Multi-specific antibodies targeting c-Met

Abstract

A multispecific antibody that targets c-Met is provided. Specifically provided are multispecific antibodies, nucleic acids encoding them, vectors comprising the nucleic acids, cells comprising the vectors, pharmaceutical compositions comprising the multispecific antibodies, and uses thereof in the treatment of related diseases.

Description

[0001] This application is a divisional application of the invention patent application filed on April 23, 2024, with Chinese application number 202480027331.6 and invention title "Multispecific Antibody Targeting c-Met". Technical Field

[0002] 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 and using the said multispecific antibodies. Background Technology

[0003] Epidermal growth factor receptor (EGFR, also known as ErbB1 or HER1) is a 170 kDa type I transmembrane glycoprotein encoded by the proto-oncogene c-erbB1, belonging 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 expression or kinase activity of EGFR is associated with a range of human tumors, making EGFR an attractive target for cancer therapy.

[0004] c-Mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase, a heterodimer of approximately 190 kDa composed of a 50 kDa extracellular chain (α chain) linked to a 145 kDa transmembrane chain (β chain). The transmembrane chain (β chain) includes a SEMA homology region (SEMA), a PSI plexin semaphorin-integrin (PSI) domain, four immunoglobulin-like regions in plexins and transcription factors (IPT), a transmembrane domain, a juxtamembrane domain (JM), a tyrosine kinase domain (TK), and a carboxyl-terminal tail region (CT). c-Met is a receptor expressed on the cell surface, in which the SEMA domain is one of the important elements for ligand binding. It is considered to be the binding site of its ligand, hepatocyte growth factor (HGF). HGF is synthesized by mesenchymal cells, fibroblasts, and smooth muscle cells, and it activates HGF / c-Met signaling through paracrine mechanisms to exert its biological functions.

[0005] In various tumors, overexpression of c-Met and HGF, along with the paracrine and autocrine positive feedback loops formed by HGF and c-Met, leads to abnormal activation of the HGF / c-Met signaling pathway, promoting tumor cell growth, invasion, migration, and angiogenesis. Abnormal expression of the c-Met gene is present in many cancer types, such as brain cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, and liver cancer. Immunotherapy agents, such as antibodies that bind to c-Met, can block the binding between HGF and c-Met.

[0006] In camels, besides the traditional four-chain IgG1, heavy-chain-only antibodies (HcAbs) IgG2 and IgG3, which lack a light chain, also naturally exist. Heavy-chain-only antibodies contain a single variable domain (V... H H) can specifically bind to antigens and has a high affinity for them. Based on its uniqueness, V is used... H The H domain, as part of an antibody or antigen-binding fragment, has significant advantages over conventional antibody fragments (such as scFv, Fab, etc.), such as requiring only a single domain to specifically bind antigens with high affinity, being easily modified into multivalent and multispecific forms, and V... HThe H domain is highly soluble and shows no tendency to aggregate; V H H molecules are small, thus possessing high tissue penetration; V H H does not need to pair with the light chain, and there is no light-heavy chain mismatch problem when forming bispecific or multispecific antibodies, etc.

[0007] Among all cancer patients resistant to EGFR tyrosine kinase inhibitors, approximately 60% showed increased c-Met expression, c-Met amplification, or increased HGF, indicating that c-Met is activated as a compensatory pathway when EGFR is inhibited.

[0008] Therefore, there is a need to develop multispecific antibodies targeting EGFR and c-Met. Invention Summary This disclosure provides multispecific antibodies targeting EGFR and c-Met, as well as related nucleic acids, vectors, cells, pharmaceutical compositions, preparation methods, and uses that can encode the provided antibodies.

[0009] On the one hand, this disclosure provides multispecific antibodies that contain (i) The first antigen binding module that binds to the first antigen; (ii) a second antigen-binding module that binds to a second antigen; and (iii) The third antigen-binding module that binds to the first antigen; Wherein the first antigen is c-Met and the second antigen is EGFR, and both the first antigen-binding module and the third antigen-binding module are single variable domains and each independently contains any one of the following: (1) CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; (2) CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T; (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; (4) CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 12; (5) CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 15; (6) CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (7) CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 21; or (8) CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 24.

[0010] In some implementations, the first antigen-binding module and the third antigen-binding module each independently include any one of the following: (2) CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T; (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; or (8) CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 24.

[0011] In some implementations, the first antigen-binding module and the third antigen-binding module each independently include any one of the following: (2) CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T; or (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9.

[0012] In some implementations, the first antigen-binding module and the third antigen-binding module each independently include any one of the following: (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; or (8) CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 24.

[0013] In some embodiments, the first antigen-binding module and the third antigen-binding module each independently comprise CDR1, CDR2, and CDR3 of a single variable domain as shown in 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 comprise CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 35, 36, or 38.

[0014] In some embodiments, the first antigen-binding module and the third antigen-binding module each independently comprise CDR1, CDR2, and CDR3, with amino acid sequences as shown in SEQ ID NO: 35 or 36, representing a single variable domain. In other embodiments, the first antigen-binding module and the third antigen-binding module each independently comprise CDR1, CDR2, and CDR3, with amino acid sequences as shown in SEQ ID NO: 36 or 38, representing a single variable domain.

[0015] In some specific embodiments, the first antigen-binding module comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO: 36, and the third antigen-binding module comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO: 35. In other specific embodiments, the first antigen-binding module comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO: 36, and the third antigen-binding module comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO: 38.

[0016] In some embodiments, the first antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; and the third antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T. Further, in some embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 41, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, 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 shown in SEQ ID NO: 36, and 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 shown in SEQ ID NO: 35. In some specific embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 35.

[0017] In other embodiments, the first antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; and the third antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24. Further, in some embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 41, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, 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 shown in SEQ ID NO: 36, and 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 shown in SEQ ID NO: 38. In some specific embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 38.

[0018] In some implementations, the first antigen-binding module and the third antigen-binding module bind to different epitopes of c-Met.

[0019] The second antigen-binding module provides the ability to bind to EGFR. 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 includes: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 63, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 64, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 65. The light chain variable region includes: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 66, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 67, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 68. Further, in some embodiments, the second antigen-binding module comprises a heavy chain variable region 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 shown in SEQ ID NO: 69, and a light chain variable region comprising 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 shown in SEQ ID NO: 70. In some specific embodiments, the second antigen-binding module comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 69, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70.

[0020] In some embodiments, the multispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.

[0021] In some specific embodiments, in the multispecific antibody, the first antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; the third antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and the second antigen-binding module comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 63, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 64, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 65; the light chain variable region comprises: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 66, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 67, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 68. LCDR3 with the amino acid sequence shown in SEQ ID NO: 68; wherein X1 is S or T. In a further specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 41, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 40; and the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 69, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70. In a further specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 35; and the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 69, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70.

[0022] In some other specific embodiments, in the multispecific antibody, the first antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; the third antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24; and 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 the amino acid sequence shown in SEQ ID NO: 63, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 64, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 65; the light chain variable region comprises: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 67, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 68. LCDR3 with the amino acid sequence shown in SEQ ID NO: 41. In a further specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 41, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 42; and the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 69, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70. In a further specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 38; and the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 69, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70.

[0023] In these embodiments, one alternative configuration is that both the first and third antigen-binding modules are single variable domains, the second antigen-binding module is Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc polypeptide in the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. In a further more specific embodiment, the multispecific antibody consists of three polypeptide chains: the first polypeptide chain contains the aforementioned first antigen-binding module, the third antigen-binding module, and one Fc polypeptide in the Fc domain; the second polypeptide chain contains the Fab heavy chain of the aforementioned second antigen-binding module and another Fc polypeptide in the Fc domain; and the third polypeptide chain is the Fab light chain of the aforementioned second antigen-binding module.

[0024] In some implementations, the multispecific antibody is trivalent.

[0025] In one aspect, this disclosure provides isolated nucleic acids containing nucleotide sequences encoding the multispecific antibodies described in this disclosure.

[0026] In one respect, this disclosure provides a carrier containing the nucleic acid described herein.

[0027] In one aspect, this disclosure provides a host cell containing the nucleic acid or the vector described herein.

[0028] On the other hand, this disclosure provides a method for preparing the multispecific antibody described herein, comprising culturing the host cells to express the multispecific antibody, and isolating and purifying the multispecific antibody in the system.

[0029] On the other hand, this disclosure provides pharmaceutical compositions comprising the aforementioned multispecific antibody and pharmaceutically acceptable excipients.

[0030] On the other hand, this disclosure provides the use of the multispecific antibody or the pharmaceutical composition described herein in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0031] On the other hand, this disclosure provides a method for treating tumors expressing c-Met and / or EGFR, comprising administering a therapeutically effective amount of the multispecific antibody or the pharmaceutical composition to the subject.

[0032] In another aspect, this disclosure provides an antibody that binds to c-Met, comprising a single variable domain, said single variable domain comprising: (1) CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; (2) CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T; (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; (4) CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 12; (5) CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 15; (6) CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (7) CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 21; or (8) CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 24.

[0033] In some embodiments, the single variable domain comprises the amino acid sequence shown in SEQ ID NO: 40, 41 or 42, while the single variable domain does not contain the amino acid sequence shown in SEQ ID NO: 29, 30 or 31.

