions have been introduced into the CL domain.

[0050] The term "scFv" refers to an immunoglobulin comprising the VH and VL domains, where these domains are present in a single polypeptide chain. In some embodiments, the scFv further includes a peptide ligand interposed between the VH and VL domains, thereby enabling the scFv to form the structure necessary for antigen binding.

[0051] The terms “Fc domain,” “Fc,” or “Fc region” are used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the natural sequence Fc and modified Fc. The C-terminal lysine (Lys447) of Fc may or may not be present. The numbering of amino acid residues in Fc or the constant region is, unless otherwise specified, based on the EU numbering system (also known as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242). As used herein, one of the “Fc polypeptides” in an Fc region refers to one of the two polypeptides that constitute the dimeric Fc domain. For example, the Fc polypeptide in the IgG Fc region includes IgG CH2 and IgG CH3.

[0052] The term “treatment” means intending to prevent, improve or eliminate one or more symptoms of a disease or associated with a disease by administering the compounds or drug compositions described herein, including, but not limited to, the following situations: (i) preventing the manifestation of a disease or disease condition in a mammal, in particular, when such a mammal is susceptible to the disease condition but has not yet been diagnosed with the disease condition; (ii) suppressing a disease or disease condition, i.e., inhibiting its development; (iii) alleviating a disease or disease condition, i.e., regressing the disease or disease condition; (iv) reducing any direct or indirect pathological consequences resulting from a disease or disease condition.

[0053] The term “therapeutic dose” means the dose of a compound relating to this disclosure that (i) treats or prevents a particular disease, condition or disorder, (ii) reduces, improves or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition or disorder described herein. The amount of a multispecific antibody, antibody-drug conjugate or drug composition relating to this disclosure that constitutes a “therapeutic dose” may vary depending on several factors, such as the compound or drug composition’s ability to elicit a desired response in an individual, the disease state and its severity, the method of administration, and the age, sex, and weight of the mammal being treated. The therapeutic dose may also be determined by a person skilled in the art based on their own knowledge and the content of this disclosure.

[0054] The term "pharmaceutically acceptable" applies to those compounds, materials, compositions and / or dosage forms, to use in contact with human and animal tissues within the bounds of reliable medical judgment, provided that they are free from excessive toxicity, irritation, allergic reactions or other problems or complications, and that the benefit / risk ratio is reasonable.

[0055] The term "excipient" refers to any component other than the active ingredient (e.g., the antibody as disclosed herein). The selection of an excipient largely depends on factors such as the specific method of administration, the effect of the excipient on solubility and stability, and the properties of the dosage form.

[0056] The term "isolation" refers to the isolation of a target compound from its natural environment, such as an antibody or its antigen-binding fragment. H H refers to nucleic acid.

[0057] The terms "Xn" and "Xaa" are equivalent, referring to unspecified amino acids, with their scope of coverage specified by subsequent definitions in related descriptions.

[0058] The term "EC" as used herein 50 " refers to the effective concentration required to elicit up to 50% of the antibody's maximum response. 50 This can be measured by ELISA, FACS analysis, or any other method known in the art.

[0059] "K D " refers to the equilibrium dissociation constant, and the dissociation rate constant (k d ) and coupling rate constant (k a The ratio of (i.e., k d / k a ) is obtained from and expressed as molar concentration (M). K of the antibody D The value can be measured by a well-established method in this field. Antibody K D A preferred method for measuring this is surface plasmon resonance (SPR) technology, which is preferably analyzed using a biosensor system, such as the Biacore surface plasmon resonance system.

[0060] The term "identity" is sometimes referred to as "consistency." The "percentage of amino acid sequence identity (%)" refers to the percentage of amino acid residues in the comparison sequence that are identical to the specific amino acid sequence shown herein, after comparing the comparison sequence to the specific amino acid sequence shown herein and introducing gaps where necessary to achieve the maximum percentage of sequence identity, without considering any conservative substitutions as part of the sequence identity. The comparison of amino acid sequences for identity can be performed by various methods within the scope of this art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters used for sequence comparison, including any algorithm necessary to achieve the maximum possible comparison from the full length of the comparison sequence.

[0061] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, including, for example, non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. Preferably, the subject according to this disclosure is human. The terms “patient” and “subject” are interchangeable unless otherwise specified. “Required subject” includes subjects who already have a disease or condition, subjects at risk of developing a disease or condition, and subjects who may develop a disease or condition and whose purpose is the prevention, delay, or mitigation of the disease or condition.

[0062] As used herein, “approximately” means that a particular value is within the acceptable margin of error as determined by an ordinary technician in the art, and this depends in part on how the value is measured or measured, i.e., the measuring system. For example, “approximately” can mean that, according to the practice in the art, it is within a standard deviation of at least 1. Alternatively, “approximately” may mean that it varies at most within ±5%, for example, within ±2%, ±1%, or ±0.5% of a given specific numerical range. Where a particular value is given within the scope of this disclosure, unless otherwise specified, “approximately” should be understood to mean that the particular value is within the acceptable margin of error. In this specification, unless otherwise specified, all values ​​of procedure parameters or conditions are modified with “approximately.”

[0063] The terms “comprise,” “contain,” and “comprising” (comprise, comprises, or comprising), and their synonyms (e.g., contain, contains, containing, include, includes, and including), should be understood as “including, but not limited to,…” and mean including other elements, components, and procedures that are not explicitly stated, in addition to the elements, components, and procedures listed.

[0064] In this specification, unless otherwise explicitly stated in the context, singular terms are also plural, and vice versa.

[0065] Single variable domain This disclosure provides a single variable domain that binds to c-Met (e.g., human c-Met). The single variable domain provides more available options for the development or drug construction of drugs that target c-Met. The single variable domain has good affinity for human c-Met and can confer targeting. In some cases, the single variable domain can confer the ability to block the binding of ligand HGF to c-Met. In particular, in some embodiments, the single variable domain cross-reacts with monkey c-Met, and cross-reaction with monkeys is advantageous for conducting drug toxicity studies, as monkeys are ideal experimental animals in drug toxicity studies.

[0066] This disclosure provides a single variable domain that binds to c-Met, and such single variable domain is (1) CDR1 containing the amino acid sequence represented by SEQ ID NO:1, CDR2 containing the amino acid sequence represented by SEQ ID NO:2, and CDR3 containing the amino acid sequence represented by SEQ ID NO:3, (2) CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, (4) CDR1 containing the amino acid sequence represented by SEQ ID NO:10, CDR2 containing the amino acid sequence represented by SEQ ID NO:11, and CDR3 containing the amino acid sequence represented by SEQ ID NO:12. (5) CDR1 containing the amino acid sequence represented by SEQ ID NO:13, CDR2 containing the amino acid sequence represented by SEQ ID NO:14, and CDR3 containing the amino acid sequence represented by SEQ ID NO:15. (6) CDR1 containing the amino acid sequence represented by SEQ ID NO:16, CDR2 containing the amino acid sequence represented by SEQ ID NO:17, and CDR3 containing the amino acid sequence represented by SEQ ID NO:18, (7) CDR1 containing the amino acid sequence represented by SEQ ID NO:19, CDR2 containing the amino acid sequence represented by SEQ ID NO:20, and CDR3 containing the amino acid sequence represented by SEQ ID NO:21, or (8) Includes CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0067] In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:1, CDR2 of the amino acid sequence represented by SEQ ID NO:2, and CDR3 of the amino acid sequence represented by SEQ ID NO:3. In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:4, CDR2 of the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 of the amino acid sequence represented by SEQ ID NO:6. In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:4, CDR2 of the amino acid sequence represented by SEQ ID NO:5 (X1 is S), and CDR3 of the amino acid sequence represented by SEQ ID NO:6. In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:4, CDR2 of the amino acid sequence represented by SEQ ID NO:5 (X1 is T), and CDR3 of the amino acid sequence represented by SEQ ID NO:6. In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:7, CDR2 of the amino acid sequence represented by SEQ ID NO:8, and CDR3 of the amino acid sequence represented by SEQ ID NO:9. In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:10, CDR2 of the amino acid sequence represented by SEQ ID NO:11, and CDR3 of the amino acid sequence represented by SEQ ID NO:12. In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:13, CDR2 of the amino acid sequence represented by SEQ ID NO:14, and CDR3 of the amino acid sequence represented by SEQ ID NO:15. In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:16, CDR2 of the amino acid sequence represented by SEQ ID NO:17, and CDR3 of the amino acid sequence represented by SEQ ID NO:18. In some embodiments, the single variable domain is derived from a camelid animal. In some embodiments, the single variable domain is humanized.In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:19, CDR2 of the amino acid sequence represented by SEQ ID NO:20, and CDR3 of the amino acid sequence represented by SEQ ID NO:21. In some embodiments, the single variable domain includes CDR1 of the amino acid sequence represented by SEQ ID NO:22, CDR2 of the amino acid sequence represented by SEQ ID NO:23, and CDR3 of the amino acid sequence represented by SEQ ID NO:24.

[0068] In some embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.

[0069] In some specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:25, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO:1, CDR2 containing the amino acid sequence represented by SEQ ID NO:2, and CDR3 containing the amino acid sequence represented by SEQ ID NO:3.

[0070] In some specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:40, and includes CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (where X1 is S or T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6.

[0071] In some more specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 26, 33, or 34, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO: 4, CDR2 containing the amino acid sequence represented by SEQ ID NO: 5 (X1 is S), and CDR3 containing the amino acid sequence represented by SEQ ID NO: 6.

[0072] In some more specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:35, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6.

[0073] In some specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:41, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9.

[0074] In some more specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 27, 36, or 37, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO: 7, CDR2 containing the amino acid sequence represented by SEQ ID NO: 8, and CDR3 containing the amino acid sequence represented by SEQ ID NO: 9.

[0075] In some specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:28, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO:10, CDR2 containing the amino acid sequence represented by SEQ ID NO:11, and CDR3 containing the amino acid sequence represented by SEQ ID NO:12.

[0076] In some specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:29, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO:13, CDR2 containing the amino acid sequence represented by SEQ ID NO:14, and CDR3 containing the amino acid sequence represented by SEQ ID NO:15.

[0077] In some specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:30, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO:16, CDR2 containing the amino acid sequence represented by SEQ ID NO:17, and CDR3 containing the amino acid sequence represented by SEQ ID NO:18.

[0078] In some specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:31, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO:19, CDR2 containing the amino acid sequence represented by SEQ ID NO:20, and CDR3 containing the amino acid sequence represented by SEQ ID NO:21.

[0079] In some specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:42, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0080] In some more specific embodiments, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:32, 38, or 39, and further includes CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0081] In some embodiments, an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, provided that the single variable domain containing such sequence maintains its ability to bind to c-Met. In some embodiments, substitutions, insertions, and / or deletions exist in a total of 1-18, 1-16, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids in amino acid sequences selected from SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42. In some embodiments, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., FR). In some embodiments, substitutions, insertions, or deletions occur in one, two, or three of the CDR regions, e.g., CDR1, CDR2, CDR3. In some embodiments, substitutions, insertions, or deletions occur in both the CDR and non-CDR regions.

[0082] In some embodiments, the amino acid sequence of the single variable domain includes an amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.

[0083] In some embodiments, the amino acid sequence of the single variable domain is represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.

[0084] In some embodiments, the single variable domain is V H H. In some embodiments, V HH represents a humanized form. A non-human single variable domain can be "humanized" by substituting one or more amino acid residues in the amino acid sequence of the original single variable domain sequence with one or more amino acid residues present in the corresponding region of the VH domain of a human antibody. Humanization is expected to reduce immunogenicity. Typically, a single variable domain has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus.

[0085] In some embodiments of this disclosure, a single variable domain is provided that binds to the same epitope as any of the single variable domains described in this disclosure. In some specific embodiments of this disclosure, a single variable domain is provided that binds to the same epitope as a single variable domain comprising an amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. In some embodiments, the single variable domain that binds to the same epitope is derived from a camelid or is humanized.

[0086] Using routine techniques known to those skilled in the art, single variable domains can be competitively screened for binding to the same epitope. Accordingly, in some embodiments of the present disclosure, single variable domains are provided that bind to c-Met in competition with any of the single variable domains described herein. In some specific embodiments, single variable domains are provided that bind to c-Met in competition with single variable domains comprising amino acid sequences represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. Binding to c-Met can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR), or any other method known in the art. In some embodiments, the single variable domains that compete to bind to c-Met are derived from camelids or are humanized.

[0087] This disclosure provides several examples of single variable domains that bind to c-Met. The CDRs (CDR1, CDR2, CDR3) and full-length amino acid sequences of the exemplary single variable domains provided in this disclosure are shown in Table S1 below. [Table S1]

[0088] This disclosure provides a c-Met-conjugated antibody containing the single variable domain. In some embodiments, the c-Met-conjugated antibody may be a monospecific antibody or a multispecific antibody.

[0089] In one embodiment, the monospecific antibody comprises an Fc domain, preferably the Fc being that of human IgG1, IgG2, IgG3, or IgG4.

[0090] In some embodiments, the c-Met-conjugated antibody contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.

[0091] In some embodiments, the c-Met-conjugated antibody contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 38, 39, 43, 44, 45%, 50, 51, 53, 55, 57, or 59.

[0092] This disclosure provides exemplary monospecific antibodies (e.g., 1B-1B6-V1, 1B-1B6-V2, 1B-1B6-V3, 1B-1C7-V1, 1B-1C7-V2, 12B04-V1, 12B04-V2, etc.) in which a single variable domain is fused to the Fc of human IgG1, and homodimers are formed via the Fc.

[0093] This disclosure provides isolated nucleic acids containing polynucleotides encoding the c-Met-binding antibodies described herein.

[0094] This disclosure provides vectors containing the nucleic acids described herein.

[0095] This disclosure provides host cells containing the nucleic acids or vectors described herein.

[0096] This disclosure provides a method for preparing a c-Met-binding antibody as described herein, which includes culturing host cells as described herein so as to express the c-Met-binding antibody, and separating and purifying the c-Met-binding antibody in a system.

[0097] This disclosure provides a drug composition comprising the c-Met-conjugated antibody described herein and pharmaceutically acceptable adjuncts.

[0098] This disclosure provides a method for treating c-Met expression-related disorders, which comprises administering a c-Met-binding antibody or the drug composition described herein to a subject as required.

[0099] This disclosure provides a method for treating c-Met expression-related disorders, which comprises administering a therapeutically effective dose of the c-Met-binding antibody or the drug composition described herein to a subject as required.

[0100] multispecific antibodies This disclosure provides a multispecific antibody containing a single variable domain, which can target c-Met and EGFR expressed on the surface of tumor cells and has tumor-killing activity.

[0101] Compared to methods employing multiple (e.g., two) Fabs, employing a single variable domain can reduce or avoid mismatches between light and heavy chains.

[0102] The multispecific antibodies described herein exhibit excellent antitumor properties, such as tumor cell degradation and inhibition of tumor cell proliferation. In some embodiments, the multispecific antibodies described herein can induce and kill multiple types of tumor cells through ADCC and / or CDC effects. The multispecific antibodies described herein exhibit good tumor-killing activity against tumor cells with different c-Met expression levels and / or different EGFR expression levels.

[0103] The multispecific antibodies described herein can suppress downstream signaling of c-Met and / or EGFR.

[0104] The multispecific antibodies according to this disclosure exhibit excellent antigen endocytosis and / or antigen degradation activity. In some embodiments, the multispecific antibodies according to this disclosure bind to c-Met and / or EGFR, then mediate endocytosis of c-Met and / or EGFR, thereby reducing c-Met and / or EGFR expression on the surface of tumor cells.

[0105] A multispecific antibody can be constructed using any of the single variable domains described in the "single variable domain" section. Therefore, a multispecific antibody is provided in this disclosure, and such a multispecific antibody is (i) A first antigen-binding module that binds to the first antigen, (ii) A second antigen-binding module that binds to the second antigen, and (iii) comprising a third antigen-binding module that binds to the first antigen, The first antigen is c-Met, the second antigen is EGFR, and both the first antigen-binding module and the third antigen-binding module are single variable domains, each independently, (1) CDR1 containing the amino acid sequence represented by SEQ ID NO:1, CDR2 containing the amino acid sequence represented by SEQ ID NO:2, and CDR3 containing the amino acid sequence represented by SEQ ID NO:3, (2) CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, (4) CDR1 containing the amino acid sequence represented by SEQ ID NO:10, CDR2 containing the amino acid sequence represented by SEQ ID NO:11, and CDR3 containing the amino acid sequence represented by SEQ ID NO:12. (5) CDR1 containing the amino acid sequence represented by SEQ ID NO:13, CDR2 containing the amino acid sequence represented by SEQ ID NO:14, and CDR3 containing the amino acid sequence represented by SEQ ID NO:15. (6) CDR1 containing the amino acid sequence represented by SEQ ID NO:16, CDR2 containing the amino acid sequence represented by SEQ ID NO:17, and CDR3 containing the amino acid sequence represented by SEQ ID NO:18, (7) CDR1 containing the amino acid sequence represented by SEQ ID NO:19, CDR2 containing the amino acid sequence represented by SEQ ID NO:20, and CDR3 containing the amino acid sequence represented by SEQ ID NO:21, or (8) Containing one of the following: CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0106] In this disclosure, the first antigen-binding module and the third antigen-binding module are both single variable domains that bind to c-Met. In some embodiments, the first antigen-binding module includes a complementary determination region as described in any one of (1) to (8) above. In some embodiments, the third antigen-binding module includes a complementary determination region as described in any one of (1) to (8) above. In the multispecific antibody, the first antigen-binding module and the third antigen-binding module may be the same or different, and any single variable domain described in this disclosure can be independently selected and combined. In some specific embodiments, the first antigen-binding module and the third antigen-binding module are the same. In some other specific embodiments, the first antigen-binding module and the third antigen-binding module are different.

[0107] Table S2 exemplifies multispecific antibodies constructed in different combinations of a first antigen-binding module and a third antigen-binding module with limited CDR features. In some embodiments, the first antigen-binding module includes the complementarity-determining region described in (3) above, and the third antigen-binding module includes the complementarity-determining region described in (2) above, that is, the first antigen-binding module includes CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, and the third antigen-binding module includes CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6. In some specific embodiments, the first antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:7, CDR2 containing an amino acid sequence represented by SEQ ID NO:8, and CDR3 containing an amino acid sequence represented by SEQ ID NO:9, and the third antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO:4, CDR2 containing an amino acid sequence represented by SEQ ID NO:5 (X1 is T), and CDR3 containing an amino acid sequence represented by SEQ ID NO:6. In a more specific embodiment, the first antigen-binding module includes CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9, and the third antigen-binding module includes CDR1 represented by SEQ ID NO:4, CDR2 represented by SEQ ID NO:5 (X1 is T), and CDR3 represented by SEQ ID NO:6.In some other embodiments, the first antigen-binding module includes the complementarity-determining region described in (3) above, and the third antigen-binding module includes the complementarity-determining region described in (8) above, that is, the first antigen-binding module includes CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, and the third antigen-binding module includes CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24. In one more specific embodiment, the first antigen-binding module includes CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9, and the third antigen-binding module includes CDR1 represented by SEQ ID NO:22, CDR2 represented by SEQ ID NO:23, and CDR3 represented by SEQ ID NO:24.

[0108] [Table S2]

[0109] Note: In the table, (X) + (Y) indicates the combination of the third antigen-binding module and the first antigen-binding module in a multispecific antibody. For example, (2) + (1) means that the third antigen-binding module in the multispecific antibody includes the complementary determination region described in (2) above, and the first antigen-binding module includes the complementary determination region described in (1) above.

[0110] Furthermore, in some embodiments, the first antigen-binding module and the third antigen-binding module each independently include an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. In some embodiments, the first antigen-binding module and the third antigen-binding module each independently include an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39.

[0111] In some embodiments, the first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. In some embodiments, the first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. In some more specific embodiments, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39.

[0112] In some embodiments, the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. In some embodiments, the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. In some more specific embodiments, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39.

[0113] In some embodiments, the first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:41, and the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:40. In some specific embodiments, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:41, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:40. In some more specific embodiments, the first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO: 27, 36, or 37, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO: 26, 33, 34, or 35.

[0114] In some embodiments, the first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:41, and the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:42. In some specific embodiments, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:41, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:42. In some more specific embodiments, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO: 27, 36, or 37, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO: 32, 38, or 39.

[0115] For example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:27, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0116] For example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:27, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0117] For example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0118] For example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0119] For example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:37, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0120] For example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:37, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0121] As an example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:27, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:26.

[0122] As an example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:27, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:26.

[0123] As an example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:33.

[0124] As an example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:33.

[0125] As an example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:34.

[0126] As an example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:34.

[0127] As an example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:35.

[0128] As an example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:35.

[0129] As an example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:32.

[0130] As an example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:32.

[0131] As an example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:38.

[0132] As an example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:38.

[0133] For example, the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:39.

[0134] As an example, the third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:39.

[0135] As an example, both the first antigen-binding module and the third antigen-binding module contain an amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39.

[0136] Table S3 exemplifies multispecific antibodies in which the first antigen-binding module and the third antigen-binding module, which possess the characteristics of the full-length amino acid sequence, are constructed in different combinations.

[0137] [Table S3]

[0138] Note: In the table, (X)+(Y) indicates the combination of the third antigen-binding module and the first antigen-binding module in a multispecific antibody. For example, "1B-1B6-V1+1B-1C7-V1 single variable domain" indicates that the third antigen-binding module in the multispecific antibody is the amino acid sequence of the 1B-1B6-V1 single variable domain, and the first antigen-binding module is the amino acid sequence of the 1B-1C7-V1 single variable domain. Refer to Table S1 for the amino acid sequences of the single variable domains related to this table.

[0139] In this disclosure, the first antigen-binding module may be derived from a camelid or be humanized. The third antigen-binding module may be derived from a camelid or be humanized. Humanization can reduce immunogenicity, and in some embodiments, both the first and third antigen-binding modules are humanized. In some embodiments, both the first and third antigen-binding modules are derived from a camelid.

[0140] In the multispecific antibodies according to this disclosure, the second antigen-binding module confers the ability to target EGFR. The second antigen-binding module may be a Fab, scFv, or ScFab (single-chain Fab). In some embodiments, the second antigen-binding module is an EGFR-binding Fab.

[0141] In some embodiments, the second antigen-binding module is derived from a mouse, a chimeric or humanized form.

[0142] In some embodiments, the second antigen-binding module includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1 containing an amino acid sequence represented by SEQ ID NO:63, HCDR2 containing an amino acid sequence represented by SEQ ID NO:64, and HCDR3 containing an amino acid sequence represented by SEQ ID NO:65, and the light chain variable region includes LCDR1 containing an amino acid sequence represented by SEQ ID NO:66, LCDR2 containing an amino acid sequence represented by SEQ ID NO:67, and LCDR3 containing an amino acid sequence represented by SEQ ID NO:68.

[0143] In one specific embodiment, the heavy chain variable region includes variable regions HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO:69, and the light chain variable region includes variable regions LCDR1, HCDR2, and LCDR3 represented by SEQ ID NO:70.

[0144] In some embodiments, the second antigen-binding module includes a heavy chain variable region of an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:69, and a light chain variable region of an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:70. In some embodiments, the heavy chain variable region of the second antigen-binding module includes the amino acid sequence represented by SEQ ID NO:69, and its light chain variable region includes the amino acid sequence represented by SEQ ID NO:70. In some embodiments, the amino acid sequence of the heavy chain variable region of the second antigen-binding module is represented by SEQ ID NO:69, and the amino acid sequence of its light chain variable region is represented by SEQ ID NO:70.

[0145] The multispecific antibody according to this disclosure may not have an Fc region, and the first antigen-binding module, the second antigen-binding module, and the third antigen-binding module are fused by an appropriate linker.

[0146] The multispecific antibodies according to this disclosure may have an Fc region, which can extend the half-life and provide functions such as effectors associated with the Fc region.

[0147] Structure of multispecific antibodies Each antigen-binding module in the multispecific antibody according to this disclosure can be fused to one another in various structures. In some embodiments, the multispecific antibody further comprises (iv) an Fc region composed of two Fc polypeptides.

[0148] In some embodiments, the third antigen-binding module and the first antigen-binding module are fused to each other, and optionally fused to each other via a peptide linker. In some more specific embodiments, the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. Furthermore, in some embodiments, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc polypeptides in the Fc region, the second antigen-binding module is a Fab, and the second antigen-binding module is fused at the C-terminus of its Fab heavy chain or Fab light chain to the N-terminus of the other Fc polypeptide in the Fc region.

[0149] In one specific embodiment, both the first antigen-binding module and the third antigen-binding module are single variable domains, the second antigen-binding module is a Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc polypeptides in the Fc region, the second antigen-binding module is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. Such a structure is schematically shown in Figure 2. In a more specific aspect of this embodiment, the multispecific antibody is composed of three polypeptide chains, the first polypeptide chain includes the first antigen-binding module, the third antigen-binding module, and one of the Fc polypeptides in the Fc region, the second polypeptide chain includes the Fab heavy chain of the second antigen-binding module and the other Fc polypeptide in the Fc region, and the third polypeptide chain is the Fab light chain of the second antigen-binding module.

[0150] In the embodiments described above, each antigen-binding module of the multispecific antibody is operably linked and fused, for example, directly or via a hinge to various peptide linkers described herein or known in the art (e.g., peptide linkers containing one or more amino acids, typically about 1 to 50 amino acids), which can be reasonably selected by those skilled in the art.

[0151] The third antigen-binding module can be fused to the first antigen-binding module directly or via a peptide linker. In one embodiment, the third antigen-binding module is fused to the first antigen-binding module via a peptide linker.

[0152] Each peptide linker can be any suitable one independently, for example, a charged linker polypeptide and / or a flexible linker polypeptide can be used. In a specific embodiment, the peptide linker consists of 1 to 50 amino acids linked by peptide bonds, and the amino acids are selected from 20 naturally occurring amino acids. In a more preferred embodiment, the 1 to 50 amino acids are selected from glycine, alanine, proline, serine, asparagine, glutamine, and lysine. Thus, exemplary peptide linkers may include polyglycine (especially (Gly)4, (Gly)5), poly(Gly-Ser), (Gly)3AsnGlySer(Gly)2, (Gly)3Cys(Gly)4, GlyProAsnGlyGly, or those disclosed in Table 4 of patent application WO2019195535.

[0153] In some embodiments, the peptide linker may be a peptide linker composed of glycine and serine. In some embodiments, the number of amino acids contained in the peptide linker may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20 or more. In some embodiments, the peptide linker is a peptide linker containing GGGGS as a unit. In some embodiments, the peptide linker with GGGGS as a unit is (GGGGS) n Herein, n is any number between 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any range limited by any two of the above digits, e.g., 1-5, 2-5, 3-6, 2-4, 1-4, etc. In some specific embodiments, the peptide linker is a linker polypeptide comprising GGGGS (SEQ ID NO: 91), (GGGGS)2, (GGGGS)3, or (GGGGS)4. In some specific embodiments, the third antigen-binding module is fused to the first antigen-binding module via the peptide linker GGGGS (SEQ ID NO: 91) or (GGGGS)2.