[0034] The multispecific antibodies targeting EGFR and c-Met disclosed herein exhibit good anti-tumor efficacy and / or safety. Attached Figure Description

[0035] Figure 1A-1G Different anti-human c-Met V antibodies detected by flow cytometry H Binding curves of H-Fc chimeric antibody with target cells at different c-Met expression levels; Figure 2 This is a schematic diagram of the structure of an exemplary multispecific antibody of this disclosure; Figures 3A-3C The binding curves of anti-EGFR / anti-c-Met multispecific antibodies against cells expressing EGFR and c-Met, as detected by flow cytometry; Figure 4 To utilize Western blotting to detect the inhibition of c-Met phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies, the leftmost lane is the marker, BM is Amivantamab, negative is hIgG1, blank is no antibody, HGF "-" or "+" represents no HGF or HGF added, respectively, and p-cMet, p-Akt, p-Erk, and p-EGFR are phosphorylated c-Met, phosphorylated Akt, phosphorylated Erk, and phosphorylated EGFR, respectively. Figure 5 To utilize Western blotting to detect the inhibition of EGFR phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies, the leftmost lane is the marker, BM is Amivantamab, negative is hIgG1, blank is no antibody, EGF "-" or "+" represents no EGF or EGF added, respectively, and p-cMet, p-Akt, p-Erk, and p-EGFR are phosphorylated c-Met, phosphorylated Akt, phosphorylated Erk, and phosphorylated EGFR, respectively. Figures 6A-6C The curves showing the competitive binding of anti-EGFR / anti-c-Met multispecific antibodies to c-Met with different concentrations of HGF ligands for ELISA detection. Figures 7A-7B The anti-EGFR / anti-c-Met multispecific antibody inhibited the proliferation of tumor cells expressing EGFR and c-Met; Figures 8A-8B The killing effect of anti-EGFR / anti-c-Met multispecific antibodies on tumor cells expressing EGFR and c-Met; Figures 9A-9C The endocytic activity of anti-EGFR / anti-c-Met multispecific antibodies against cells expressing EGFR and c-Met was measured by flow cytometry. Invention Details Definitions and Explanations Unless otherwise stated, the following terms as used in this disclosure have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0037] The term "antibody" is used in the broadest sense to encompass natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), as well as various antibody structures of single-chain antibodies, as long as they exhibit the desired antigen-binding activity.

[0038] The term "multispecific" refers to an antibody's ability to specifically bind to multiple different antigenic determinants, such as two or more different antigenic determinants. In this article, antigenic determinant is synonymous with antigenic epitope. Typically, a bispecific antibody contains two antigen-binding sites, each specific to a different antigenic determinant. Different antigenic determinants can be expressed on the same or different cells. Different antigenic determinants can differ depending on the antigen (e.g., binding to antigens EGFR and c-Met) or can be present on the same antigen. An antigenic determinant is a specific chemical group with a certain composition and structure on the surface or other sites of an antigen molecule, capable of specifically binding to its corresponding antibody or sensitized lymphocyte. An example of an antigenic determinant is c-Met, which has multiple antigenic determinants with known or unknown structures. In this article, any antibody that can bind to two different antigenic determinants on an antigen is called a bispecific antibody. A specific bispecific antibody, for example, can bind to EGFR and c-Met.

[0039] The term "N-valent antibody" indicates that the antibody has N antigen-binding sites. For example, "bivalent antibody" or "antibody is bivalent" means that the antibody has two antigen-binding sites, and "trivalent antibody" or "antibody is trivalent" means that the antibody has three antigen-binding sites. Natural human immunoglobulin molecules typically have two antigen-binding sites, Fab molecules typically have a single antigen-binding site, and monovariable domain and scFv molecules typically have a single antigen-binding site.

[0040] The term "antigen-binding module" refers to a polypeptide molecule that specifically binds to an antigenic determinant. A specific antigen-binding module can be Fab, scFv, or a single variable domain.

[0041] The terms “first,” “second,” or “third” used in this disclosure to refer to antigen-binding modules, antigens, Fc peptides, peptide linkers, polypeptide chains, etc., are used for ease of distinction when more than one type of part is present. Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order or orientation.

[0042] The term "fusion" means that components (such as antigen-binding modules, Fc peptides, etc.) are linked directly or via one or more peptide linkers through peptide bonds. For example, some peptide linkers consist of 1 to 50 amino acids linked by peptide bonds, wherein said amino acids may be 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.

[0043] The term "variable domain" or "variable region" refers to a domain of an antibody involved in the binding of the antibody to an antigen. For example, natural four-chain antibodies (e.g., derived from humans, mice, etc.) have a heavy chain variable domain (also called a heavy chain variable domain, VH, or VH domain) and a light chain variable domain (also called a light chain variable domain, VL, or VL domain), while antibodies derived from animals such as camels or sharks have only a single heavy chain variable domain. In most cases, each variable domain of a natural antibody consists essentially of four "frame regions (FRs)" and three "complementarity-determining regions (CDRs)". The four frame regions are referred to as frame region 1 (or FR1), frame region 2 (or FR2), frame region 3 (or FR3), and frame region 4 (or FR4), respectively; the frame regions are separated by three complementarity-determining regions, referred to in the art and hereinafter as complementarity-determining regions 1 (or CDR1), 2 (or CDR2), and 3 (or CDR3), respectively. Therefore, the general structure of the variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domain, by possessing an antigen-binding site, endows the antibody with specificity for the antigen.

[0044] The term "monovariable domain" refers to a variable domain capable of specifically binding to an antigenic epitope without pairing with other variable domains. A monovariable domain typically has three CDRs (CDR1, CDR2, and CDR3) present on a single domain. In some cases, the monovariable domain can be a heavy-chain variable domain (e.g., VH); as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit essentially composed of the monovariable domain, so that the monovariable domain does not need to interact with another variable domain to form a functional antigen-binding unit). Another example of a monovariable domain is the "V" of the camel family. H H-domain (or simply "V") H H or VHH).

[0045] Use the term "V" H The term "H domain" is used to distinguish these variable domains from the heavy chain variable domains and light chain variable domains present in conventional four-chain antibodies. H The H domain specifically binds to the epitope without the need for other antigen-binding domains (this differs from the VH or VL domains in conventional four-chain antibodies, where the epitope is recognized by both the VL and VH domains). H The H domain is a small, stable, and efficient antigen recognition unit formed by a single domain.

[0046] The term "complementarity-determining region" (CDR) is also known as the "hypervariant region" (HVR). Naturally occurring four-chain antibodies typically contain six CDRs: three in the heavy chain variable region (HCDR1, HCDR2, and HCDR3) and three in the light chain variable region (LCDR1, LCDR2, and LCDR3). Heavy chain-only antibodies or those with a single variable domain typically have three CDRs (CDR1, CDR2, and CDR3).

[0047] Currently, there are many methods for defining CDRs. The Kabat definition, based on sequence variability, is the most commonly used (Elvin A. Kabat, et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); while the Chothia definition is based on the location 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 a compromise between the Kabat and Chothia definitions and is used by the AbM antibody modeling software from Oxford Molecular. The "contact" definition of CDRs is based on the analysis of available complex crystal structures. Additionally, there is the CCG definition. However, it should be noted that the boundaries of the CDRs (Continuous Derivatives) of the same antibody variable region obtained by different methods may differ; that is, the CDR sequences of the same antibody variable region defined by different methods may be different. Therefore, when referring to antibodies defined by a specific CDR sequence, the scope of the antibody also includes antibodies defined by CDR sequences of other arbitrary definitions (e.g., one or more combinations of definitions such as Kabat, IMGT, Chothia, Contact, AbM, CCG, etc.).

[0048] The term "Fab" refers to a protein composed of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of an immunoglobulin. In this text, Fab refers to the Fab molecule in its native form or modified form, i.e., a Fab heavy chain (VH-CH1, N-to-C-terminal) consisting of the variable region VH and the constant region CH1 of the heavy chain, and a Fab light chain (VL-CL, N-to-C-terminal) consisting of the variable region and the constant region CL of the light chain. A modified Fab can be, for example, a Fab with amino acid substitutions introduced into the CH1 / CL domain and / or the VH / VL domain. A specific example of a modified Fab is a Fab with amino acid substitutions introduced into the CL domain.

[0049] The term "scFv" encompasses the VH and VL domains of an immunoglobulin, wherein these domains are present within a single polypeptide chain. In some embodiments, the scFv also includes a peptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.

[0050] The terms “Fc domain,” “Fc,” or “Fc region” are used herein to define the C-terminal region of the immunoglobulin heavy chain, which contains at least a portion of the constant region. This term includes both native sequence Fc and variant Fc. The C-terminal lysine residue (Lys447) of the Fc may or may not be present. Unless otherwise stated, the amino acid residues in the Fc or constant region are numbered according to the EU numbering system, also known as the EU index, described in Kabat, E.A. 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 “Fc polypeptide” of the Fc domain refers to one of the two polypeptides that form the dimer Fc domain. For example, the Fc polypeptide of the IgG Fc domain contains IgG CH2 and IgG CH3.

[0051] The term “treatment” means administering the compounds or pharmaceutical compositions described in this disclosure to prevent, improve or eliminate a disease or one or more symptoms associated with said disease, including but not limited to: (i) preventing the occurrence of a disease or disease state in mammals, particularly when such mammals are susceptible to the disease state but have not yet been diagnosed with it; (ii) suppressing a disease or disease state, i.e., halting its development; (iii) alleviating a disease or disease state, even if the disease or disease state subsides; and (iv) reducing any direct or indirect pathological consequences of the disease or disease state.

[0052] The term "therapeutic effective amount" means (i) the amount of the disclosed compound used to treat or prevent a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the disclosed multispecific antibody, antibody-drug conjugate, or pharmaceutical composition constituting a "therapeutic effective amount" can vary depending on factors such as the compound or pharmaceutical composition and its ability to elicit a desired response in an individual, the disease state and its severity, the route of administration, and the age, sex, and weight of the mammal to be treated. Therapeutic effective amounts can also routinely be determined by those skilled in the art based on their own knowledge and the content of this disclosure.

[0053] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0054] The term "excipient" refers to any component other than the active ingredient (e.g., the antibody of this disclosure). The selection of excipients will depend to a great extent on factors such as the specific method of administration, the efficacy of the excipient in terms of solubility and stability, and the nature of the dosage form.

[0055] The term "isolated" refers to a target compound that has been isolated from its natural environment, such as an antibody or its antigen-binding fragment, V... H H or nucleic acid.

[0056] The terms “Xn” and “Xaa” are equivalent and refer to unspecified amino acids, the scope of which is specified by subsequent definitions in the relevant descriptions.