[0154] Typically, fusion to Fc occurs via a hinge region. In one embodiment, the first antigen-binding module is fused to one Fc polypeptide in the Fc region via a first hinge, and the second antigen-binding module is fused to the other Fc polypeptide in the Fc region via a second hinge. In some embodiments, the first and second hinges can form a covalent bond, such as a disulfide bond, with each other. The first hinge and / or the second hinge may contain amino acids from the hinge region of human IgG, and the hinge region of human IgG may include the natural hinge region or a variant thereof. In some embodiments, the first hinge and / or the second hinge contain amino acids from the hinge region of human IgG1. In some embodiments, the first hinge and / or the second hinge contain amino acids from the hinge region of human IgG4. In some specific embodiments, the first hinge includes GEPKSSDKTHTCPPCP (SEQ ID NO: 89), and the second hinge includes EPKSCDKTHTCPPCP (SEQ ID NO: 90).

[0155] Fc area The Fc region of a multispecific antibody is composed of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc region of an immunoglobulin G (IgG) molecule is a dimer, and each Fc polypeptide contains CH2 and CH3 of the constant region of the IgG heavy chain. Two Fc polypeptides in the Fc region can stably associate with each other. In one embodiment, the multispecific antibody according to this disclosure contains one Fc region.

[0156] In some embodiments, the Fc region of the multispecific antibody is the IgG Fc region. In some embodiments, the Fc region is the IgG1 Fc region. In some embodiments, the Fc region is the human Fc region. In some specific embodiments, the Fc region is the human IgG1 Fc region.

[0157] In some embodiments, the Fc region includes modifications such as amino acid substitutions. These modifications may include, for example, modifications that promote heterodimerization or modifications that alter the ability to bind to protein A.

[0158] In some embodiments, the Fc includes modifications that promote heterodimerization.

[0159] The multispecific antibodies according to this disclosure include different antigen-binding modules fused to one or the other of two Fc polypeptides in the Fc region, and therefore the two Fc polypeptides are typically contained in two different polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization generate several possible combinations of the two polypeptides. To increase the yield and purity of the multispecific antibody in recombinant production, it is advantageous to introduce modifications to the Fc region of the multispecific antibody that promote the desired polypeptide binding. Accordingly, in specific embodiments, the Fc region includes amino acid substitutions that promote the association of the two Fc polypeptides in the Fc region.

[0160] The most extensive protein-protein interaction site between two Fc polypeptides in the human IgG Fc region is located in the CH3 domain of the Fc region. Therefore, in one embodiment, this modification is located in the CH3 domain of the Fc region.

[0161] In specific embodiments, this modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two Fc polypeptides in the Fc region and a "hole" modification in the other of the two Fc polypeptides in the Fc region. Typically, this method can position the protrusion in the hole by introducing a protrusion ("knob") at the interface of one Fc polypeptide and a corresponding depression ("hole") at the interface of the other Fc polypeptide, thereby promoting heterodimer formation while hindering homodimer formation. The protrusion is constructed by substituting a smaller amino acid side chain from the interface of one Fc polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary depression of the same or similar size as the protrusion is created at the interface of the other Fc polypeptide by substituting the larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0162] Therefore, in a specific embodiment, by substituting an amino acid residue in the CH3 domain of one Fc polypeptide of the multispecific antibody with an amino acid residue having a larger side chain volume, a projection that can be positioned in a depression in the CH3 domain of the other Fc polypeptide is generated within the CH3 domain of this Fc polypeptide, and by substituting an amino acid residue in the CH3 domain of the other Fc polypeptide with an amino acid residue having a smaller side chain volume, a depression is generated within the CH3 domain of this Fc polypeptide.

[0163] In some embodiments, one Fc polypeptide in the Fc region includes the EU numbering amino acid substitutions 354C and / or 366Y / W, and the other Fc polypeptide includes the EU numbering amino acid substitutions 349C, 366S, 368A and / or 407T / V. In some specific embodiments, one Fc polypeptide in the Fc region includes the EU numbering amino acid substitutions 354C and 366Y / W, and the other Fc polypeptide includes the EU numbering amino acid substitutions 349C, 366S, 368A and 407T / V. In some more specific embodiments, one Fc polypeptide in the Fc region includes the EU numbering amino acid substitutions 354C and 366W, and the other Fc polypeptide includes the EU numbering amino acid substitutions 349C, 366S, 368A and 407V. In the embodiments described above, Fc may be human IgG1 Fc. In one specific embodiment, one of the Fc polypeptides in the Fc region includes the amino acid substitutions S354C and T366W according to EU numbering, and the other Fc polypeptide includes the amino acid substitutions Y349C, T366S, L368A and Y407V according to EU numbering.

[0164] In some embodiments, the Fc region includes modifications that reduce or eliminate the binding between the CH3 region of one Fc polypeptide in the Fc region and protein A (Protein A from Staphylococcus aureus). In some embodiments, the Fc region includes amino acid substitutions that reduce or eliminate the binding between the CH3 region of one Fc polypeptide in the Fc region and protein A. In some embodiments, the Fc region includes the EU numbering amino acid substitution (a)435R or (b)435R and 436F, which occurs in only one Fc polypeptide and not the other. In some embodiments, the Fc region includes the EU numbering amino acid substitution (a)435R or (b)435R and 436F, which occurs in only one Fc polypeptide. In some specific embodiments, the Fc region includes the EU numbering amino acid substitutions H435R and Y436F, which occur in only one Fc polypeptide. In some specific embodiments, the Fc region contains the EU numbering-based amino acid substitution H435R, which occurs in only one of the Fc polypeptides. In the embodiments described above, this Fc is IgG1 Fc, particularly human IgG1 Fc.

[0165] In the multispecific antibodies according to this disclosure, the Fc region may include (i) modifications that promote heterodimerization and / or (ii) modifications that reduce or eliminate the binding between the CH3 region of one Fc polypeptide in the Fc region and protein A. In some embodiments, the Fc includes (i) modifications that promote heterodimerization and (ii) modifications that reduce or eliminate the binding between the CH3 region of one Fc polypeptide in the Fc region and protein A. For example, in one specific embodiment, the Fc region includes the following group of amino acid substitutions by EU numbering.

[0166] i. 349C, 366S, 368A, 407T / V, 354C, 366Y / W (amino acid substitutions 354C and 366Y / W are located on the same Fc polypeptide, but are not located on the same Fc polypeptide as any of the other amino acid substitutions in (i)) and, ii. (a) 435R or (b) 435R and 436F generated in only one of the Fc polypeptides.

[0167] In a more specific embodiment, one of the Fc polypeptides in the Fc region includes the EU numbering amino acid substitutions Y349C, T366S, L368A, Y407V, H435R, and Y436F, while the other Fc polypeptide includes the EU numbering amino acid substitutions S354C and T366W. In such a specific embodiment, this Fc is IgG1 Fc, particularly human IgG1 Fc.

[0168] In another, more specific embodiment, one of the Fc polypeptides in the Fc region includes the EU numbering amino acid substitutions Y349C, T366S, L368A, Y407V, and H435R, while the other Fc polypeptide includes the EU numbering amino acid substitutions S354C and T366W. In such a specific embodiment, this Fc is IgG1 Fc, particularly human IgG1 Fc.

[0169] In the context of this disclosure, amino acid substitutions are represented as original amino acid-site-substituted amino acid, amino acid residues are represented by a three-letter code (Xaa) or a one-letter code (X), and the original amino acid may be omitted. Therefore, for example, "H435R" or "435R" means that amino acid H or the original amino acid at site 435 is substituted with amino acid R, and the substituted amino acid may include more than one; for example, "T366Y / W" means that amino acid T at site 366 is substituted with amino acid Y or W.

[0170] In some embodiments, the Fc region does not contain fucose.

[0171] In some embodiments, the multispecific antibodies according to this disclosure are non-fucosylated. Defucosylation enhances the interaction between the multispecific antibody and FcγRIIIa and further enhances the ADCC effect of the antibody. Methods for generating multispecific antibodies in which a small amount of fucose is present or absent on the glycosylation site in the Fc region without altering the amino acid sequence are known in the art, and include, for example, methods for adjusting the components of the culture medium in which the expressing cells are located, or methods for knocking out fucose expression-related genes in the expressing cells, such as FUT8.

[0172] In some embodiments, the multispecific antibodies described herein are trivalent, meaning that the first antigen-binding module, the second antigen-binding module, and the third antigen-binding module each provide monovalent binding to the corresponding antigen.

[0173] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 71, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85. It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87.

[0174] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 73, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence with SEQ ID It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87.

[0175] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having SEQ ID It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87.

[0176] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 87, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having SEQ ID It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87.

[0177] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 79, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence with SEQ ID It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87.

[0178] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 81, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence with SEQ ID It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87.

[0179] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 83, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence with SEQ ID It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87.

[0180] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain containing an amino acid sequence represented by SEQ ID NO:71, another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:87.

[0181] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain containing an amino acid sequence represented by SEQ ID NO:73, another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:87.

[0182] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain containing an amino acid sequence represented by SEQ ID NO:75, another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:87.

[0183] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain containing an amino acid sequence represented by SEQ ID NO:77, another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:87.

[0184] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain containing an amino acid sequence represented by SEQ ID NO:79, another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:87.

[0185] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain containing an amino acid sequence represented by SEQ ID NO:81, another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:87.

[0186] In some embodiments, the multispecific antibody is composed of three polypeptide chains, specifically, one polypeptide chain containing an amino acid sequence represented by SEQ ID NO:83, another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain containing an amino acid sequence represented by SEQ ID NO:87.

[0187] This disclosure provides exemplary trivalent multispecific antibodies.

[0188] For example, a multispecific antibody is composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains are represented by SEQ ID NO:71, SEQ ID NO:85, and SEQ ID NO:87, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are represented by SEQ ID NO:72, SEQ ID NO:86, and SEQ ID NO:88, respectively.

[0189] For example, a multispecific antibody is composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains are represented by SEQ ID NO:73, SEQ ID NO:85, and SEQ ID NO:87, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are represented by SEQ ID NO:74, SEQ ID NO:86, and SEQ ID NO:88, respectively.

[0190] For example, a multispecific antibody is composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains are represented by SEQ ID NO:75, SEQ ID NO:85, and SEQ ID NO:87, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are represented by SEQ ID NO:76, SEQ ID NO:86, and SEQ ID NO:88, respectively.

[0191] For example, a multispecific antibody is composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains are represented by SEQ ID NO:77, SEQ ID NO:85, and SEQ ID NO:87, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are represented by SEQ ID NO:78, SEQ ID NO:86, and SEQ ID NO:88, respectively.

[0192] For example, a multispecific antibody is composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains are represented by SEQ ID NO:79, SEQ ID NO:85, and SEQ ID NO:87, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are represented by SEQ ID NO:80, SEQ ID NO:86, and SEQ ID NO:88, respectively.

[0193] For example, a multispecific antibody is composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains are represented by SEQ ID NO:81, SEQ ID NO:85, and SEQ ID NO:87, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are represented by SEQ ID NO:82, SEQ ID NO:86, and SEQ ID NO:88, respectively.

[0194] For example, a multispecific antibody is composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains are represented by SEQ ID NO:83, SEQ ID NO:85, and SEQ ID NO:87, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are represented by SEQ ID NO:84, SEQ ID NO:86, and SEQ ID NO:88, respectively.

[0195] Isolated nucleic acids This disclosure provides isolated nucleic acids containing nucleotide sequences encoding the multispecific antibodies described herein. The sequence listing exemplifies several nucleotide sequences encoding the multispecific antibodies.

[0196] vector This disclosure provides vectors containing the nucleic acids. In some embodiments, the vector is a clonal vector. In some other embodiments, the vector is an expression vector, specifically, the expression vector being pcDNA3.1(+). The expression vector is any expression vector capable of selectively expressing the multispecific antibodies described herein.

[0197] host cell This disclosure provides host cells containing nucleic acids or vectors described herein. In some embodiments, the host cells are suitable for cloning or expressing multispecific antibodies. In some embodiments, the host cells are prokaryotic cells. In some other embodiments, the host cells are eukaryotic cells. In some embodiments, the host cells are selected from the group consisting of yeast cells, mammalian cells, or other cells suitable for the preparation of multispecific antibodies. Mammalian cells are, for example, Chinese hamster ovary (CHO) cells, CHO-S cells.

[0198] Antibody-drug conjugate (ADC) This disclosure provides an antibody-drug conjugate containing the multispecific antibody described herein. In some embodiments, the antibody-drug conjugate contains the multispecific antibody and a cytotoxic drug. The multispecific antibody and the cytotoxic drug are preferably linked via a linker, which is either a degradable or indegradable linker.

[0199] Drug composition This disclosure provides drug compositions comprising, in addition to the multispecific antibody described herein, one or more pharmaceutically acceptable auxiliary materials. This disclosure also provides drug compositions comprising, in addition to the antibody-drug conjugate according to this disclosure, one or more pharmaceutically acceptable auxiliary materials. Examples of pharmaceutically acceptable auxiliary materials include excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0200] In the drug compositions according to this disclosure, the multispecific antibody may be completely non-fucosylated (i.e., not containing detectable fucosyl) or partially non-fucosylated.

[0201] Method for preparing multispecific antibodies In some embodiments of this disclosure, methods for preparing multispecific antibodies are provided, which include culturing host cells to express the multispecific antibodies and optionally isolating and purifying the multispecific antibodies in a system. To generate the multispecific antibodies, a nucleic acid encoding the multispecific antibodies is isolated and one or more vectors are inserted for further cloning and / or expression in host cells. The nucleic acids can be obtained by various methods well known in the art, such as gene conjugation and chemical synthesis.