[0057] As used in this article, the term "EC" 50 "Effective concentration" refers to the concentration that elicits a maximum antibody response of 50%. EC 50 It can be measured by ELISA or FACS analysis or any other method known in the art.

[0058] “K D "Refers to the equilibrium dissociation constant, which is the self-dissociation rate constant (k)" d ) relative to the binding rate constant (k a (i.e., k) d / k a The ratio of K to α is expressed as molar concentration (M). The K of the antibody DThe value can be determined using methods well-established in the art. The K value used for antibody determination... D The preferred method is to use surface plasmon resonance (SPR) technology, preferably using a biosensor system such as the Biacore surface plasmon resonance system for analysis.

[0059] The term "identity," also known as consistency, refers to the percentage of amino acid residues in the sequence to be aligned that are identical to those in the specific amino acid sequence shown herein, after aligning the sequence to be aligned with it and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Amino acid sequence alignment for identity can be performed using various methods within the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the aligned sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences.

[0060] 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 non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Preferably, the subject according to this disclosure is a human. Unless otherwise stated, the terms "patient" and "subject" may be used interchangeably. "Subject in need" includes subjects who already have a disease or condition, subjects at risk of developing a disease or condition, and subjects who may have a disease or condition and whose purpose is to prevent, delay, or reduce a disease or condition.

[0061] As used herein, “about” means within the acceptable range of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” may, in accordance with art practice, mean within one or more standard deviations. Alternatively, “about” may mean a range of up to ±5%, such as fluctuations within ±2%, ±1%, or ±0.5% of a given specific numerical range. When a particular value is given in the scope of this disclosure, unless otherwise stated, “about” shall be understood to mean within the acceptable range of error for that particular value. In this document, unless otherwise stated, the values ​​of step parameters or conditions are implicitly modified by “about”.

[0062] The terms “comprise,” “containing,” and “comprising” and their equivalents (e.g., contain, contains, containing, include, includes, and including) shall be understood as “including but not limited to,” meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be covered.

[0063] In this document, unless the context clearly indicates otherwise, singular terms encompass plural referents, and vice versa.

[0064] Single variable structure 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 construction of drugs targeting c-Met. The single variable domain has a good affinity for human c-Met, providing targeting specificity. In some cases, the single variable domain can provide the ability to block the binding of the ligand HGF to c-Met. In particular, in some embodiments, the single variable domain cross-reacts with monkey c-Met; since monkeys are ideal experimental animals for drug toxicology studies, cross-reactivity with monkeys will facilitate the conduct of drug toxicology experiments.

[0065] This disclosure provides a single mutable structural field incorporating c-Met, which includes: (1) CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; (2) CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T; (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; (4) CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 12; (5) CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 15; (6) CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (7) CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 21; or (8) CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 24.

[0066] In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO: 1, CDR2 of the amino acid sequence shown in SEQ ID NO: 2, and CDR3 of the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO: 4, CDR2 of the amino acid sequence shown in SEQ ID NO: 5, and CDR3 of the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T. In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO: 4, CDR2 of the amino acid sequence shown in SEQ ID NO: 5, and CDR3 of the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S. In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO: 4, CDR2 of the amino acid sequence shown in SEQ ID NO: 5, and CDR3 of the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is T. In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO: 7, CDR2 of the amino acid sequence shown in SEQ ID NO: 8, and CDR3 of the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO: 10, CDR2 of the amino acid sequence shown in SEQ ID NO: 11, and CDR3 of the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO: 13, CDR2 of the amino acid sequence shown in SEQ ID NO: 14, and CDR3 of the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the single variable domain comprises: CDR1 as shown in SEQ ID NO: 16, CDR2 as shown in SEQ ID NO: 17, and CDR3 as shown in SEQ ID NO: 18. In some embodiments, the single variable domain is of camel origin. In some embodiments, the single variable domain is humanized. In some embodiments, the single variable domain comprises: CDR1 as shown in SEQ ID NO: 19, CDR2 as shown in SEQ ID NO: 20, and CDR3 as shown in SEQ ID NO: 21.In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO: 22, CDR2 of the amino acid sequence shown in SEQ ID NO: 23, and CDR3 of the amino acid sequence shown in SEQ ID NO: 24.

[0067] In some embodiments, the single variable domain 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 shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.

[0068] In some specific embodiments, the single variable domain 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 shown in SEQ ID NO: 25, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 3.

[0069] In some specific embodiments, the single variable domain 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 shown in SEQ ID NO: 40, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T.

[0070] In some more specific embodiments, the single variable domain 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 shown in SEQ ID NO: 26, 33, or 34, and comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S.

[0071] In some more specific embodiments, the single variable domain 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 shown in SEQ ID NO: 35, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is T.

[0072] In some specific embodiments, the single variable domain 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 shown in SEQ ID NO: 41, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9.

[0073] In some more specific embodiments, the single variable domain 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 shown in SEQ ID NO: 27, 36, or 37, and comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9.

[0074] In some specific embodiments, the single variable domain 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 shown in SEQ ID NO: 28, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 12.

[0075] In some specific embodiments, the single variable domain 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 shown in SEQ ID NO: 29, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 15.

[0076] In some specific embodiments, the single variable domain 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 shown in SEQ ID NO: 30, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 18.

[0077] In some specific embodiments, the single variable domain 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 shown in SEQ ID NO: 31, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 21.

[0078] In some specific embodiments, the single variable domain 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 shown in SEQ ID NO: 42, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 24.

[0079] In some more specific embodiments, the single variable domain 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 shown in SEQ ID NO: 32, 38, or 39, and comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24.

[0080] 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 with a reference sequence contains substitutions (e.g., conserved substitutions), insertions, or deletions, but the single variable domain containing that sequence retains the ability to bind to c-Met. In some embodiments, 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 are substituted, inserted, and / or deleted in the 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 implementations, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). In some implementations, substitution, insertion, or deletion occurs in the CDR region, for example, one, two, or three of CDR1, CDR2, and CDR3. In some implementations, substitution, insertion, or deletion occurs in both CDR regions and non-CDR regions.

[0081] In some embodiments, the amino acid sequence of the single variable domain comprises the amino acid sequence shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42.

[0082] In some embodiments, the amino acid sequence of the single variable domain is as shown in 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 implementations, the single variable structural domain is V H H. In some implementations, V H H is humanized. A non-human single variable domain can be "humanized" by replacing one or more amino acid residues in the original single variable domain sequence with one or more amino acid residues present at the corresponding position in the human antibody VH domain. Humanization can be expected to reduce immunogenicity. Typically, the single variable domain has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0084] In some embodiments, this disclosure provides a single variable domain that binds to the same epitope as any of the single variable domains described herein. In some specific embodiments, this disclosure provides a single variable domain that binds to the same epitope as a single variable domain comprising the amino acid sequence 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 binding to the same epitope is of camel-like origin or humanized.

[0085] Conventional techniques known to those skilled in the art can be used to competitively screen for binding to the same epitope. Therefore, in some embodiments, this disclosure provides a single variable domain that competes with any of the single variable domains described herein for binding to c-Met. In some specific embodiments, a single variable domain that competes with a single variable domain containing the amino acid sequence SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 for binding to c-Met is provided. Binding to c-Met can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art. In some embodiments, the single variable domain competing for binding to c-Met is of camel origin or humanized.

[0086] This disclosure provides some exemplary single variable domains that bind c-Met. The exemplary single variable domain CDRs (CDR1, CDR2, and CDR3) and their full-length amino acid sequences provided in this disclosure are given in Table S1 below.

[0087] Table S1. CDRs and full-length amino acid sequences of single variable domains (SEQ ID NO.)

[0088] This disclosure provides an antibody that binds to c-Met and includes the aforementioned single variable domain.

[0089] In some implementations, the antibody binding c-Met may be a monospecific antibody or a multispecific antibody.

[0090] In one embodiment, the monospecific antibody comprises an Fc domain; preferably, the Fc is the Fc of human IgG1, IgG2, IgG3 or IgG4.

[0091] In some embodiments, in the above-described c-Met-binding antibody, the single variable domain 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 shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.

[0092] In some embodiments, the c-Met-binding antibody 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 shown in SEQ ID NO: 38, 39, 43, 44, 45, 46, 47, 48, 49, 50, 51, 53, 55, 57, or 59.

[0093] 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.) that fuse a single variable domain with the Fc of human IgG1 to form a homodimer via the Fc.

[0094] This disclosure provides isolated nucleic acids containing polynucleotides encoding the antibody that binds to c-Met as described in this disclosure.

[0095] This disclosure provides a vector containing the nucleic acid described herein.

[0096] This disclosure provides a host cell that contains the nucleic acid or vector described in this disclosure.

[0097] This disclosure provides a method for preparing the c-Met-binding antibody of this disclosure, comprising culturing the host cells of this disclosure to express the c-Met-binding antibody, and isolating and purifying the c-Met-binding antibody in the system.

[0098] This disclosure provides pharmaceutical compositions comprising the c-Met-binding antibody described herein and pharmaceutically acceptable excipients.

[0099] This disclosure provides a method for treating c-Met expression-related diseases, comprising administering to a subject in need an antibody that binds to c-Met as described in this disclosure or a pharmaceutical composition thereof.

[0100] This disclosure provides a method for treating c-Met expression-related diseases, comprising administering a therapeutically effective amount of the c-Met-binding antibody or the pharmaceutical composition described herein to a subject in need.

[0101] Multispecific antibodies This disclosure provides a multispecific antibody containing a single variable domain that can target c-Met and EGFR expressed on the surface of tumor cells and has tumor-killing activity.

[0102] Using a single variable structural domain, compared to using multiple (e.g., two) Fabs, can reduce or avoid mismatches between light and heavy chains.

[0103] The multispecific antibodies disclosed herein exhibit excellent antitumor properties, such as lysing tumor cells or inhibiting tumor cell proliferation. In some embodiments, the multispecific antibodies of this disclosure can induce the killing of various tumor cells through ADCC and / or CDC effects. The multispecific antibodies of this disclosure exhibit good tumor-killing activity against tumor cells with different c-Met expression levels and / or different EGFR expression levels.

[0104] The multispecific antibody disclosed herein can inhibit downstream signaling of c-Met and / or EGFR.

[0105] The multispecific antibodies disclosed herein exhibit excellent antigen endocytosis and / or antigen degradation activity. In some embodiments, upon binding to c-Met and / or EGFR, the multispecific antibodies of this disclosure mediate c-Met and / or EGFR endocytosis, thereby reducing the expression of c-Met and / or EGFR on the surface of tumor cells.

[0106] Multispecific antibodies can be constructed using any of the single variable domains described in the "single variable domain" section. Therefore, this disclosure provides a multispecific antibody comprising... (i) The first antigen binding module that binds to the first antigen; (ii) a second antigen-binding module that binds to a second antigen; and (iii) The third antigen-binding module that binds to the first antigen; Wherein the first antigen is c-Met and the second antigen is EGFR, and both the first antigen-binding module and the third antigen-binding module are single variable domains and each independently contains any one of the following: (1) CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; (2) CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T; (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; (4) CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 12; (5) CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 15; (6) CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (7) CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 21; or (8) CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 24.

[0107] In this disclosure, both the first antigen-binding module and the third antigen-binding module are single variable domains that bind c-Met. In some embodiments, the first antigen-binding module includes the complementarity-determining region described in any one of (1)-(8) above. In some embodiments, the third antigen-binding module includes the complementarity-determining region described in any one of (1)-(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 may be selected independently for combination. In some specific embodiments, the first antigen-binding module and the third antigen-binding module are the same. In other specific embodiments, the first antigen-binding module and the third antigen-binding module are different.

[0108] Table S2 exemplarily lists multispecific antibodies constructed using different combinations of the first antigen-binding module and the third antigen-binding module that define the CDR characteristics. 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, i.e., the first antigen-binding module includes: CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; and the third antigen-binding module includes: CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; wherein X1 is S or T. In some specific embodiments, the first antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; and the third antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is T. More specifically, in another embodiment, the first antigen-binding module comprises CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9; and the third antigen-binding module comprises CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6, wherein X1 is T. In 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, i.e., the first antigen-binding module includes: CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; and the third antigen-binding module includes: CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 24.In a more specific embodiment, the first antigen-binding module comprises CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9; and the third antigen-binding module comprises CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24.

[0109] Table S2. Exemplary multispecific antibodies defining CDR characteristics of the first antigen-binding module and the third antigen-binding module

[0110] Note: In the table, (X)+(Y) represents 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 contains the complementary determination region described in (2) above, and the first antigen binding module contains the complementary determination region described in (1) above.

[0111] Furthermore, in some embodiments, the first antigen-binding module and the third antigen-binding module each independently comprise 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 shown in 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 comprise 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 shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39.

[0112] In some embodiments, 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 shown in 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 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 shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. In some specific embodiments, the first antigen-binding module comprises an amino acid sequence shown in 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 comprises the amino acid sequence shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39.

[0113] In some embodiments, 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 shown in 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 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 shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42. In some specific embodiments, the third antigen-binding module comprises an amino acid sequence shown in 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 comprises the amino acid sequence shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39.

[0114] In some embodiments, 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 shown in SEQ ID NO: 41; and 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 shown in SEQ ID NO: 40. In some specific embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 41, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 40. In some more specific embodiments, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 27, 36 or 37, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 26, 33, 34 or 35.

[0115] In some embodiments, 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 shown in SEQ ID NO: 41, and 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 shown in SEQ ID NO: 42. In some specific embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 41, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 42. In some more specific embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 27, 36, or 37, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 32, 38, or 39.

[0116] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 27, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 26, 32, 33, 34, 35, 38, or 39.

[0117] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 27, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 26, 32, 33, 34, 35, 38, or 39.

[0118] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 26, 32, 33, 34, 35, 38, or 39.

[0119] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 26, 32, 33, 34, 35, 38, or 39.

[0120] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 37, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 26, 32, 33, 34, 35, 38, or 39.

[0121] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 37, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 26, 32, 33, 34, 35, 38, or 39.

[0122] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 27, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 26.

[0123] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 27, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 26.

[0124] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 33.

[0125] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 33.

[0126] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 34.

[0127] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 34.

[0128] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 35.

[0129] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 35.

[0130] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO:32.

[0131] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:32.

[0132] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 38.

[0133] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 38.

[0134] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO: 39.

[0135] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 39.

[0136] In one example, both the first antigen-binding module and the third antigen-binding module contain the amino acid sequence shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39.

[0137] Table S3 provides exemplary examples of multispecific antibodies constructed using different combinations of the first and third antigen-binding modules, which feature the full-length amino acid sequence.

[0138] Table S3. Exemplary multispecific antibodies defining the amino acid sequences of the first and third antigen-binding modules.

[0139] Note: In the table, (X)+(Y) represents 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" means 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. The amino acid sequences of the single variable domains involved in this table are shown in Table S1.

[0140] In this disclosure, the first antigen-binding module may be of camel origin or humanized. The third antigen-binding module may be of camel origin or humanized. Humanization can reduce immunogenicity; in some embodiments, both the first and third antigen-binding modules shown are humanized. In some embodiments, both the first and third antigen-binding modules are of camel origin.

[0141] In the multispecific antibody disclosed herein, the second antigen-binding module provides the ability to target EGFR. The second antigen-binding module can be Fab, scFv, or ScFab (single-chain Fab). In some embodiments, the second antigen-binding module is an EGFR-binding Fab.

[0142] In some implementations, the second antigen-binding module is murine, chimeric, or humanized.

[0143] 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 the amino acid sequence shown in SEQ ID NO: 63, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 64, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 65; and the light chain variable region includes: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 66, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 67, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 68.

[0144] In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable regions shown in SEQ ID NO: 69; and the light chain variable region comprises LCDR1, HCDR2, and LCDR3 of the variable regions shown in SEQ ID NO: 70.

[0145] In some embodiments, the second antigen-binding module comprises a heavy chain variable region having 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 shown in SEQ ID NO: 69, and a light chain variable region having 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 shown in SEQ ID NO: 70. In some embodiments, the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 69, and its light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70. In some embodiments, the amino acid sequence of the heavy chain variable region of the second antigen-binding module is as shown in SEQ ID NO: 69, and the amino acid sequence of its light chain variable region is as shown in SEQ ID NO: 70.

[0146] The multispecific antibody disclosed herein may be without an Fc domain, and the first antigen-binding module, the second antigen-binding module, and the third antigen-binding module are fused together through a suitable linker.

[0147] The multispecific antibody disclosed herein may have an Fc domain, which can extend the half-life and provide Fc domain-related effectors, etc.

[0148] Multispecific antibody configuration The antigen-binding modules in the multispecific antibody disclosed herein can be fused together in various configurations. In some embodiments, the multispecific antibody further comprises (iv) an Fc domain consisting of two Fc polypeptides.

[0149] In some embodiments, the third antigen-binding module is fused to the first antigen-binding module, optionally via a peptide linker. More specifically, the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. Further, in some embodiments, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides in the Fc domain, and the second antigen-binding module is Fab, with its C-terminus of either its heavy or light Fab chain fused to the N-terminus of another Fc peptide in the Fc domain.

[0150] In one specific implementation, both the first and third antigen-binding modules 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 Fc polypeptide in the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc polypeptide in the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. This type of construction is schematically described in... Figure 2 In this embodiment, and more specifically, the multispecific antibody has three polypeptide chains. The first polypeptide chain contains the aforementioned first antigen-binding module, the third antigen-binding module, and one of the Fc polypeptides of the Fc domain. The second polypeptide chain contains the aforementioned Fab heavy chain of the second antigen-binding module and another Fc polypeptide of the Fc domain. The third polypeptide chain is the aforementioned Fab light chain of the second antigen-binding module.

[0151] In the above-described embodiments, the antigen-binding modules of the multispecific antibody can be operatively linked, either directly or via various peptide linkers (e.g., peptide linkers containing one or more amino acids, typically about 1-50 amino acids), hinge fusion, as can be reasonably chosen by those skilled in the art.

[0152] 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.

[0153] Each peptide linker can be independently adopted from any suitable one, for example, a charged and / or flexible linker polypeptide can be used. In a specific embodiment, the peptide linker consists of 1 to 50 amino acids linked by peptide bonds, wherein said amino acids may be 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 be 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, etc.

[0154] In some embodiments, the peptide linker may be a peptide linker composed of glycine and serine. In some embodiments, the peptide linker may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acids. In some embodiments, the peptide linker comprises peptide linkers in units of GGGGS. In some embodiments, the peptide linker in units of GGGGS is (GGGGS). n Where n is any number between 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or any range defined by any two of the aforementioned numbers, such as 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.

[0155] When fused with Fc, fusion is typically via the hinge region. In one embodiment, the first antigen-binding module fuses with one Fc polypeptide of the Fc domain via a first hinge, and the second antigen-binding module fuses with another Fc polypeptide of the Fc domain via a second hinge. In some embodiments, the first and second hinges are capable of forming covalent bonds, such as disulfide bonds, with each other. The first and / or second hinges may contain amino acids from the hinge region of human IgG, which contains a native hinge region or a variant thereof. In some embodiments, the first and / or second hinges contain amino acids from the hinge region of human IgG1. In some embodiments, the first and / or second hinges contain amino acids from the hinge region of human IgG4. In some specific embodiments, the first hinge contains GEPKSSDKTHTCPPCP (SEQ ID NO: 89), and the second hinge contains EPKSCDKTHTCPPCP (SEQ ID NO: 90).

[0156] Fc domain The Fc domain of a multispecific antibody consists of a pair of polypeptide chains containing heavy chain domains of immunoglobulin molecules. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, with each Fc polypeptide containing CH2 and CH3 of the constant region of the IgG heavy chain. The two Fc polypeptides of the Fc domain can stably associate with each other. In one embodiment, the multispecific antibody of this disclosure comprises one Fc domain.