[0202] The prepared multispecific antibodies can be purified by techniques known in the art, such as high-efficiency liquid chromatography, ion-exchange chromatography, gel chromatography, affinity chromatography, size exclusion chromatography, and ceramic hydroxyapatite (CHT) chromatography. The actual conditions for purifying a particular protein are partially determined by factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. In the case of affinity chromatography, the multispecific antibody can be purified using an antibody, ligand, receptor, or antigen that binds to the multispecific antibody. For example, the affinity chromatography of the multispecific antibody according to this disclosure can be performed using a matrix containing protein A or protein G. The multispecific antibody according to this disclosure can be purified using affinity chromatography, ion-exchange chromatography, gel chromatography, and / or CHT chromatography in sequence.

[0203] The purity of multispecific antibodies can be determined by any of the various well-known analytical methods, such as gel electrophoresis, high-efficiency liquid chromatography, and size exclusion chromatography.

[0204] The physicochemical properties and / or biological activities of multispecific antibodies described herein can be identified, screened, or characterized by various measurement methods known in the art.

[0205] use This disclosure provides the use of multispecific antibodies as described herein. In some specific embodiments, the multispecific antibodies used may be V12, V41, V35, V42, V67, V73 and / or V74.

[0206] This disclosure provides the use of the multispecific antibody, the antibody-drug conjugate, or the drug composition in the preparation of drugs for treating c-Met and / or EGFR expression disorders. In some embodiments, the drug is prepared by combining the multispecific antibody, the antibody-drug conjugate, or the drug composition with one or more other therapeutic agents. The other therapeutic agents may be oncological agents known in the art.

[0207] This disclosure provides a method for treating c-Met and / or EGFR-expressing diseases, which comprises administering the multispecific antibody, the antibody-drug conjugate, or the drug composition to a subject of interest. This disclosure provides a method for treating c-Met and / or EGFR-expressing diseases, which comprises administering the multispecific antibody, the antibody-drug conjugate, or the drug composition to a subject of interest in a therapeutically effective amount. In some embodiments, the method further comprises administering one or more other therapeutic agents to a subject of interest. The other therapeutic agents may be oncological agents known in the art. In some embodiments, the disease is a tumor.

[0208] In some embodiments, the tumors are epithelial cell carcinoma, mammary gland carcinoma, ovarian carcinoma, lung carcinoma, lung adenocarcinoma, colorectal carcinoma, anal carcinoma, prostate carcinoma, kidney carcinoma, liver carcinoma, bladder carcinoma, head and neck carcinoma, stomach carcinoma, pancreatic carcinoma, skin carcinoma, oral carcinoma, pharyngeal carcinoma, nasal carcinoma, tongue carcinoma, esophageal carcinoma, testicular carcinoma, vaginal carcinoma, cervical carcinoma, splenic carcinoma, testicular carcinoma, thyroid carcinoma, salivary gland carcinoma, and / or thymic carcinoma. In some embodiments, the lung carcinoma includes non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC).

[0209] Specific Embodiments While this disclosure provides several specific embodiments, the scope of protection provided by this disclosure is not limited to these.

[0210] Embodiment 1. A multispecific antibody, (i) A first antigen-binding module that binds to the first antigen, (ii) A second antigen-binding module that binds to the second antigen, and (iii) comprising a third antigen-binding module that binds to the first antigen, The first antigen is c-Met, the second antigen is EGFR, and both the first antigen-binding module and the third antigen-binding module are single variable domains, each independently, (1) CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T, preferably T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6, (2) CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO:1, CDR2 containing the amino acid sequence represented by SEQ ID NO:2, and CDR3 containing the amino acid sequence represented by SEQ ID NO:3, (4) CDR1 containing the amino acid sequence represented by SEQ ID NO:10, CDR2 containing the amino acid sequence represented by SEQ ID NO:11, and CDR3 containing the amino acid sequence represented by SEQ ID NO:12. (5) CDR1 containing the amino acid sequence represented by SEQ ID NO:13, CDR2 containing the amino acid sequence represented by SEQ ID NO:14, and CDR3 containing the amino acid sequence represented by SEQ ID NO:15. (6) CDR1 containing the amino acid sequence represented by SEQ ID NO:16, CDR2 containing the amino acid sequence represented by SEQ ID NO:17, and CDR3 containing the amino acid sequence represented by SEQ ID NO:18, (7) CDR1 containing the amino acid sequence represented by SEQ ID NO:19, CDR2 containing the amino acid sequence represented by SEQ ID NO:20, and CDR3 containing the amino acid sequence represented by SEQ ID NO:21, or (8) A multispecific antibody comprising one of the following: CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0211] Embodiment 2. With the multispecific antibody described in Embodiment 1, the first antigen-binding module and the third antigen-binding module each independently, (1) CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T, preferably T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6, (2) CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, or (8) Containing one of the following: CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0212] Embodiment 3. Using the multispecific antibody described in Embodiment 2, the first antigen-binding module comprises CDR1 containing an amino acid sequence represented by SEQ ID NO:7, CDR2 containing an amino acid sequence represented by SEQ ID NO:8, and CDR3 containing an amino acid sequence represented by SEQ ID NO:9, and the third antigen-binding module comprises CDR1 containing an amino acid sequence represented by SEQ ID NO:4, CDR2 containing an amino acid sequence represented by SEQ ID NO:5 (X1 is S or T, preferably T), and CDR3 containing an amino acid sequence represented by SEQ ID NO:6.

[0213] Embodiment 4. With the multispecific antibody described in Embodiment 2, the first antigen-binding module includes CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, and the third antigen-binding module includes CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and the amino acid sequence represented by SEQ ID NO:24.

[0214] Embodiment 5. A multispecific antibody, (i) A first antigen-binding module that binds to the first antigen, (ii) A second antigen-binding module that binds to the second antigen, and (iii) comprising a third antigen-binding module that binds to the first antigen, A multispecific antibody in which the first antigen is c-Met, the second antigen is EGFR, and both the first antigen-binding module and the third antigen-binding module are single variable domains, each independently containing CDR1, CDR2, and CDR3, which are single variable domains whose amino acid sequences are represented by SEQ ID NO: 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, or 39.

[0215] Embodiment 6. With the multispecific antibody described in Embodiment 5, the first antigen-binding module and the third antigen-binding module each independently contain CDR1, CDR2, and CDR3, which are single variable domains whose amino acid sequences are represented by SEQ ID NO: 35, 36, or 38.

[0216] Embodiment 7. With the multispecific antibody described in Embodiment 6, the first antigen-binding module comprises CDR1, CDR2, and CDR3, single variable domains whose amino acid sequence is represented by SEQ ID NO:36, and the third antigen-binding module comprises CDR1, CDR2, and CDR3, single variable domains whose amino acid sequence is represented by SEQ ID NO:35.

[0217] Embodiment 8. With the multispecific antibody described in Embodiment 6, the first antigen-binding module comprises CDR1, CDR2, and CDR3, which are single variable domains whose amino acid sequence is represented by SEQ ID NO:36, and the third antigen-binding module comprises CDR1, CDR2, and CDR3, which are single variable domains whose amino acid sequence is represented by SEQ ID NO:38.

[0218] Embodiment 9. With a multispecific antibody according to any one of Embodiments 1 to 8, the first antigen-binding module and the third antigen-binding module each independently contain an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 40, 41, 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, or 42.

[0219] Embodiment 10. The first antigen-binding module, with respect to the multispecific antibody described in any one of Embodiments 1 to 9, includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:41.

[0220] Embodiment 11. With the multispecific antibody described in Embodiment 10, the first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 27, 36, or 37. Preferably, the first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 36.

[0221] Embodiment 12. The third antigen-binding module, with respect to the multispecific antibody described in any one of Embodiments 1 to 11, includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 40.

[0222] Embodiment 13. With the multispecific antibody described in Embodiment 12, the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 26, 33, 34, or 35. Preferably, the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 35.

[0223] Embodiment 14. The third antigen-binding module, with respect to the multispecific antibody described in any one of Embodiments 1 to 11, includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:42.

[0224] Embodiment 15. With the multispecific antibody described in Embodiment 14, the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 32, 38, or 39. Preferably, the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 38.

[0225] Embodiment 16. The first antigen-binding module and the third antigen-binding module are selected from any one of the following, using the multispecific antibody described in any one of Embodiments 1 to 8: (1) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:35. (2) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:27, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:26. (3) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:33. (4) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:34. (5) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:36, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:32. (6) The third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:38, or (7) The third antigen-binding module includes an amino acid sequence represented by SEQ ID NO:36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO:39.

[0226] Embodiment 17. The single variable domain is derived from a camelid animal or humanized by any one of Embodiments 1 to 16 using a multispecific antibody.

[0227] Embodiment 18. The second antigen-binding module is Fab, scFv, or scFab, using the multispecific antibody described in any one of Embodiments 1 to 17.

[0228] Embodiment 19. With a multispecific antibody according to any one of Embodiments 1 to 18, the second antigen-binding module includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1 containing an amino acid sequence represented by SEQ ID NO: 63, HCDR2 containing an amino acid sequence represented by SEQ ID NO: 64, and HCDR3 containing an amino acid sequence represented by SEQ ID NO: 65, and the light chain variable region includes LCDR1 containing an amino acid sequence represented by SEQ ID NO: 66, LCDR2 containing an amino acid sequence represented by SEQ ID NO: 67, and LCDR3 containing an amino acid sequence represented by SEQ ID NO: 68.

[0229] Embodiment 20. The second antigen-binding module comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises variable regions HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO: 69, and the light chain variable region comprises variable regions LCDR1, HCDR2, and HCDR3 represented by SEQ ID NO: 70.

[0230] Embodiment 21. By the multispecific antibody according to any one of Embodiments 1 to 20, the second antigen-binding module includes a heavy-chain variable region having an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence represented by SEQ ID NO: 69, and a light-chain variable region having an amino acid sequence with at least 85%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence represented by SEQ ID NO: 70.

[0231] Embodiment 22. By the multispecific antibody according to Embodiment 21, the heavy-chain variable region of the second antigen-binding module includes the amino acid sequence represented by SEQ ID NO: 69, and its light-chain variable region includes the amino acid sequence represented by SEQ ID NO: 70.

[0232] Embodiment 23. By the multispecific antibody according to any one of Embodiments 1 to 22, the second antigen-binding module is of mouse origin, chimeric or humanized.

[0233] Embodiment 24. By the multispecific antibody according to any one of Embodiments 1 to 23, the third antigen-binding module and the first antigen-binding module are fused to each other, and optionally fused to each other via a peptide linker.

[0234] Embodiment 25. By the multispecific antibody according to Embodiment 24, the third antigen-binding module is fused to the N-terminus of the first antigen-binding module at its C-terminus.

[0235] Embodiment 26. By the multispecific antibody according to any one of Embodiments 1 to 25, the multispecific antibody further includes an Fc region composed of (iv) two Fc polypeptides.

[0236] Embodiment 27. With the multispecific antibody described in Embodiment 26, the first antigen-binding module is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, and the second antigen-binding module is Fab, and the second antigen-binding module is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region.

[0237] Embodiment 28. With the multispecific antibody described in Embodiment 26 or 27, the Fc region is an IgG Fc region, preferably an IgG1 Fc region.

[0238] Embodiment 29. With the multispecific antibody described in Embodiment 28, the IgG Fc region is a human IgG Fc region, preferably a human IgG1 Fc region.

[0239] Embodiment 30. The Fc region includes an amino acid substitution that promotes the association of two Fc polypeptides in the Fc region, provided that the multispecific antibody described in any one of Embodiments 26 to 29 is used.

[0240] Embodiment 31. With the multispecific antibody described in Embodiment 30, one Fc polypeptide in the Fc region contains amino acid substitutions 354C and 366Y / W according to EU numbering, and the other Fc polypeptide contains amino acid substitutions 349C, 366S, 368A and 407T / V according to EU numbering.

[0241] Embodiment 32. The Fc region includes an amino acid substitution that reduces or eliminates the binding between the CH3 region of one of the Fc polypeptides in the Fc region and protein A, by any one of the multispecific antibodies described in Embodiments 26 to 31.

[0242] Embodiment 33. With the multispecific antibody described in Embodiment 32, the Fc region includes an EU numbering amino acid substitution (a)435R or (b)435R and 436F that occurred in only one of the Fc polypeptides.

[0243] Embodiment 34. With the multispecific antibody described in any one of Embodiments 26 to 29, one Fc polypeptide in the Fc region contains the EU numbering amino acid substitutions 349C, 366S, 368A, 407V, 435R and 436F, and the other Fc polypeptide contains the EU numbering amino acid substitutions 354C and 366W.

[0244] Embodiment 35. With the multispecific antibody described in any one of Embodiments 26 to 29, one Fc polypeptide in the Fc region contains the EU numbering amino acid substitutions 349C, 366S, 368A, 407V and 435R, and the other Fc polypeptide contains the EU numbering amino acid substitutions 354C and 366W. Embodiment 36. The multispecific antibody is trivalent, as described in any one of Embodiments 1 to 35.

[0245] Embodiment 37. The multispecific antibody described in any one of Embodiments 1 to 36 is composed of three polypeptide chains, specifically, (1) One polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 87, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having SEQ ID It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87. (2) One polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:71, another polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:85 It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87. (3) One polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:73, another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:85 It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87. (4) One polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:75, another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:85, and yet75 It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87. (5) One polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 89, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having SEQ ID It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87. (6) One polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:81, another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:85, and yet It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87, or, (7) One polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:83, another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO:85 It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:87.

[0246] Embodiment 38. A multispecific antibody composed of three polypeptide chains, (1) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO:77, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:87. (2) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO:71, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:87. (3) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO:73, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:87. (4) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO:75, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:87. (5) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO:79, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:87. (6) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO:81, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:85, yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:87, or (7) A multispecific antibody in which one polypeptide chain contains the amino acid sequence represented by SEQ ID NO:83, another polypeptide chain contains the amino acid sequence represented by SEQ ID NO:85, and yet another polypeptide chain contains the amino acid sequence represented by SEQ ID NO:87.