[0157] In some embodiments, the Fc domain of the multispecific antibody is an IgG Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the Fc domain is a human Fc domain. In some specific embodiments, the Fc domain is a human IgG1 Fc domain.

[0158] In some embodiments, the Fc domain contains modifications, such as amino acid substitutions. These modifications may, for example, be modifications that promote heterodimerization or modifications that alter the binding affinity to protein A.

[0159] In some implementations, the Fc includes modifications that promote heterodimerization.

[0160] The multispecific antibody disclosed herein comprises a different antigen-binding module fused to one or the other of two Fc polypeptides in the Fc domain; therefore, the two Fc polypeptides are typically contained in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides yield several possible combinations of the two polypeptides. To improve the yield and purity of the multispecific antibody in recombinant production, it is advantageous to introduce modifications into the Fc domain of the multispecific antibody that promote the binding of the desired polypeptide. Therefore, in a specific embodiment, the Fc domain comprises amino acid substitutions that promote association between the two Fc polypeptides in the Fc domain.

[0161] The most extensive protein-protein interaction between the two Fc polypeptides of the human IgG Fc domain occurs in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is performed in the CH3 domain of the Fc domain.

[0162] In a specific implementation, this modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two Fc polypeptides in the Fc domain and a "mortar" modification in the other of the two Fc polypeptides in the Fc domain. Typically, this method involves introducing a protrusion ("knob") at the interface of one Fc polypeptide and a corresponding depression ("mortar") at the interface of the other Fc polypeptide, such that the protrusion can be positioned within the depression to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing a small 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 replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0163] Therefore, in a specific implementation, in the CH3 domain of one Fc peptide of the multispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the Fc peptide that can be positioned in the CH3 domain of another Fc peptide. In the CH3 domain of the other Fc peptide, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a depression in the CH3 domain of the Fc peptide.

[0164] In some embodiments, according to EU designations, one Fc polypeptide of the Fc domain comprises amino acid substitutions of 354C and / or 366Y / W, and the other Fc polypeptide comprises amino acid substitutions of 349C, 366S, 368A, and / or 407T / V. In some specific embodiments, according to EU designations, one Fc polypeptide of the Fc domain comprises amino acid substitutions of 354C and 366Y / W, and the other Fc polypeptide comprises amino acid substitutions of 349C, 366S, 368A, and 407T / V. In some more specific embodiments, according to EU designations, one Fc polypeptide of the Fc domain comprises amino acid substitutions of 354C and 366W, and the other Fc polypeptide comprises amino acid substitutions of 349C, 366S, 368A, and 407V. In the above embodiments, the Fc may be the Fc of human IgG1. In one specific implementation, according to the EU designation, one of the Fc polypeptides in the Fc domain comprises amino acid substitutions S354C and T366W, and the other Fc polypeptide comprises amino acid substitutions Y349C, T366S, L368A, and Y407V.

[0165] In some embodiments, the Fc domain comprises a modification that reduces or eliminates the binding of the CH3 region of one Fc polypeptide to protein A (from Staphylococcus aureus). In some embodiments, the Fc domain comprises an amino acid substitution that reduces or eliminates the binding of the CH3 region of one Fc polypeptide to protein A. In some embodiments, according to EU designations, the Fc domain comprises an amino acid substitution (a) 435R or (b) 435R and 436F, which occurs only on one Fc polypeptide and not on the other. In some embodiments, according to EU designations, the Fc domain comprises an amino acid substitution (a) 435R or (b) 435R and 436F occurring only on one Fc polypeptide. In some specific embodiments, according to EU designations, the Fc domain comprises an amino acid substitution H435R and Y436F occurring only on one Fc polypeptide. In some specific embodiments, according to EU designations, the Fc domain comprises an amino acid substitution H435R occurring only on one Fc polypeptide. In the above implementation scheme, the Fc is IgG1 Fc, especially human IgG1 Fc.

[0166] In the multispecific antibodies disclosed herein, the Fc domain may include (i) modifications that promote heterodimerization and / or (ii) modifications that reduce or eliminate the binding of the CH3 region of an Fc polypeptide within the Fc domain to protein A. In some embodiments, the Fc domain includes (i) modifications that promote heterodimerization and (ii) modifications that reduce or eliminate the binding of the CH3 region of an Fc polypeptide within the Fc domain to protein A. For example, in one specific embodiment, according to EU designation, the Fc domain includes amino acid substitutions from the following group: i. 349C, 366S, 368A, 407T / V, 354C, and 366Y / W; wherein the amino acid substitutions of 354C and 366Y / W are on the same Fc polypeptide, and are not on the same Fc polypeptide as the other amino acid substitutions in (i); and ii. (a) 435R or (b) 435R and 436F, occurring only on one of the Fc peptides.

[0167] In a more specific embodiment, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: Y349C, T366S, L368A, Y407V, H435R, and Y436F, and the other Fc polypeptide comprises amino acid substitutions: S354C and T366W. In this specific embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc.

[0168] In another, more specific embodiment, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: Y349C, T366S, L368A, Y407V, and H435R, and the other Fc polypeptide comprises amino acid substitutions: S354C and T366W. In this particular embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc.

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

[0170] In some implementations, the Fc domain does not contain fucose.

[0171] In some embodiments, the multispecific antibodies of this disclosure are defucosylated. Defucosylation can enhance the interaction between the multispecific antibodies and FcγRIIIa, thereby enhancing the antibody's ADCC activity. Methods for generating multispecific antibodies with little or no fucose at the Fc domain glycosylation site without altering the amino acid sequence are known in the art, such as adjusting the composition of the culture medium in which the expressing cells are located, or knocking out fucose expression-related genes such as FUT8 in the expressing cells.

[0172] In some embodiments, the multispecific antibody described in this disclosure is 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 comprises three polypeptide chains, wherein one 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 shown in 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87.

[0174] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one 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 shown in 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87.

[0175] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one 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 shown in SEQ ID NO: 75; 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87.

[0176] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one 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 shown in SEQ ID NO: 77; 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87.

[0177] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one 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 shown in 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87.

[0178] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one 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 shown in 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87.

[0179] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one 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 shown in 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87.

[0180] In some embodiments, the multispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 71, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0181] In some embodiments, the multispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 73, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0182] In some embodiments, the multispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 75, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0183] In some embodiments, the multispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 77, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0184] In some embodiments, the multispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 79, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0185] In some embodiments, the multispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 81, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0186] In some embodiments, the multispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 83, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0187] This disclosure provides exemplary trivalent multispecific antibodies.

[0188] As an example, a multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in 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 shown in SEQ ID NO: 72, SEQ ID NO: 86, and SEQ ID NO: 88, respectively.

[0189] As an example, a multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in 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 shown in SEQ ID NO: 74, SEQ ID NO: 86, and SEQ ID NO: 88, respectively.

[0190] As an example, a multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in 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 shown in SEQ ID NO: 76, SEQ ID NO: 86, and SEQ ID NO: 88, respectively.

[0191] As an example, a multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in 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 shown in SEQ ID NO: 78, SEQ ID NO: 86, and SEQ ID NO: 88, respectively.

[0192] As an example, a multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in 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 shown in SEQ ID NO: 80, SEQ ID NO: 86, and SEQ ID NO: 88, respectively.

[0193] As an example, a multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in 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 shown in SEQ ID NO: 82, SEQ ID NO: 86, and SEQ ID NO: 88, respectively.

[0194] As an example, a multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in 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 shown in 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 provides exemplary examples of nucleotide sequences encoding multispecific antibodies.

[0196] carrier This disclosure provides vectors comprising the described nucleic acids. In some embodiments, the vector is a cloning vector; in other embodiments, the vector is an expression vector, and as a specific example, the expression vector is pcDNA3.1(+). The expression vector may optionally be any expression vector capable of expressing the multispecific antibodies described herein.

[0197] host cells This disclosure provides host cells comprising the nucleic acids or vectors of this disclosure. In some embodiments, the host cell is a suitable host cell for cloning or expressing multispecific antibodies. In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing multispecific antibodies. Mammalian cells include, for example, Chinese hamster ovary (CHO) cells and CHO-S cells.

[0198] Antibody-drug conjugates (ADCs) This disclosure provides antibody-drug conjugates comprising the multispecific antibody of this disclosure. In some embodiments, the antibody-drug conjugate comprises the multispecific antibody and a cytotoxic drug. The multispecific antibody and the cytotoxic drug are preferably linked by a linker, which may be a cleavable linker or a non-cleavable linker.

[0199] Pharmaceutical Composition This disclosure provides pharmaceutical compositions comprising the multispecific antibody of this disclosure and further comprising one or more pharmaceutically acceptable excipients. This disclosure also provides pharmaceutical compositions comprising the antibody-drug conjugate of this disclosure and further comprising one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0200] In the pharmaceutical compositions disclosed herein, the multispecific antibody may be completely unfucosylated (i.e., free of detectable fucose) or partially unfucosylated.

[0201] Methods for preparing multispecific antibodies In some embodiments, this disclosure provides a method for preparing a multispecific antibody, the method comprising: culturing the host cells to express the multispecific antibody, and optionally isolating and purifying the multispecific antibody in the system. To generate the multispecific antibody, a nucleic acid encoding the multispecific antibody is isolated and inserted into one or more vectors for further cloning and / or expression in the host cells. The nucleic acid can be obtained using various methods well known in the art, such as gene splicing and chemical synthesis.

[0202] The prepared multispecific antibodies can be purified using techniques known in the art, such as high-performance liquid chromatography, ion exchange chromatography, gel chromatography, affinity chromatography, size exclusion chromatography, ceramic hydroxyapatite (CHT) chromatography, etc. The specific conditions used to purify a particular protein depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and these are obvious to those skilled in the art. For affinity chromatography, purification can be performed using an antibody, ligand, receptor, or antigen that binds to the multispecific antibody. For example, for affinity chromatography of the multispecific antibodies of this disclosure, purification can be performed using a matrix containing protein A or protein G. The multispecific antibodies of this disclosure can be purified sequentially using affinity chromatography, ion exchange chromatography, gel chromatography, and / or CHT chromatography.

[0203] The purity of multispecific antibodies can be determined using any of a variety of well-known analytical methods, such as gel electrophoresis, high-performance liquid chromatography, size exclusion chromatography, etc.

[0204] The physicochemical properties and / or biological activities of the multispecific antibodies described herein can be identified, screened, or characterized using a variety of assays known in the art.

[0205] use This disclosure provides the use of the multispecific antibodies disclosed 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 pharmaceutical composition in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR. In some embodiments, the multispecific antibody, the antibody-drug conjugate, or the pharmaceutical composition is prepared with one or more additional therapeutic agents. The additional therapeutic agents may be oncology therapeutic agents known in the art.

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

[0208] In some embodiments, 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, thyroid cancer, salivary gland cancer, and / or thymic cancer. In some embodiments, the lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Detailed Implementation

[0209] This disclosure also provides the following specific implementation schemes, but the scope of protection of this disclosure is not limited thereto: Implementation Scheme 1. A multispecific antibody comprising (i) a first antigen-binding module that binds to a first antigen; (ii) a second antigen-binding module that binds to a second antigen; and (iii) a third antigen-binding module that binds to the first antigen; wherein the first antigen is c-Met and the second antigen is EGFR, the first antigen-binding module and the third antigen-binding module are both single variable domains and each independently comprises any one of the following: (1) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is selected from S or T, preferably T; (2) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; (4) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 12; (5) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 15; (6) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (7) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 21; or (8) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. NO: 24 shows the CDR3 of the amino acid sequence.

[0210] Implementation Scheme 2. The multispecific antibody according to Implementation Scheme 1, wherein the first antigen binding module and the third antigen binding module each independently comprise any one of the following: (1) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is selected from S or T, preferably T; (2) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; or (8) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24.

[0211] Implementation Scheme 3. The multispecific antibody according to Implementation Scheme 2, wherein the first antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; and the third antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is selected from S or T; preferably, X1 is T.

[0212] Implementation Scheme 4. The multispecific antibody according to Implementation Scheme 2, wherein the first antigen binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; and the third antigen binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24.

[0213] Implementation Scheme 5. A multispecific antibody comprising (i) a first antigen-binding module that binds to a first antigen; (ii) a second antigen-binding module that binds to a second antigen; and (iii) a third antigen-binding module that binds to the first antigen; wherein the first antigen is c-Met and the second antigen is EGFR, and both the first and third antigen-binding modules are single variable domains and each independently contains CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, or 39.

[0214] Implementation Scheme 6. The multispecific antibody according to Implementation Scheme 5, wherein the first antigen-binding module and the third antigen-binding module each independently comprise CDR1, CDR2 and CDR3 of a single variable domain as shown in SEQ ID NO: 35, 36 or 38.

[0215] Implementation Scheme 7. The multispecific antibody according to Implementation Scheme 6, wherein the first antigen binding module comprises CDR1, CDR2 and CDR3 with a single variable domain as shown in SEQ ID NO: 36, and the third antigen binding module comprises CDR1, CDR2 and CDR3 with a single variable domain as shown in SEQ ID NO: 35.

[0216] Implementation Scheme 8. The multispecific antibody according to Implementation Scheme 6, wherein the first antigen binding module comprises CDR1, CDR2 and CDR3 with a single variable domain as shown in SEQ ID NO: 36, and the third antigen binding module comprises CDR1, CDR2 and CDR3 with a single variable domain as shown in SEQ ID NO: 38.

[0217] Implementation Scheme 9. A multispecific antibody according to any one of Implementation Schemes 1-8, wherein the first antigen-binding module and the third antigen-binding module each independently comprise 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 shown in SEQ ID NO: 40, 41, 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, or 42.

[0218] Implementation Scheme 10. A multispecific antibody according to any one of Implementation Schemes 1-9, 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 shown in SEQ ID NO: 41.

[0219] Implementation Scheme 11. The multispecific antibody according to Implementation Scheme 10, 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 shown in SEQ ID NO: 27, 36, or 37; preferably, 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 shown in SEQ ID NO: 36.

[0220] Implementation Scheme 12. The multispecific antibody according to any one of Implementation Schemes 1-11, wherein 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 shown in SEQ ID NO: 40.

[0221] Implementation Scheme 13. The multispecific antibody according to Implementation Scheme 12, wherein 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 shown in SEQ ID NO: 26, 33, 34, or 35; 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 shown in SEQ ID NO: 35.

[0222] Implementation Scheme 14. A multispecific antibody according to any one of Implementation Schemes 1-11, wherein 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 shown in SEQ ID NO: 42.

[0223] Implementation Scheme 15. The multispecific antibody according to Implementation Scheme 14, wherein 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 shown in SEQ ID NO: 32, 38, or 39; 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 shown in SEQ ID NO: 38.

[0224] Implementation Scheme 16. A multispecific antibody according to any one of Implementation Schemes 1-8, wherein the first antigen-binding module and the third antigen-binding module are selected from any one of the following: (1) The first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 35; (2) The first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 27, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 26; (3) The first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 33; (4) The first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 34; (5) The first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 32; (6) The third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 36, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 38; or (7) The third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 38. The amino acid sequence shown in SEQ ID NO: 36, and the first antigen binding module contains the amino acid sequence shown in SEQ ID NO: 39.

[0225] Implementation Scheme 17. A multispecific antibody according to any one of Implementation Schemes 1-16, wherein the single variable domain is of camel origin or humanized.

[0226] Implementation Scheme 18. A multispecific antibody according to any one of Implementation Schemes 1-17, wherein the second antigen-binding module is Fab, scFv, or scFab.

[0227] Implementation Scheme 19. A multispecific antibody according to any one of Implementation Schemes 1-18, 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 the amino acid sequence shown in SEQ ID NO: 63, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 64, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 65; and the light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 67, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 68.

[0228] Implementation Scheme 20. A multispecific antibody according to any one of Implementation Schemes 1-18, 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, HCDR2 and HCDR3 of the variable region shown in SEQ ID NO: 69; and the light chain variable region comprising LCDR1, HCDR2 and LCDR3 of the variable region shown in SEQ ID NO: 70.

[0229] Implementation Scheme 21. A multispecific antibody according to any one of Implementation Schemes 1-20, wherein the second antigen-binding module comprises a heavy chain variable region having 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 shown in SEQ ID NO: 69, and a light chain variable region having 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 shown in SEQ ID NO: 70.

[0230] Implementation Scheme 22. The multispecific antibody according to Implementation Scheme 21, wherein the heavy chain variable region of the second antigen binding module contains the amino acid sequence shown in SEQ ID NO: 69, and its light chain variable region contains the amino acid sequence shown in SEQ ID NO: 70.

[0231] Implementation Scheme 23. A multispecific antibody according to any one of Implementation Schemes 1-22, wherein the second antigen-binding module is murine, chimeric, or humanized.

[0232] Implementation Scheme 24. A multispecific antibody according to any one of Implementation Schemes 1-23, wherein the third antigen-binding module and the first antigen-binding module are fused to each other, optionally fused to each other via a peptide linker.

[0233] Implementation Scheme 25. The multispecific antibody according to Implementation Scheme 24, wherein the third antigen-binding module is fused to the N-terminus of the first antigen-binding module at its C-terminus.

[0234] Implementation Scheme 26. The multispecific antibody according to any one of Implementation Schemes 1-25, wherein the multispecific antibody further comprises (iv) an Fc domain consisting of two Fc polypeptides.

[0235] Implementation Scheme 27. The multispecific antibody according to Implementation Scheme 26, wherein the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, 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 another Fc peptide of the Fc domain.

[0236] Implementation Scheme 28. The multispecific antibody according to Implementation Scheme 26 or 27, wherein the Fc domain is an IgG Fc domain, preferably an IgG1 Fc domain.

[0237] Implementation Scheme 29. The multispecific antibody according to Implementation Scheme 28, wherein the IgG Fc domain is a human IgG Fc domain, preferably a human IgG1 Fc domain.

[0238] Implementation Scheme 30. A multispecific antibody according to any one of Implementation Schemes 26-29, wherein the Fc domain comprises an amino acid substitution that promotes association between two Fc polypeptides of the Fc domain.

[0239] Implementation Scheme 31. The multispecific antibody according to Implementation Scheme 30, wherein, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions 354C and 366Y / W, and the other Fc polypeptide comprises amino acid substitutions 349C, 366S, 368A, and 407T / V.

[0240] Implementation Scheme 32. The multispecific antibody according to any one of Implementation Schemes 26-31, wherein the Fc domain comprises an amino acid substitution that reduces or eliminates the binding of the CH3 region of an Fc polypeptide in the Fc domain to protein A.

[0241] Implementation Scheme 33. The multispecific antibody according to Implementation Scheme 32, wherein, according to the EU number, the Fc domain comprises an amino acid substitution (a) 435R or (b) 435R and 436F occurring only in one of the Fc polypeptides.

[0242] Implementation Scheme 34. A multispecific antibody according to any one of Implementation Schemes 26-29, wherein, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: 349C, 366S, 368A, 407V, 435R, and 436F, and the other Fc polypeptide comprises amino acid substitutions: 354C and 366W.

[0243] Implementation Scheme 35. A multispecific antibody according to any one of Implementation Schemes 26-29, wherein, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: 349C, 366S, 368A, 407V, and 435R, and the other Fc polypeptide comprises amino acid substitutions: 354C and 366W.

[0244] Implementation Scheme 36. The multispecific antibody according to any one of Implementation Schemes 1-35, wherein the multispecific antibody is trivalent.