[0247] Embodiment 39. The multispecific antibody according to any one of Embodiments 1 to 38, wherein the multispecific antibody is non-fucosylated.

[0248] Embodiment 40. An isolated nucleic acid comprising a nucleotide sequence encoding the multispecific antibody according to any one of Embodiments 1 to 39.

[0249] Embodiment 41. A vector containing the nucleic acid according to Embodiment 40.

[0250] Embodiment 42. A host cell containing the nucleic acid according to Embodiment 40 or the vector according to Embodiment 41.

[0251] Embodiment 43. A method for preparing the multispecific antibody according to any one of Embodiments 1 to 39, comprising culturing the host cell according to Embodiment 42 so that the multispecific antibody is expressed, and isolating and purifying the multispecific antibody in the system.

[0252] Embodiment 44. An antibody-drug conjugate containing the multispecific antibody according to any one of Embodiments 1 to 39 and a cytotoxic drug.

[0253] Embodiment 45. A drug composition containing the multispecific antibody according to any one of Embodiments 1 to 39 or the antibody-drug conjugate according to Embodiment 44, and a pharmaceutically acceptable adjuvant.

[0254] Embodiment 46. Use of the multispecific antibody according to any one of Embodiments 1 to 39, the antibody-drug conjugate according to Embodiment 44 or the drug composition according to Embodiment 45 in the preparation of a drug for treating a c-Met or / and EGFR expression disease.

[0255] Embodiment 47. By the use according to Embodiment 46, the disease is a tumor.

[0256] Embodiment 48. By use according to Embodiment 47, the tumor is an epithelial cell carcinoma, mammary gland cancer, ovarian cancer, lung cancer, lung adenocarcinoma, colorectal cancer, anal cancer, prostate cancer, kidney cancer, liver cancer, bladder cancer, head and neck cancer, stomach cancer, pancreatic cancer, skin cancer, oral cancer, pharyngeal cancer, nasal cancer, tongue cancer, esophageal cancer, testicular cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, thyroid cancer, salivary gland cancer, or thymic cancer.

[0257] Embodiment 49. By use as described in Embodiment 48, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0258] Embodiment 50. By use as described in any one of Embodiments 46 to 49, the drug is prepared by combining the multispecific antibody, the antibody-drug conjugate, or the drug composition with one or more other therapeutic agents.

[0259] Embodiment 51. A method for treating c-Met and / or EGFR expression diseases, A therapeutic method comprising administering to a required subject a therapeutically effective amount of a multispecific antibody described in any one of Embodiments 1 to 39, an antibody-drug conjugate described in Embodiment 44, or a drug composition described in Embodiment 45.

[0260] Embodiment 52. The disease is a tumor according to the method described in Embodiment 51.

[0261] Embodiment 53. By the method described in Embodiment 52, the tumor is epithelial cell carcinoma, mammary gland cancer, ovarian cancer, lung cancer, lung adenocarcinoma, colorectal cancer, anal cancer, prostate cancer, kidney cancer, liver cancer, bladder cancer, head and neck cancer, stomach cancer, pancreatic cancer, skin cancer, oral cancer, pharyngeal cancer, nasal cancer, tongue cancer, esophageal cancer, testicular cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, thyroid cancer, salivary gland cancer, or thymic cancer.

[0262] Embodiment 54. By the method described in Embodiment 53, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0263] Embodiment 55. A c-Met-binding antibody comprising a single variable domain, wherein the single variable domain is (1) CDR1 containing the amino acid sequence represented by SEQ ID NO:4, CDR2 containing the amino acid sequence represented by SEQ ID NO:5 (X1 is S or T), and CDR3 containing the amino acid sequence represented by SEQ ID NO:6, (2) CDR1 containing the amino acid sequence represented by SEQ ID NO:7, CDR2 containing the amino acid sequence represented by SEQ ID NO:8, and CDR3 containing the amino acid sequence represented by SEQ ID NO:9, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO:1, CDR2 containing the amino acid sequence represented by SEQ ID NO:2, and CDR3 containing the amino acid sequence represented by SEQ ID NO:3, (4) CDR1 containing the amino acid sequence represented by SEQ ID NO:10, CDR2 containing the amino acid sequence represented by SEQ ID NO:11, and CDR3 containing the amino acid sequence represented by SEQ ID NO:12. (5) CDR1 containing the amino acid sequence represented by SEQ ID NO:13, CDR2 containing the amino acid sequence represented by SEQ ID NO:14, and CDR3 containing the amino acid sequence represented by SEQ ID NO:15. (6) CDR1 containing the amino acid sequence represented by SEQ ID NO:16, CDR2 containing the amino acid sequence represented by SEQ ID NO:17, and CDR3 containing the amino acid sequence represented by SEQ ID NO:18, (7) CDR1 containing the amino acid sequence represented by SEQ ID NO:19, CDR2 containing the amino acid sequence represented by SEQ ID NO:20, and CDR3 containing the amino acid sequence represented by SEQ ID NO:21, or (8) A c-Met-conjugated antibody comprising CDR1 containing the amino acid sequence represented by SEQ ID NO:22, CDR2 containing the amino acid sequence represented by SEQ ID NO:23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:24.

[0264] Embodiment 56. With the c-Met-binding antibody described in Embodiment 55, the single variable domain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 40, 41, 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, or 42.

[0265] Embodiment 57. With the c-Met-conjugated antibody described in Embodiment 56, the single variable domain contains an amino acid sequence represented by SEQ ID NO: 33, 34, 35, 36, 37, 38, or 39.

[0266] Embodiment 58. With the c-Met-conjugated antibody described in Embodiment 56, the single variable domain contains an amino acid sequence represented by SEQ ID NO: 40, 41, or 42, but does not contain an amino acid sequence represented by SEQ ID NO: 29, 30, or 31.

[0267] Embodiment 59. The single variable domain is derived from a camelid animal or humanized by the c-Met-binding antibody described in any one of Embodiments 55 to 58.

[0268] Embodiment 60. The c-Met-binding antibody described in any one of Embodiments 55 to 59 contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 44, 45, 43, 46, 47, 48, 49, 50, 51, 53, 55, 57, or 59.

[0269] For clarity, the present disclosure will be further illustrated with examples, but these examples will not limit the scope of the present disclosure. The reagents used in the present disclosure are commercially available and can be used without further purification. The amivantamab used in the examples was purchased from Janssen, and its NDC number is 57894-501-01. [Examples]

[0270] Example 1: Anti-human c-Met V H Construction of an H-phage display library Recombinant human c-Met-Fc fusion protein (purchased from SinoBiological, catalog number 10692-H02H) and complete Freund's adjuvant were emulsified in a 1:1 volume ratio, and Bactrian camels were immunized by initial subcutaneous multi-point injection. Subsequently, every two weeks, additional immunization was performed using a 1:1 volume ratio emulsified mixture of recombinant human c-Met-Fc fusion protein and incomplete Freund's adjuvant. After the fourth or fifth immunization, serum was collected and the amount of anti-human c-Met antibody was examined. After multiple cycles of immunization, peripheral blood was collected from Bactrian camels, and peripheral blood mononuclear cells (PBMCs) were isolated. The total RNA of the PBMCs was extracted and reverse transcribed into cDNA, and the variable region (V) of the camel antibody was identified by nested PCR. H H) The sequence was amplified.

[0271] Amplified V HAfter cleaving the H-coding fragment with PstI / NotI endonuclease, the recombinant vector was constructed by inserting it into the phage vector pMECS (purchased from the NTCC Plasmid Vector Species Cell Gene Depositary Center, catalog number No. pMECS). This recombinant vector was then electroporated into E. coli TG1 (purchased from Lucigen, catalog number No. 60502-1) to construct the library species. The library species was amplified to the logarithmic growth phase, and the auxiliary phage M13KO7 (purchased from New England Biolabs, catalog number No. N0315S) was added to further amplify the library. The mixture was then shaken overnight at 28°C and 200 rpm. The bacterial suspension is centrifuged and the supernatant is collected. 1 / 4 volume of PEG6000 / NaCl solution (20% PEG6000 (w / v), 2.5M NaCl) is added to the supernatant, and the mixture is incubated on ice for 1-2 hours to precipitate the phages. The phage precipitate is collected by centrifugation, resuspended in PBS, and then 20% glycerin is added. H The H phage display library was stored at -80°C.

[0272] Example 2: Anti-human c-Met V H H screening V H H phage display libraries were selected, the selected monoclones were cultured, and expression was induced with isopropyl-β-D-thiogalactoside (IPTG) to prepare the supernatant.

[0273] Positive identification of selected clones was performed using an indirect ELISA method against human c-Met-His (purchased from SinoBiological, catalog number 10692-H08H). Positive clones that bound only to human c-Met-His and had relatively high signal values ​​were selected, preserved, and sequenced. Screening yielded positive clones 1B-1B2, 1B-3B11, 1B-1C7, 1B-1B6, 1B-1A8, 3B-1C7, 4&5B-2F01, and 4&5C-12B04. Sequence analysis revealed that 1B-3B11 was V H The amino acid sequence of H is represented by SEQ ID NO:25, with V at 1B-1B6. HThe amino acid sequence of H is represented by SEQ ID NO:26, and V is 1B-1C7. H The amino acid sequence of H is represented by SEQ ID NO:27, and V is 1B-1A8. H The amino acid sequence of H is represented by SEQ ID NO:28, and V is 1B-1B2. H The amino acid sequence of H is represented by SEQ ID NO:29, and V is 3B-1C7. H The amino acid sequence of H is represented by SEQ ID NO:30, and V is 4&5B-2F01. H The amino acid sequence of H is represented by SEQ ID NO:31, and V is 4&5C-12B04. H The amino acid sequence of H is represented by SEQ ID NO:32.

[0274] Example 3: Anti-human c-Met V H Preparation of H-Fc chimeric antibodies V of selected positive clones H The H sequence is concatenated to the human Fc region and V H An H-Fc chimeric antibody was constructed. Specifically, the V sequence determined in Example 2 was used. H The H sequence was inserted into a pcDNA3.1(+) eukaryotic expression vector containing the human IgG1 Fc region, and these V sequences were transfected using the transient transfection expression system Expifectamine® CHO Transfection Kit (purchased from Thermo Fisher Scientific Inc., catalog number A29129). H H-Fc chimeric antibodies were expressed. Simultaneously, the sequences VL1016-069 and VH1016-069 from patent application US20200079872A1 were inserted into pcDNA3.1(+) eukaryotic expression vectors containing the human IgG1 constant region (amino acid sequence, SEQ ID NO:92), respectively, and the chimeric antibody 1016-069 was expressed as a control using the same method.

[0275] Sequence analysis revealed that the amino acid sequence of 1B-3B11-Fc is represented by SEQ ID NO:43, the amino acid sequence of 1B-1B6-Fc is represented by SEQ ID NO:44, the amino acid sequence of 1B-1C7-Fc is represented by SEQ ID NO:45, the amino acid sequence of 1B-1A8-Fc is represented by SEQ ID NO:46, the amino acid sequence of 1B-1B2-Fc is represented by SEQ ID NO:47, the amino acid sequence of 3B-1C7-Fc is represented by SEQ ID NO:48, the amino acid sequence of chimeric antibody 4&5B-2F01-Fc is represented by SEQ ID NO:49, and the amino acid sequence of chimeric antibody 4&45C-12B04-Fc is represented by SEQ ID NO:50.

[0276] Example 4: Anti-human V H Affinity of H-Fc chimeric antibodies with human and cynomolgus monkey c-Met 4.1 Affinity measurement of antibodies to human and cynomolgus monkey c-Met using surface plasmon resonance technology Biomolecular interaction analysis system (purchased from GE, Biacore T200 or Biacore 8K) is used to analyze for anti-human c-Met V H Affinity measurements were performed on H-Fc chimeric antibodies. Anti-hIgG(Fc) antibody (purchased from GE, catalog number BR-1008-39) was bound to the sensor chip CM5 via amino linkage, and anti-human c-MetV HThe H-Fc chimeric antibody was diluted to 1 μg / mL in running buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4, 0.05% surfactant P-20 (w / v), pH 7.4) and captured by passing it through the experimental channel at a flow rate of 30 μL / min. Human c-Met-His (purchased from SinoBiological, catalog number 10692-H08H) or cynomolgus monkey c-Met-His (purchased from SinoBiological, catalog number 90304-C08H) were diluted in running buffer to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM. These were then bound at a flow rate of 50 μL / min, with a binding time set to 200 seconds. After that, injection was stopped to allow dissociation, with a dissociation time set to 600-1400 seconds. Data signals were collected in real time using BiaControl Software 2.0 or Biacore 8K Control Software 3.0, and data analysis was performed using BiaEvaluation Software 2.0 or Biacore Insight Evaluation Software 3.0. A Langmuir 1:1 model was fitted, and the binding rate constant k was determined. a (1 / Ms), dissociation rate constant k d (1 / s), equilibrium dissociation constant K D The value of (M) was calculated. As shown in Tables 1-1 and 1-2, the measurement results show that 1B-1B2-Fc, 1B-3B11-Fc, 1B-1C7-Fc, 1B-1B6-Fc, 1B-1A8-Fc, 3B-1C7-Fc, 4&5B-2F01-Fc, and 4&5C-12B04-Fc all have relatively high affinity for human c-Met protein and cross-reactivity with cynomolgus monkey c-Met protein.

[0277] [Table 1]

[0278] 4.2 Measurement of antibody binding to cells by flow cytometry Flow cytometry revealed anti-human c-Met V H The binding of H-Fc chimeric antibodies to target cells with different c-Met expression levels was measured. Specifically, NCI-H1993 cells (purchased from BeNa Culture Collection, catalog number BNCC342186) are a human lung adenocarcinoma cell lineage with high c-Met expression levels; MKN45 cells (purchased from Nanjing Cobioer Biosciences, catalog number CBP60541) are a human gastric cancer cell lineage with moderate c-Met expression levels; KP4 cells (purchased from Nanjing Cobioer Biosciences, catalog number CBP60541) are a human pancreatic cancer cell lineage with low c-Met expression levels; NCI-H1975 cells (purchased from BeNa Culture Collection, catalog number BNCC100690) are a human non-small cell lung adenocarcinoma cell lineage with moderate / low c-Met expression levels; and NCI-H292 cells (purchased from BeNa Culture Collection, catalog number BNCC100671) are a human epithelial lung cancer cell lineage with moderate / low c-Met expression levels. 2 x 10 5 Individual target cells were subjected to gradient dilution (initial concentration 100 nM, 5-fold gradient dilution, 7 concentration levels) of anti-human c-Met V. HCells were incubated with H-Fc chimeric antibody, incubated on ice for 1 hour, washed, and PE-labeled anti-human IgG Fc antibody (purchased from Jackson Immuno Research, catalog number 109-116-170) was added. The cells were incubated on ice for 0.5 hours, washed, and then measured using a flow cytometer (purchased from Thermo Fisher Scientific Inc., Attune NXT). As a result, as shown in Figures 1A to 1G, Tables 2-1 and 2-2, 1B-1B2-Fc, 1B-3B11-Fc, 1B-1C7-Fc, 1B-1B6-Fc, 1B-1A8-Fc, 3B-1C7-Fc, 4&5B-2F01-Fc, and 4&5C-12B04-Fc all exhibit relatively high binding ability to target cells with different c-Met expression levels, and 4&5B-2F01-Fc and 4&5C-12B04-Fc are superior to the control 1016-069.

[0279] [Table 2]

[0280] Example 5: Anti-human c-Met V H Epitope difference analysis between different clones of H Epitope competition was analyzed using a biomolecular interaction analysis system (purchased from Fortebio, catalog number Octet RED96). Using an Anti-Penta-HIS (HIS1K) sensor (purchased from Fortebio, catalog number 18-5120), the c-Met-His protein (purchased from SinoBiological, product catalog number 10692-H08H) was diluted to approximately 5 μg / mL in running buffer. The sensor was immersed in the diluted antigen sample, and the hardening height was controlled to approximately 1 nm by adjusting the binding time. The sensor was then sequentially interacted with the first antibody A and the second antibody B, and the binding signal of antibody B was detected to determine whether the two antibodies recognized the same epitope. The results are shown in Tables 3-1, 3-2, and 3-3. The criteria for judgment are as follows: a value above 60% indicates no competition between the two antibodies; a value between 20% and 60% indicates partial competition between the two antibodies (with the possibility of epitope cross-reaction); and a value below 20% indicates complete competition between the two antibodies. If the autoreactivity signal (underlined portion) was less than 20%, the data was considered valid.

[0281] As is clear from the data in Table 3-1, the two antibodies 1B-3B11-Fc and 1B-1A8-Fc are in complete competition, and 1B-3B11-Fc and 1B-1A8-Fc do not have a clear competitive relationship with the other three candidate antibodies, each belonging to a different epitope. Therefore, the antibodies 1B-3B11-Fc and 1B-1B2-Fc, 1B-3B11-Fc and 1B-1C7-Fc, 1B-3B11-Fc and 1B-1B6-Fc, 1B-1A8-Fc and 1B-1B2-Fc, 1B-1A8-Fc and 1B-1C7-Fc, 1B-1A8-Fc and 1B-1B6-Fc, 1B-1B2-Fc and 1B-1C7-Fc, 1B-1B2-Fc and 1B-1B6-Fc, and 1B-1C7-Fc and 1B-1B6-Fc can simultaneously bind to different epitopes of the c-Met antigen. As is clear from the data in Table 3-2, the two antibodies 3B-1C7-Fc and 1B-1B2-Fc are completely competitive. Therefore, considering these results in combination with the data in Table 3-1, it is thought that antibodies 1B-1A8-Fc and 3B-1C7-Fc, 3B-1C7-Fc and 1B-1C7-Fc, and 3B-1C7-Fc and 1B-1B6-Fc can simultaneously bind to different epitopes of the c-Met antigen.

[0282] As is clear from the data in Table 3-3, 4&5B-2F01-Fc and 4&5C-12B04-Fc are in direct competition with 1016-069.

[0283] [Table 3]

[0284] Example 6: Anti-human c-Met V H Construction, expression, purification, and affinity measurement of H-Fc humanized antibodies Anti-human c-Met V H Humanization modifications were performed on each of the H-Fc chimeric antibodies: 1B-1B6-Fc, 1B-1C7-Fc, and 4&5C-12B04-Fc. Humanized V HThe H sequence was inserted into a pcDNA3.1(+) eukaryotic expression vector containing the human IgG1 Fc constant region, and these humanized V sequences were transfected using the transient transfection expression system Expifectamine® CHO Transfection Kit (purchased from Thermo Fisher Scientific Inc., catalog number A29129). H H-Fc antibodies were expressed. After humanization modification of the chimeric antibody 1B-1B6-Fc, three humanized antibodies were obtained: 1B-1B6-V1, 1B-1B6-V2, and 1B-1B6-V3. Their full-length amino acid sequences are represented by SEQ ID NO: 51, 53, and 55, respectively, and their full-length nucleotide sequences are represented by SEQ ID NO: 52, 54, and 56, respectively. After humanization modification of the chimeric antibody 1B-1C7-Fc, two humanized antibodies were obtained: 1B-1C7-V1 and 1B-1C7-V2. Their full-length amino acid sequences are represented by SEQ ID NO: 57 and 59, respectively, and their full-length nucleotide sequences are represented by SEQ ID NO: 58 and 60, respectively. After humanization modification of the chimeric antibody 4&5C-12B04-Fc, two humanized V H H was obtained, and these were 12B04-V1 and 12B04-V2, respectively, and their amino acid sequences are represented by SEQ ID NO:38 and 39, respectively.

[0285] The affinity of the obtained humanized and chimeric antibodies to human c-Met protein was measured using a biomolecular interaction analysis system (purchased from GE, Biacore 8K) by surface plasmon resonance technology. Anti-hIgG(Fc) antibody (purchased from GE, catalog number BR-1008-39) was conjugated to the sensor chip CM5 via amino linkage, and anti-human c-Met V HThe H-Fc chimeric antibody was diluted to 2 μg / mL in running buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4, 0.05% surfactant P-20 (w / v), pH 7.4) and passed through the experimental channel at a flow rate of 30 μL / min for 90 seconds. Human c-Met-His protein (purchased from SinoBiological, catalog number 10692-H08H) was diluted to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM in running buffer and bound at a flow rate of 50 μL / min, and the binding signal curve was observed.

[0286] Affinity data for humanized antibodies (1B-1B6-V1, 1B-1B6-V2, 1B-1B6-V3, 1B-1C7-V1, 1B-1C7-V2) and chimeric antibodies (1B-1B6-Fc, 1B-1C7-Fc) with human c-Met protein are shown in Table 4.

[0287] [Table 4]

[0288] The measurement results showed that both 1B-1B6-Fc and its humanized antibody, and 1B-1C7-Fc and its humanized antibody, can specifically bind to the c-Met-His protein.

[0289] Example 7: Construction, expression, and purification of anti-EGFR / anti-c-Met multispecific antibodies Anti-EGFR / anti-c-Met multispecific antibodies target two c-Met molecules, including humanized V HH atoms were linked in series. For the modification of the Fc region of the multispecific antibody, a knob-into-hole approach was used (one Fc polypeptide used Y349C, T366S, L368A, Y407V mutations according to EU numbering, and the other Fc polypeptide used S354C, T366W mutations according to EU numbering), and one of the Fc polypeptides in the Fc region further included a mutation that does not bind to binding protein A (H435R and Y436F or H435R according to EU numbering).

[0290] Anti-EGFR / anti-c-Met multispecific antibodies were constructed according to the structure shown in Figure 2, and named V12, V41, V35, V42, V67, V73, and V74, respectively. Here, the second antigen-binding module is a Fab-type EGFR-binding antigen-binding domain. Specifically, the anti-EGFR / anti-c-Met multispecific antibody is composed of three polypeptide chains, specifically one polypeptide chain (anti-c-Met-V H The H-Fc (named H-Fc) contains two series-linked c-Met-binding antigen-binding domains (derived from 1B-1B6-Fc, 1B-1C7-Fc, and the chimeric antibody 4&5C-12B04-Fc and its humanized antibody, as shown in Table 5). The amino acid sequences of the polypeptide chains V12, V41, V35, V42, V67, V73, and V74 are represented by SEQ ID NOs: 71, 73, 75, 77, 79, 81, and 83, respectively, and their nucleotide sequences are represented by SEQ ID NOs: 72, 74, 76, 78, 80, 82, and 84, respectively. The other two polypeptide chains contain Fab-type EGFR antigen-binding domains (the variable region sequences that bind to EGFR are derived from Patent Document CN100497389C), one of which is named anti-EGFR-HC-Fc (amino acid sequence represented by SEQ ID NO: 85, nucleotide sequence represented by SEQ ID NO: 86), and the other polypeptide chain is named anti-EGFR-LC (amino acid sequence represented by SEQ ID NO: 87, nucleotide sequence represented by SEQ ID NO: 88). anti-c-Met-V HThe nucleotide sequences encoding H-Fc, anti-EGFR-HC-Fc, and anti-EGFR-LC were inserted into the pcDNA3.1(+) eukaryotic expression vector, respectively, to obtain expression vectors expressing the corresponding polypeptide chains. The above expression vectors were then expressed using the CHOgro® high-yield expression system (Catalog No.: MIR 6270) to obtain the anti-c-Met-V expression vector. H CHO-S cells (CHO FUT 8) with the FUT8 gene knocked out were transfected using a transfection ratio of H-Fc:vector anti-EGFR-HC:vector anti-EGFR-LC = 1.5:1:1.5. - / - The cells (named "cells") were co-transfected, and the cell density at the time of transfection was 4 × 10⁶. 6 The concentration was 1 cell / mL. Cells were continuously cultured for 10 days after transfection, and the cell culture supernatant was collected by centrifugation. Protein purification was performed using the AKTA pure protein purification system (GE Healthcare) via protein A affinity chromatography, CHT chromatography, and gel chromatography. Protein concentration was measured using a UV-Vis spectrophotometer (NanoDrop One C, purchased from Thermo Scientific). The multispecific antibody with the desired structure and sequence was confirmed by electrophoresis (reduced SDS-PAGE), molecular weight (TOF MS), and sequencing analysis.

[0291] [Table 5]

[0292] Example 8: Affinity of anti-EGFR / anti-c-Met multispecific antibody with human c-Met Referring to the method in Example 6, the binding status of multispecific chimeric antibodies V12, V67 and multispecific humanized antibodies V35, V41, V42, V73, and V74 to human c-Met protein was measured by surface plasmon resonance technology. As shown in Table 6, all antibodies were able to specifically bind to human c-Met-His protein, and their affinity for the multispecific humanized antibodies was comparable to that of the multispecific chimeric antibodies.

[0293] [Table 6]

[0294] Example 9: Binding of anti-EGFR / anti-c-Met multispecific antibody to EGFR and c-Met expressing tumor cells Flow cytometry was used to analyze the binding of anti-EGFR / anti-c-Met multispecific antibodies (including amivantamab, V42, and V73) to A431 cells (high EGFR expression, low c-Met expression, source: Shanghai Cell Laboratory), NCI-H1975 cells (medium EGFR expression, medium-low c-Met expression, source: BeNa Culture Collection), and NCI-H1993 cells (medium EGFR expression, high c-Met expression, source: BeNa Culture Collection), which express EGFR and c-Met. The negative control was hIgG1 (purchased from Biointron, catalog number B117901).

[0295] A431, NCI-H1975, and NCI-H1993 cells were collected during the logarithmic growth phase, and their viable cell density was measured at 5 × 10⁶ using RPMI medium containing 2% fetal bovine serum (purchased from Hyclon, catalog number SH30809.01). 6 ~1 × 10 7The culture medium was adjusted to cells / mL and inoculated at 50 μL / well into a 96-well U-shaped cell culture plate (purchased from Costar, catalog number 3799). Using the above medium, anti-EGFR / anti-c-Met multispecific antibodies of different concentrations were prepared, resulting in a maximum concentration of 500 nM and a 5-fold gradient dilution, creating a total of 10 concentration gradients. 50 μL / well of each antibody concentration was added to the 96-well cell culture plate, mixed well, and incubated at 4°C for 1 hour. After washing the cells with pre-cooled running buffer (purchased from MACS, catalog number 130-091-221), the supernatant was discarded, pre-cooled fluorescently labeled sheep anti-human IgG antibody (purchased from Jackson, catalog number 109-116-170) was added, and the mixture was resuspended at 100 μL / well, mixed well, and incubated at 4°C for 30 minutes. After washing, pre-cooled running buffer was added, the cells were resuspended at 40 μL / well, and thoroughly mixed. Flow collection was then performed on an iQue3 flow cytometer, and data analysis was performed using software.

[0296] Table 7 and Figures 3A-3C show the binding ability of anti-EGFR / anti-c-Met multispecific antibodies to A431, NCI-H1975, and NCI-H1993 cells. The results indicated that V42 and V73 were nearly comparable to amivantamab in terms of binding ability to the three cell lines.