[0245] Implementation Scheme 37. A multispecific antibody according to any one of Implementation Schemes 1-36, wherein the multispecific antibody is composed of three polypeptide chains, wherein: (1) One 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 shown in SEQ ID NO: 77; 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87; (2) One 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 shown in SEQ ID NO: 87; The amino acid sequence shown in SEQ ID NO: 71 has 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 shown in 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 shown in SEQ ID NO: 87; (3) one 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 shown in SEQ ID NO: 87; The amino acid sequence shown in SEQ ID NO: 83 has 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 shown in 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 shown in SEQ ID NO: 87;(4) One 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 shown in SEQ ID NO: 75; 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87; (5) One 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 shown in SEQ ID NO: 87; The amino acid sequence shown in SEQ ID NO: 79 has 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 shown in 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 shown in SEQ ID NO: 87; (6) a 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 shown in SEQ ID NO: 87; The amino acid sequence shown in SEQ ID NO: 81 has 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 shown in 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 shown in SEQ ID NO: 87;Or (7) one 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 shown in 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 shown in SEQ ID NO: 85; and a third 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 shown in SEQ ID NO: 87.

[0246] Implementation Scheme 38. A multispecific antibody, said multispecific antibody being composed of three polypeptide chains, wherein: (1) One polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 77, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87; (2) One polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 71, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87; (3) One polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 73, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87; (4) One polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 75, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87; (5) One polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 79, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 71. (6) One polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 81, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87; or (7) One polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 83, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0247] Implementation Scheme 39. The multispecific antibody according to any one of Implementation Schemes 1-38, wherein the multispecific antibody is unfucosylated.

[0248] Implementation Scheme 40. An isolated nucleic acid comprising a nucleotide sequence encoding a multispecific antibody according to any one of Implementation Schemes 1-39.

[0249] Implementation Scheme 41. A vector comprising the nucleic acid according to Implementation Scheme 40.

[0250] Implementation Scheme 42. A host cell comprising the nucleic acid according to Implementation Scheme 40, or the vector according to Implementation Scheme 41.

[0251] Implementation Scheme 43. A method for preparing a multispecific antibody according to any one of Implementation Schemes 1-39, comprising culturing a host cell according to Implementation Scheme 42 to express the multispecific antibody, and isolating and purifying the multispecific antibody in the system.

[0252] Implementation Scheme 44. An antibody-drug conjugate comprising a multispecific antibody according to any one of Implementation Schemes 1-39, and a cytotoxic drug.

[0253] Implementation Scheme 45. A pharmaceutical composition comprising a multispecific antibody according to any one of Implementation Schemes 1-39 or an antibody-drug conjugate according to Implementation Scheme 44, and pharmaceutically acceptable excipients.

[0254] Implementation Scheme 46. Use of the multispecific antibody according to any one of Implementation Schemes 1-39, the antibody-drug conjugate according to Implementation Scheme 44, or the pharmaceutical composition according to Implementation Scheme 45 in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0255] Implementation Scheme 47. The use according to Implementation Scheme 46, wherein the disease is a tumor.

[0256] Implementation Scheme 48. The use according to Implementation Scheme 47, wherein 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.

[0257] Implementation Scheme 49. The use according to Implementation Scheme 48, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0258] Implementation Scheme 50. The use according to any one of Implementation Schemes 46-49, wherein the multispecific antibody, the antibody-drug conjugate, or the pharmaceutical composition is prepared with one or more additional therapeutic agents to produce the medicament.

[0259] Implementation Scheme 51. A method for treating a disease expressing c-Met and / or EGFR, comprising administering to a subject in need a therapeutically effective amount of a multispecific antibody according to any one of Implementation Schemes 1-39, an antibody-drug conjugate according to Implementation Scheme 44, or a pharmaceutical composition according to Implementation Scheme 45.

[0260] Implementation Scheme 52. The method according to Implementation Scheme 51, wherein the disease is a tumor.

[0261] Implementation Scheme 53. The method according to Implementation Scheme 52, wherein 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.

[0262] Implementation Scheme 54. The method according to Implementation Scheme 53, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

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

[0264] Implementation Scheme 56. The c-Met binding antibody according to Implementation Scheme 55, wherein the single variable domain 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 shown in SEQ ID NO: 40, 41, 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, or 42.

[0265] Implementation Scheme 57. The antibody binding c-Met according to Implementation Scheme 56, wherein the single variable domain comprises the amino acid sequence shown in SEQ ID NO: 33, 34, 35, 36, 37, 38 or 39.

[0266] Implementation Scheme 58. The antibody binding c-Met according to Implementation Scheme 56, wherein the single variable domain comprises the amino acid sequence shown in SEQ ID NO: 40, 41 or 42, while the single variable domain does not comprise the amino acid sequence shown in SEQ ID NO: 29, 30 or 31.

[0267] Implementation Scheme 59. An antibody binding to c-Met according to any one of Implementation Schemes 55-58, wherein the single variable domain is of camel origin or humanized.

[0268] Implementation Scheme 60. An antibody binding c-Met according to any one of Implementation Schemes 55-59, wherein the antibody binding c-Met 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 shown in SEQ ID NO: 44, 45, 43, 46, 47, 48, 49, 50, 51, 53, 55, 57, or 59.

[0269] For clarity, this disclosure is further illustrated by examples, but these examples are not intended to limit the scope of this disclosure. The reagents used in this disclosure are generally commercially available and can be used without further purification. The Amivantamab used in the examples is from Janssen, NDC number 57894-501-01.

[0270] Example 1: Anti-human c-Met V H Construction of H phage display library Recombinant human c-Met-Fc fusion protein (SinoBiological, catalog number 10692-H02H) was emulsified with complete Freund's adjuvant at a 1:1 volume ratio and administered as the first subcutaneous multi-site immunization to Bactrian camels. Subsequently, booster immunizations were administered every two weeks by emulsifying recombinant human c-Met-Fc fusion protein with incomplete Freund's adjuvant at a 1:1 volume ratio. Serum titers of anti-human c-Met antibodies were measured after the fourth or fifth immunization. Peripheral blood was collected from Bactrian camels after multiple rounds of immunization, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted from PBMCs and converted to cDNA. Nested PCR was used to amplify the variable region (V) of the camel antibody. H H) sequence.

[0271] The amplified V H The H-coding fragment was digested with PstI / NotI restriction enzymes and inserted into the phage vector pMECS (NTCC Plasmid Vector Bacterial Cell Gene Preservation Center, catalog number pMECS) to construct a recombinant vector. This vector was then electroporated into *E. coli* TG1 (Lucigen, catalog number 60502-1) to obtain the original library. The original library was amplified to the logarithmic growth phase, and M13KO7 helper phage (New England Biolabs, catalog number N0315S) was added for further amplification. The amplification was performed overnight at 28°C and 200 rpm with shaking. The bacterial culture was centrifuged, and the supernatant was collected. 1 / 4 volume of PEG6000 / NaCl solution (20% PEG6000 (w / v), 2.5 M NaCl) was added to the supernatant. The mixture was incubated on ice for 1-2 hours to precipitate the phage. The phage pellet was collected by centrifugation, resuspended in PBS, and stored at -80°C with 20% glycerol as V. H H phage display library.

[0272] Example 2: Anti-human c-Met V H H screening V was subjected to solid-phase panning. H H phage display library was panned, and the selected single clones were cultured and expressed by isopropyl-β-D-thiogalactoside (IPTG) to prepare supernatant.

[0273] Selected clones were identified positively using an indirect ELISA method targeting human c-Met-His (SinoBiological, catalog number 10692-H08H). Positive clones that bound only human c-Met-His and had high signal values ​​were selected for preservation and sequencing. Positive clones 1B-1B2, 1B-3B11, 1B-1C7, 1B-1B6, 1B-1A8, 3B-1C7, 4&5B-2F01, and 4&5C-12B04 were obtained. Sequence analysis showed that 1B-3B11 had a V... H The amino acid sequence of H is shown in SEQ ID NO: 25, and V of 1B-1B6. H The amino acid sequence of H is shown in SEQ ID NO: 26, and V of 1B-1C7. H The amino acid sequence of H is shown in SEQ ID NO: 27; V of 1B-1A8 H The amino acid sequence of H is shown in SEQ ID NO: 28; V of 1B-1B2 H The amino acid sequence of H is shown in SEQ ID NO:29; V of 3B-1C7 H The amino acid sequence of H is shown in SEQ ID NO: 30, and V of 4&5B-2F01. H The amino acid sequence of H is shown in SEQ ID NO: 31; V of 4&5C-12B04 H The amino acid sequence of H is shown in SEQ ID NO: 32.

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

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

[0276] Example 4: Anti-human V H Affinity of H-Fc chimeric antibody to human and cynomolgus monkey c-Met 4.1 Determination of the affinity of the antibody for human and cynomolgus monkey c-Met using surface plasmon resonance (SPR) technique Anti-human c-Met V was analyzed using a biomolecular interaction analysis system (GE, Biacore T200 or Biacore 8K). H Affinity detection of H-Fc chimeric antibodies. Amino-conjugated anti-hIgG (Fc) antibody (GE, catalog no. BR-1008-39) was fed into the CM5 sensor chip and diluted with 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) to detect the affinity of anti-human c-Met V. HH-Fc chimeric antibody was diluted to 1 μg / mL and captured via the experimental channel at a flow rate of 30 μL / min. Human c-Met-His (SinoBiological, catalog number 10692-H08H) or cynomolgus c-Met-His (SinoBiological, catalog number 90304-C08H) was diluted with running buffer to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM, respectively, and bound at a flow rate of 50 μL / min for 200 s. Dissociation was then initiated after 600–1400 s. Data signals were acquired in real-time using BiaControl Software 2.0 or Biacore 8K Control Software 3.0, and analyzed using BiaEvaluation Software 2.0 or Biacore Insight Evaluation Software 3.0. The binding rate constant k was calculated using a Langmuir 1:1 model. a (1 / Ms), dissociation rate constant k d (1 / s), equilibrium dissociation constant K D (M) value. The detection results are shown in Tables 1-1 and 1-2. 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 have high affinity for human c-Met protein, and all of them cross-react with cynomolgus monkey c-Met protein.