[0297] [Table 7]

[0298] Example 10: Suppression of HGF-induced c-Met phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies. Western blotting was used to analyze the suppression of HGF-induced c-Met phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies (including amivantamab, V42, and V73). The negative control was hIgG1 (purchased from Biointron, catalog number B117901). The coding sequences for HGF-α (amino acid sequence represented by SEQ ID NO: 61) and HGF-β (amino acid sequence represented by SEQ ID NO: 62), respectively, were inserted into a pcDNA3.1(+) eukaryotic expression vector. These vectors were then expressed using the Expifectamine® CHO Transfection Kit (purchased from Thermo Fisher Scientific Inc., catalog number A29129), and purified using a nickel column Ni Sepharose excel (purchased from GE, catalog number 17-3712-01) to obtain HGF.

[0299] Logarithmic growth phase cells A549 (expressed in EGFR and c-Met, non-small cell lung cancer, source: Center for Basic Medical Cells, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were collected, counted, and then the viable cell density was measured at 4 × 10⁶ in RPMI medium containing 10% fetal bovine serum (purchased from Hyclon, catalog number SH30809.01). 5 The culture medium was adjusted to cells / mL, added to a 6-well cell culture plate at 2 mL / well, and incubated in a 37°C, 5% CO2 cell culture chamber for 6 hours. After incubation, the medium was discarded, RPMI medium was added at 1 mL / well, and the cells were starved overnight. The overnight starved 6-well cell culture plates were removed, the original medium was discarded, and either a final concentration of 100 nM anti-EGFR / anti-c-Met multispecific antibody was added, or a final concentration of 100 ng / mL of HGF and a final concentration of 100 nM anti-EGFR / anti-c-Met multispecific antibody were added simultaneously. The cells were incubated in a 37°C, 5% CO2 cell culture chamber for 15 minutes. The cell culture plates were placed on ice, washed with pre-cooled PBS, and then degraded for 30 minutes with a degradation solution containing protease inhibitors and phosphatase inhibitors. Proteins were collected and quantified by BCA, and c-Met phosphorylation and downstream signaling pathways were examined by Western blotting.

[0300] Figure 4 shows the inhibitory effects of anti-EGFR / anti-c-Met multispecific antibodies on c-Met phosphorylation and downstream signaling pathways. The results indicate that V42 and V73 are comparable to amivantamab (BM in Figure 4) in their inhibitory effects on HGF-induced c-Met phosphorylation and downstream signaling pathways.

[0301] Example 11: Suppression of EGF-induced EGFR phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies Western blotting was used to analyze the suppression of EGF-induced EGFR phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies (including amivantamab, V42, and V73). The negative control was hIgG1 (purchased from Biointron, catalog number B117901).

[0302] Cells A549 in the logarithmic growth phase were collected, counted, and then cultured in complete medium to achieve a viable cell density of 4 × 10⁻⁶. 5 The culture medium was adjusted to cells / mL, added to a 6-well cell culture plate at 2 mL / well, and incubated in a 37°C, 5% CO2 cell culture chamber for 6 hours. Afterward, the complete medium was discarded, and RPMI medium (purchased from Hyclon, catalog number SH30809.01) was added at 1 mL / well, followed by overnight starvation. The overnight starved 6-well cell culture plate was removed, the original medium was discarded, and either a final concentration of 200 nM anti-EGFR / anti-c-Met multispecific antibody was added, or 40 ng / mL final concentration EGF (purchased from R&D, catalog number 236-EG) and a final concentration of 200 nM anti-EGFR / anti-c-Met multispecific antibody were added simultaneously. The cells were then incubated in a 37°C, 5% CO2 cell culture chamber for 15 minutes. Cell culture plates were placed on ice, washed with pre-cooled PBS, and then digested for 30 minutes with a digestion solution containing protease inhibitors and phosphatase inhibitors. Proteins were collected and quantified by BCA, and EGFR phosphorylation and downstream signaling pathways were examined by Western blotting.

[0303] Figure 5 shows the inhibitory effects of anti-EGFR / anti-c-Met multispecific antibodies on EGFR phosphorylation and downstream signaling pathways. The results indicate that V42 and V73 are comparable to amivantamab (BM in Figure 5) in their inhibitory effects on EGF-induced EGFR phosphorylation and downstream signaling pathways.

[0304] Example 12: Activity of anti-EGFR / anti-c-Met multispecific antibody to bind to c-Met in competition with HGF ligand. ELISA was used to analyze the activity of anti-EGFR / anti-c-Met multispecific antibodies (including amivantamab, V42, and V73) in competing with HGF ligands to bind to c-Met. The negative control was hIgG1 (purchased from Biointron, catalog number B117901).

[0305] 2.0 mg of biotin (purchased from Thermo, catalog number 20217) was weighed out, and 590 μL of DMSO (purchased from Sigma, catalog number D2650) was added to dissolve and mix well. Biotin solution was added in a ratio of 2.7 μL to 100 μL of HGF protein (prepared according to Example 10, with a concentration of 2 mg / mL), mixed, and incubated at room temperature for 40 minutes to prepare biotin-labeled HGF. A highly adsorbent 96-well plate coated with c-Met-His (purchased from Corning, catalog number 9018) was removed after incubation overnight at 4°C, the solution in the plate was shaken off, the plate was washed three times with PBST, and the plate was closed with 3% BSA at room temperature for 2 hours. The plate was then washed three times with PBST, and an equivolute mixture of biotin-labeled HGF and anti-EGFR / anti-c-Met multispecific antibody (final concentration of anti-EGFR / anti-c-Met multispecific antibody: 100 nM, final concentration of HGF protein: 50 ng / mL, 10 ng / mL, or 1 ng / mL) was added and incubated at room temperature for 2 hours. The plate was washed three times with PBST, HRP-Avidin antibody (purchased from Invitrogen, catalog number 18-4100-51) was added, and incubated at room temperature for 1 hour. The plate was washed five times with PBST, TMB (purchased from Thermo, 00-4201-56) was added, and the mixture was incubated at room temperature away from light for 10 minutes. A 1 M reaction stop solution containing sulfuric acid was added, and the mixture was left at room temperature for 5 minutes. The absorbance value at a wavelength of 450 nm was read using a microplate reader with 630 nm as the reference wavelength.

[0306] Figures 6A to 6C show the activity of anti-EGFR / anti-c-Met multispecific antibodies in competing with HGF ligands to bind to c-Met. As a result, it was shown that V42 and V73 were nearly comparable to amivantamab in terms of activity in competing with HGF ligands to bind to c-Met.

[0307] Example 13: Inhibitory activity of tumor cell proliferation by anti-EGFR / anti-c-Met multispecific antibody The tumor cell proliferation inhibitory activity of anti-EGFR / anti-c-Met multispecific antibodies (including amivantamab, V42, and V73) was analyzed using a microplate reader, and the negative control was hIgG1 (purchased from Biointron, catalog number B117901). Logarithmic growth phase cells of NCI-H292 (expressing moderately in EGFR, moderately to low c-Met expression, source: BeNa Culture Collection) and KP4 (expressing moderately in EGFR, low c-Met expression, source: Cobioer) were collected and the viable cell density was increased to 1.5 × 10⁶ in RPMI medium containing 2% fetal bovine serum (purchased from Hyclon, catalog number SH30809.01). 4 ~2×10 4 The antibody was adjusted to cells / mL and added at a rate of 100 μL / well to a 96-well plate (purchased from Costar, catalog number 3599). Anti-EGFR / anti-c-Met multispecific antibodies were prepared using RPMI medium. For NCI-H292 cells, the maximum concentration was 2500 nM, with a 5-fold gradient dilution resulting in a total of 10 concentration steps. For KP4 cells, the maximum concentration was 555 nM, with a 3-fold gradient dilution resulting in a total of 8 concentration steps. 100 μL / well of the antibodies at different concentrations were added to the 96-well cell culture plate. In a 37°C, 5% CO2 incubator, 50 μL of HGF at a final concentration of 1 ng / mL was added to the medium for NCI-H292 cells, and the culture was incubated for 144 hours. For KP4 cells, the culture was incubated for 120 hours. 20 μL / well of CCK8 activator was added to the cell culture plate, and the cells were incubated at 37°C, 5% CO2 incubator for 2 hours. The absorbance value at a wavelength of 450 nm was read using a microplate reader with a reference wavelength of 630 nm.

[0308] Figures 7A-7B and Table 8 show the tumor cell proliferation inhibitory activity of anti-EGFR / anti-c-Met multispecific antibodies. The results indicated that V73 and V42 were superior to amivantamab in tumor cell proliferation inhibitory activity in NCI-H292 and KP4 cells.

[0309] [Table 8]

[0310] Example 14: ADCC effect on tumor cells by anti-EGFR / anti-c-Met multispecific antibody We studied the antibody-dependent cytotoxicity (ADCC) effect on tumor cells using human PBMCs (peripheral blood mononuclear cells) with anti-EGFR / anti-c-Met multispecific antibodies (including amivantamab, V42, and V73). The negative control was hIgG1 (purchased from Biointron, catalog number B117901). NCI-H292 and KP4 cells were collected during the logarithmic growth phase and incubated in RPMI medium containing 2% fetal bovine serum (purchased from Hyclon, catalog number SH30809.01) to a viable cell density of 3 × 10⁻⁶. 5 Target cells were prepared at a concentration of 10 cells / mL. Anti-EGFR / anti-c-Met multispecific antibodies were prepared using RPMI medium, with a maximum concentration of 8 nM to 200 nM, using a 5-fold gradient dilution to create a total of 8 to 10 concentration gradients. After resuscitation of PBMC cells, they were counted, and the PBMC live cell density was set to 1.5 × 10⁶ using RPMI medium. 6 Effector cells were prepared by adjusting the cell / mL concentration. The following treatment groups were established: administration group (50 μL target cells + 100 μL effector cells + 50 μL antibody), target cell group (50 μL target cells + 150 μL medium), effector cell group (100 μL effector cells + 100 μL medium), target cell + effector cell group (50 μL target cells + 100 μL effector cells + 50 μL medium), blank control group (200 μL medium), decomposition control group (200 μL medium + 20 μL decomposition), and maximum target cell release group (50 μL target cells + 150 μL medium + 20 μL decomposition). These were added to 96-well plates (purchased from Costar, catalog number 3599) so that the ratio of effector cells to target cells was 10:1. The cells were incubated at 37°C, 5% CO2 for 24 hours. The non-radioactive cytotoxicity test kit CytoTox96 (registered trademark) (purchased from Promega, catalog number G1780) was used for testing. Finally, the absorbance value at a wavelength of 490 nm was measured using a microplate reader.

[0311]

number

[0312] Figures 8A to 8B and Table 9 show the ADCC effect of anti-EGFR / anti-c-Met multispecific antibodies on tumor cells.

[0313] [Table 9]

[0314] Example 15: Endocytosis activity of anti-EGFR / anti-c-Met multispecific antibody against tumor cells Flow cytometry was used to analyze the endocytotic activity of anti-EGFR / anti-c-Met multispecific antibodies (including amivantamab, V42, and V73) in A431, NCI-H1975, and NCI-H441 cells expressing EGFR and c-Met (expression in EGFR and c-Met, source: BeNa Culture Collection). The negative control was hIgG1 (purchased from Biointron, catalog number B117901).

[0315] A431 cells, NCI-H1975 cells, and NCI-H441 cells were collected during the logarithmic growth phase, and their respective viable cell densities were measured in complete culture medium at 2 × 10⁻⁶. 6The concentration was adjusted to cells / mL and inoculated at 20 μL / well into a 96-well V-shaped cell culture plate (purchased from Costar, catalog number 3894). Conjugates of anti-EGFR / anti-c-Met multispecific antibody and Antibody Internalization Human Reagent were prepared according to the instructions for Antibody Internalization Human Reagent (purchased from Sartorius, catalog number 90564) so ​​that the final concentration of the anti-EGFR / anti-c-Met multispecific antibody was 100 nM to 167 nM, and incubated at 37°C for 15 minutes. Subsequently, a 5-fold dilution was performed to create a total of 10 concentration gradients. The conjugate solution was added to the cell culture plate at 20 μL / well, mixed thoroughly, and incubated at 37°C in a 5% CO2 incubator for 2 hours. Changes in fluorescence values ​​were measured using the RL1 channel of a flow cytometer iQue3, and data analysis was performed using software.

[0316] Figures 9A to 9C and Table 10 show the endocytotic activity of anti-EGFR / anti-c-Met multispecific antibodies against A431 cells, NCI-H1975 cells, and NCI-H441 cells.

[0317] [Table 10]

[0318] Example 16: Pharmacological evaluation of anti-EGFR / anti-c-Met multispecific antibody in a U-87MG human glioma nude mouse subcutaneous tumor model. SPF-grade female BALB / c-nu nude mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were subcutaneously inoculated with U-87MG cells (ATCC HTB-14®), and tumors of 100-150 mm were observed. 3 After proliferation, the mice were divided into a model group (physiological saline), a V42 group, and a V73 group, with 8 mice in each group.

[0319] The first administration day was day 0 (D0), and the drug (0.25 mg / kg or 0.75 mg / kg) was intravenously injected (i.v.). The injection was administered twice a week for a total of 6 times, with a volume of 0.1 mL injected per 10 g of mouse body weight. The tumors were measured twice a week using a caliper with a secondary scale. The therapeutic effect was evaluated based on the tumor growth inhibition rate (TGI).

[0320] The calculation formula for the test index is as follows: Tumor volume (mm 3 ) = 1 / 2 × (a × b 2 ), where a represents the major diameter of the tumor and b represents the minor diameter of the tumor. Relative tumor growth rate T / C(%) = (T - T0) / (C - C0) × 100%, TGI(%) = 100% - T / C, where T and C are the tumor volumes of the treatment group and the model group at the end of the experiment, respectively, and T0 and C0 are the tumor volumes of the treatment group and the model group at the start of the experiment, respectively.

[0321] If the tumor volume of the tumor is smaller than that at the start of the experiment, that is, T < T0 or C < C0, the tumor is defined as partially regressed (PR). When tumor regression occurs, TGI(%) = 100 - (T - T0) / T0 × 100%.

[0322] The results of the test index are shown in Table 11. In the U-87MG human glioma nude mouse subcutaneous transplantation tumor model, both V42 and V73 have significant tumor suppression effects.

[0323]

Table 11

[0324] The sequence information according to the present disclosure is summarized in Table S4 below.

[0325]

Table S4-1

Table S4-2

Table S4-3

[0326] For explanatory and disclosure purposes, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided simply because their disclosures were made prior to the filing date of this disclosure. All statements regarding the dates of these documents or expressions regarding the contents of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the dates or contents of these documents. Furthermore, in no country shall any reference to these publications herein constitute an endorsement that such publications are part of the common knowledge of the art.

[0327] Although the Disclosure has been described in detail above through a general description and specific embodiments, several modifications or improvements can be made based on the Disclosure, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the Disclosure are included within the scope of protection of the Disclosure.

Claims

1. A multispecific antibody, (i) First antigen-binding module that binds to the first antigen, (ii) A second antigen-binding module that binds to the second antigen, and (iii) comprising a third antigen-binding module that binds to the first antigen, The first antigen is c-Met, the second antigen is EGFR, and both the first antigen-binding module and the third antigen-binding module are single variable domains, each independently, (1) CDR1 containing the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 5 (X 1 CDR2 contains an amino acid sequence represented by S or T, preferably T), and CDR3 contains an amino acid sequence represented by SEQ ID NO:

6. (2) CDR1 containing the amino acid sequence represented by SEQ ID NO: 7, CDR2 containing the amino acid sequence represented by SEQ ID NO: 8, and CDR3 containing the amino acid sequence represented by SEQ ID NO: 9, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO: 1, CDR2 containing the amino acid sequence represented by SEQ ID NO: 2, and CDR3 containing the amino acid sequence represented by SEQ ID NO: 3, (4) CDR1 containing the amino acid sequence represented by SEQ ID NO: 10, CDR2 containing the amino acid sequence represented by SEQ ID NO: 11, and CDR3 containing the amino acid sequence represented by SEQ ID NO:

12. (5) CDR1 containing the amino acid sequence represented by SEQ ID NO: 13, CDR2 containing the amino acid sequence represented by SEQ ID NO: 14, and CDR3 containing the amino acid sequence represented by SEQ ID NO:

15. (6) CDR1 containing the amino acid sequence represented by SEQ ID NO: 16, CDR2 containing the amino acid sequence represented by SEQ ID NO: 17, and CDR3 containing the amino acid sequence represented by SEQ ID NO:

18. (7) CDR1 containing the amino acid sequence represented by SEQ ID NO: 19, CDR2 containing the amino acid sequence represented by SEQ ID NO: 20, and CDR3 containing the amino acid sequence represented by SEQ ID NO: 21, or (8) A multispecific antibody comprising one of the following: CDR1 containing the amino acid sequence represented by SEQ ID NO: 22, CDR2 containing the amino acid sequence represented by SEQ ID NO: 23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:

24.

2. The first antigen-binding module and the third antigen-binding module are each independent of each other. (1) CDR1 containing the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 5 (X 1 CDR2 contains an amino acid sequence represented by S or T, preferably T), and CDR3 contains an amino acid sequence represented by SEQ ID NO:

6. (2) CDR1 containing the amino acid sequence represented by SEQ ID NO: 7, CDR2 containing the amino acid sequence represented by SEQ ID NO: 8, and CDR3 containing the amino acid sequence represented by SEQ ID NO: 9, or (8) A CDR containing any one of the following: CDR1 containing the amino acid sequence represented by SEQ ID NO: 22, CDR2 containing the amino acid sequence represented by SEQ ID NO: 23, and CDR3 containing the amino acid sequence represented by SEQ ID NO:

24. Preferably, the first antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO: 7, CDR2 containing an amino acid sequence represented by SEQ ID NO: 8, and CDR3 containing an amino acid sequence represented by SEQ ID NO: 9, and the third antigen-binding module includes CDR1 containing an amino acid sequence represented by SEQ ID NO: 4, and SEQ ID NO: 5 (X 1 It comprises CDR2 containing an amino acid sequence represented by S or T, preferably T, and CDR3 containing an amino acid sequence represented by SEQ ID NO: 6, or, Preferably, the first antigen-binding module comprises CDR1 containing an amino acid sequence represented by SEQ ID NO: 7, CDR2 containing an amino acid sequence represented by SEQ ID NO: 8, and CDR3 containing an amino acid sequence represented by SEQ ID NO: 9, and the third antigen-binding module comprises CDR1 containing an amino acid sequence represented by SEQ ID NO: 22, CDR2 containing an amino acid sequence represented by SEQ ID NO: 23, and CDR3 containing an amino acid sequence represented by SEQ ID NO: 24, the multispecific antibody according to claim 1.

3. The multispecific antibody according to claim 1 or 2, wherein the first antigen-binding module and the third antigen-binding module each independently contain an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence represented by SEQ ID NO: 40, 41, 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, or 42.

4. The first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:

41. Preferably, the first antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 27, 36, or 37. More preferably, the multispecific antibody according to any one of claims 1 to 3, wherein the first antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:

36.

5. The third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:

40. Preferably, the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 26, 33, 34, or 35; more preferably, the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 35, or, Preferably, the third antigen-binding module includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:

42. More preferably, the third antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 32, 38, or 39, and even more preferably, the third antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 38, according to any one of claims 1 to 4.

6. The first antigen-binding module and the third antigen-binding module are selected from any one of the following items: (1) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO: 36, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:

35. (2) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO: 27, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:

26. (3) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO: 36, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:

33. (4) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO: 36, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:

34. (5) The first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO: 36, and the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:

32. (6) The third antigen-binding module includes an amino acid sequence represented by SEQ ID NO: 36, and the first antigen-binding module includes an amino acid sequence represented by SEQ ID NO: 38, or (7) The multispecific antibody according to any one of claims 1 to 5, wherein the third antigen-binding module comprises an amino acid sequence represented by SEQ ID NO: 36, and the first antigen-binding module comprises an amino acid sequence represented by SEQ ID NO:

39.

7. The multispecific antibody according to any one of claims 1 to 6, wherein the single variable domain is derived from a camelid or is humanized.

8. The multispecific antibody according to any one of claims 1 to 7, wherein the second antigen-binding module comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 comprising an amino acid sequence represented by SEQ ID NO: 63, HCDR2 comprising an amino acid sequence represented by SEQ ID NO: 64, and HCDR3 comprising an amino acid sequence represented by SEQ ID NO: 65, and the light chain variable region comprising LCDR1 comprising an amino acid sequence represented by SEQ ID NO: 66, LCDR2 comprising an amino acid sequence represented by SEQ ID NO: 67, and LCDR3 comprising an amino acid sequence represented by SEQ ID NO:

68.

9. The second antigen-binding module includes a heavy chain variable region of an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 69, and a light chain variable region of an amino acid sequence having at least 85%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO:

70. Preferably, the multispecific antibody according to any one of claims 1 to 8, wherein the heavy chain variable region of the second antigen-binding module comprises an amino acid sequence represented by SEQ ID NO: 69, and the light chain variable region comprises an amino acid sequence represented by SEQ ID NO:

70.

10. The multispecific antibody according to any one of claims 1 to 9, wherein the second antigen-binding module is derived from mouse, is a chimeric or humanized.

11. The multispecific antibody further comprises (iv) an Fc region composed of two Fc polypeptides, Preferably, the Fc region is an IgG Fc region, more preferably an IgG1 Fc region. Preferably, the IgG Fc region is a human IgG Fc region, more preferably a human IgG1 Fc region, according to any one of claims 1 to 10, a multispecific antibody according to any one of claims 1 to 10.

12. The Fc region includes amino acid substitutions that promote the association of two Fc polypeptides in the Fc region, and / or amino acid substitutions that reduce or eliminate the binding between the CH3 region of one of the Fc polypeptides in the Fc region and protein A. Preferably, one Fc polypeptide in the Fc region contains amino acid substitutions 349C, 366S, 368A, 407V, 435R, and 436F according to EU numbering, and the other Fc polypeptide contains amino acid substitutions 354C and 366W according to EU numbering, or Preferably, one Fc polypeptide in the Fc region contains amino acid substitutions 349C, 366S, 368A, 407V, and 435R according to EU numbering, and the other Fc polypeptide contains amino acid substitutions 354C and 366W according to EU numbering, the multispecific antibody according to any one of claims 1 to 11.

13. The third antigen-binding module and the first antigen-binding module are fused with each other, and optionally fused with each other via a peptide linker. Preferably, the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. Preferably, the first antigen-binding module is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, and the second antigen-binding module is Fab, wherein the second antigen-binding module is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region.

14. The aforementioned multispecific antibody is composed of three polypeptide chains, specifically, (1) One polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having SEQ ID It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:

87. (2) One polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 71, another polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85 It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:

87. (3) One polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 73, another polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85 It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:

87. (4) One polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 75, another polypeptide chain contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet75 It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:

87. (5) One polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence having SEQ ID It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:

87. (6) One polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO: 81, another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO: 85 It contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO: 87, or, (7) One polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 94%, 94%, 95%, 96%, 97%, 97%, 97%, 98%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO: 83, another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence represented by SEQ ID NO: 85 It includes an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by NO:

87. More preferably, the multispecific antibody is composed of three polypeptide chains, specifically, (1) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 77, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 87, (2) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 71, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:

87. (3) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 73, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:

87. (4) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 75, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:

87. (5) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 79, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:

87. (6) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 81, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 85, yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 87, or (7) One polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 83, another polypeptide chain contains an amino acid sequence represented by SEQ ID NO: 85, and yet another polypeptide chain contains an amino acid sequence represented by SEQ ID NO:

87. Preferably, the multispecific antibody is non-fucosylated, according to any one of claims 1 to 13.

15. An isolated nucleic acid comprising a nucleotide sequence encoding a multispecific antibody according to any one of claims 1 to 14.

16. A method for preparing a multispecific antibody according to any one of claims 1 to 14, A preparation method comprising culturing host cells containing the nucleic acid described in claim 15 so as to express the multispecific antibody, and isolating and purifying the multispecific antibody in a system.

17. An antibody-drug conjugate containing a multispecific antibody and a cytotoxic drug according to any one of claims 1 to 14.

18. A drug composition comprising a multispecific antibody according to any one of claims 1 to 14 or an antibody-drug conjugate according to claim 17, and a pharmaceutically acceptable auxiliary material.

19. A method for treating cMet and / or EGFR expression disorders, The method comprises administering to a required subject a therapeutically effective amount of a multispecific antibody according to any one of claims 1 to 14, an antibody-drug conjugate according to claim 17, or a drug composition according to claim 18. Preferably, the disease is a tumor. Preferably, the tumor is epithelial cell carcinoma, breast cancer, ovarian cancer, lung cancer, lung adenocarcinoma, colorectal cancer, anal cancer, prostate cancer, kidney cancer, liver cancer, bladder cancer, head and neck cancer, stomach cancer, pancreatic cancer, skin cancer, oral cancer, pharyngeal cancer, nasal cancer, tongue cancer, esophageal cancer, testicular cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, thyroid cancer, salivary gland cancer, or thymic cancer. Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer. Optionally, the method further comprises administering one or more other therapeutic agents to a subject as needed.

20. A c-Met-binding antibody having a single variable domain, wherein the single variable domain is (1) CDR1 containing the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 5 (X 1 CDR2 contains an amino acid sequence represented by S or T, and CDR3 contains an amino acid sequence represented by SEQ ID NO:

6. (2) CDR1 containing the amino acid sequence represented by SEQ ID NO: 7, CDR2 containing the amino acid sequence represented by SEQ ID NO: 8, and CDR3 containing the amino acid sequence represented by SEQ ID NO: 9, (3) CDR1 containing the amino acid sequence represented by SEQ ID NO: 1, CDR2 containing the amino acid sequence represented by SEQ ID NO: 2, and CDR3 containing the amino acid sequence represented by SEQ ID NO: 3, (4) CDR1 containing the amino acid sequence represented by SEQ ID NO: 10, CDR2 containing the amino acid sequence represented by SEQ ID NO: 11, and CDR3 containing the amino acid sequence represented by SEQ ID NO:

12. (5) CDR1 containing the amino acid sequence represented by SEQ ID NO: 13, CDR2 containing the amino acid sequence represented by SEQ ID NO: 14, and CDR3 containing the amino acid sequence represented by SEQ ID NO:

15. (6) CDR1 containing the amino acid sequence represented by SEQ ID NO: 16, CDR2 containing the amino acid sequence represented by SEQ ID NO: 17, and CDR3 containing the amino acid sequence represented by SEQ ID NO:

18. (7) CDR1 containing the amino acid sequence represented by SEQ ID NO: 19, CDR2 containing the amino acid sequence represented by SEQ ID NO: 20, and CDR3 containing the amino acid sequence represented by SEQ ID NO: 21, or (8) A CDR1 containing the amino acid sequence represented by SEQ ID NO: 22, a CDR2 containing the amino acid sequence represented by SEQ ID NO: 23, and a CDR3 containing the amino acid sequence represented by SEQ ID NO: 24, Preferably, the single variable domain includes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 40, 41, 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, or 42. More preferably, the single variable domain comprises an amino acid sequence represented by SEQ ID NO: 35, 36, 33, 34, 37, 38, or 39. More preferably, the single variable domain contains an amino acid sequence represented by SEQ ID NO: 40, 41, or 42, but does not contain an amino acid sequence represented by SEQ ID NO: 29, 30, or 31. More preferably, the single variable domain is derived from a camelid or is humanized. More preferably, the c-Met-binding antibody contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO: 44, 45, 43, 46, 47, 48, 49, 50, 51, 53, 55, 57, or 59.

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