[0277] Table 1-1. V H Affinity of H-Fc chimeric antibody to human and cynomolgus monkey c-Met

[0278] Table 1-2. V H Affinity of H-Fc chimeric antibody to human and cynomolgus monkey c-Met

[0279] 4.2 Flow cytometry determination of antibody-cell binding Flow cytometry was used to detect anti-human c-Met V. HThe H-Fc chimeric antibody was used to bind to target cells with different c-Met expression levels. Specifically, NCI-H1993 cells (Beina Biotechnology, catalog number BNCC342186) are a human lung adenocarcinoma cell line with high c-Met expression; MKN45 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60541) are a human gastric cancer cell line with moderate c-Met expression; KP4 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60541) are a human pancreatic cancer cell line with low c-Met expression; NCI-H1975 cells (Beina Biotechnology, catalog number BNCC100690) are a human non-small cell lung adenocarcinoma cell line with low to moderate c-Met expression; and NCI-H292 cells (Beina Biotechnology, catalog number BNCC100671) are a human epidermal lung cancer cell line with low to moderate c-Met expression. The antibody was serially diluted (initial concentration 100 nM, 5-fold serial dilutions, 7 concentrations) with anti-human c-Met V. H H-Fc chimeric antibody incubation 2 × 10 5 Target cells were incubated on ice for 1 hour, then washed and incubated on ice for 0.5 hours with PE-labeled anti-human IgG Fc antibody (Jackson Immuno Research, catalog number 109-116-170). After washing, the cells were analyzed using flow cytometry (Thermo Fisher Scientific Inc., Attune NXT). Results are as follows: Figure 1A-1G As shown in 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 showed high binding ability to target cells with different c-Met expression levels. 4&5B-2F01-Fc and 4&5C-12B04-Fc were superior to the control 1016-069.

[0280] Table 2-1. Anti-human c-Met V H H-Fc chimeric antibody binding to target cells

[0281] Table 2-2. Anti-human c-Met V H H-Fc chimeric antibody binding to target cells

[0282] Example 5: Anti-human c-Met V H Epitope Differences Among Different Clones Epitope competition analysis was performed using a biomolecular interaction system (Fortebio, catalog number Octet RED96). An Anti-Penta-HIS (HIS1K) sensor (Fortebio, catalog number 18-5120) was used. The c-Met-His protein (SinoBiological, catalog number 10692-H08H) was diluted to approximately 5 μg / mL with running buffer. The sensor was immersed in the diluted antigen sample, and the immobilization height was controlled to approximately 1 nm by adjusting the binding time. The sensor was then sequentially interacted with antibody A and antibody B. 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 were: a value >60% indicated no competition between the two antibodies; a value between 20% and 60% indicated partial competition (possible epitope overlap); and a value <20% indicated complete competition between the two antibodies. A self-reaction signal (underlined portion) <20% was considered valid.

[0283] The data in Table 3-1 show that antibodies 1B-3B11-Fc and 1B-1A8-Fc are in complete competition; there is no obvious competition between 1B-3B11-Fc and 1B-1A8-Fc and the other three candidate antibodies, as they belong to different epitopes. Therefore, 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. From the data in Table 3-2, it can be seen that antibodies 3B-1C7-Fc and 1B-1B2-Fc are in complete competition. Therefore, based on the data results in Table 3-1, it can be concluded 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.

[0284] The data in Table 3-3 shows that 4&5B-2F01-Fc and 4&5C-12B04-Fc are in full competition with 1016-069.

[0285] Table 3-1. Epitope Difference Analysis

[0286] Table 3-2. Epitope Difference Analysis

[0287] Table 3-3. Epitope Difference Analysis

[0288] Example 6: Anti-human c-Met V H Construction, expression, purification, and affinity detection of H-Fc humanized antibodies Respectively against human c-Met V H The H-Fc chimeric antibodies 1B-1B6-Fc, 1B-1C7-Fc, and 4&5C-12B04-Fc were humanized. The humanized V... H The H sequence was inserted into the pcDNA3.1(+) eukaryotic expression vector containing the human IgG1 constant region, and these V sequences were expressed using the Expifectamine™ CHO Transfection Kit transient expression system (Thermo Fisher Scientific Inc., catalog number A29129). H H-Fc humanized antibodies. Specifically, humanization of the chimeric antibody 1B-1B6-Fc yielded three humanized antibodies: 1B-1B6-V1, 1B-1B6-V2, and 1B-1B6-V3, with full-length amino acid sequences as shown in SEQ ID NO: 51, 53, and 55, and full-length nucleotide sequences as shown in SEQ ID NO: 52, 54, and 56, respectively. Humanization of the chimeric antibody 1B-1C7-Fc yielded two humanized antibodies: 1B-1C7-V1 and 1B-1C7-V2, with full-length amino acid sequences as shown in SEQ ID NO: 57 and 59, and full-length nucleotide sequences as shown in SEQ ID NO: 58 and 60, respectively. Humanization of the chimeric antibody 4&5C-12B04-Fc yielded two humanized antibodies. H H represents 12B04-V1 and 12B04-V2, respectively, with amino acid sequences shown in SEQ ID NO: 38 and 39, respectively.

[0289] The affinity of humanized and chimeric antibodies for human c-Met protein was detected using surface plasmon resonance (SPR) technology and a biomolecular interaction analysis system (GE, Biacore 8K). An amino-conjugated anti-hIgG (Fc) antibody (GE, catalog no. BR-1008-39) was added to the CM5 sensor chip and diluted with 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) to dilute anti-human c-Met V. H H-Fc chimeric antibody was diluted to 2 μg / mL and captured for 90 s at a flow rate of 30 μL / min through the experimental channel. Human c-Met-His protein (SinoBiological, catalog number 10692-H08H) was diluted with run buffer to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM, and bound at a flow rate of 50 μL / min. The binding signal curves were observed.

[0290] The affinity data of humanized antibodies (1B-1B6-V1, 1B-1B6-V2, 1B-1B6-V3, 1B-1C7-V1 and 1B-1C7-V2), as well as chimeric antibodies (1B-1B6-Fc and 1B-1C7-Fc) to human c-Met protein are shown in Table 4.

[0291] Table 4. Anti-human c-Met V H Binding affinity of H-Fc chimeric antibodies and humanized antibodies to human c-Met-His

[0292] Tests showed that 1B-1B6-Fc and its humanized antibody, as well as 1B-1C7-Fc and its humanized antibody, can specifically bind to the c-Met-His protein.

[0293] Example 7: Construction, expression, and purification of anti-EGFR / anti-c-Met multispecific antibodies The anti-EGFR / anti-c-Met multispecific antibody uses two humanized V antibodies targeting c-Met. H H-tandem. Modification of the Fc domain of multispecific antibodies: knock-into-hole (according to EU designations, one Fc peptide uses the Y349C, T366S, L368A, Y407V mutation; the other Fc peptide uses the S354C, T366W mutation), one of the Fc peptides in the Fc domain also contains a mutation that does not bind to protein A (according to EU designations, H435R and Y436F; or H435R).

[0294] according to Figure 2 The configurations shown construct anti-EGFR / anti-c-Met multispecific antibodies, named V12, V41, V35, V42, V67, V73, and V74, respectively. The second antigen-binding module is a Fab-type antigen-binding domain that binds to EGFR. Specifically, the anti-EGFR / anti-c-Met multispecific antibodies have three polypeptide chains, one of which (named anti-c-Met-V) H The H-Fc contains two tandem antigen-binding domains that bind c-Met (derived from chimeric antibodies and humanized antibodies of 1B-1B6-Fc, 1B-1C7-Fc, and 4&5C-12B04-Fc, as shown in Table 5). The amino acid sequences of this polypeptide chain of V12, V41, V35, V42, V67, V73, and V74 are shown in SEQ ID NO: 71, 73, 75, 77, 79, 81, and 83, respectively, and the nucleotide sequences are shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, and 84, respectively. Two other polypeptide chains form an EGFR-binding domain in Fab form (the variable region sequence for EGFR binding is derived from patent document CN100497389C), one of which is named anti-EGFR-HC-Fc (amino acid sequence shown in SEQ ID NO: 85, nucleotide sequence shown in SEQ ID NO: 84). As shown in SEQ ID NO: 86), another polypeptide chain is named anti-EGFR-LC (amino acid sequence as shown in SEQ ID NO: 87, nucleotide sequence as shown in SEQ ID NO: 88). This will encode anti-c-Met-V. H The nucleotide sequences of H-Fc, anti-EGFR-HC-Fc, and anti-EGFR-LC were inserted into the pcDNA3.1(+) eukaryotic expression vector to obtain expression vectors expressing the corresponding polypeptide chains. These expression vectors were then processed using the CHOgro® high-yield expression system (catalog number: MIR 6270) according to the anti-c-Met-V vector formula. H The H-Fc:vector anti-EGFR-HC-Fc:vector anti-EGFR-LC transfection ratio was 1.5:1:1.5, and the cells were co-transfected into CHO-S cells with the FUT8 gene knocked out (named CHO FUT8). - / - In the cells, the transfected cell density was 4 × 10⁻⁶. 6Cells / mL. Cell culture supernatant was collected by centrifugation on day 10 post-transfection. Protein purification was performed using the ÄKTA 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, Thermo Scientific). Electrophoresis (reducing SDS-PAGE), time-of-flight MS, and sequence analysis confirmed the acquisition of the aforementioned multispecific antibody with the expected structure and sequence.

[0295] Table 5. Compositional sources of the antigen-binding domains for c-Met in anti-EGFR / anti-c-Met multispecific antibodies

[0296] Example 8: Affinity of anti-EGFR / anti-c-Met multispecific antibody to human c-Met Referring to the method in Example 6, surface plasmon resonance (SPR) technology was used to detect the binding of chimeric multispecific antibodies V12 and V67, and humanized multispecific antibodies V35, V41, V42, V73, and V74 to human c-Met protein. As shown in Table 6, all antibodies specifically bound to human c-Met-His protein, and the affinity of the humanized multispecific antibodies was comparable to that of the chimeric multispecific antibodies.

[0297] Table 6. Binding affinity of anti-EGFR / anti-c-Met multispecific antibodies to human c-Met-His

[0298] Example 9: Binding of anti-EGFR / anti-c-Met multispecific antibodies to tumor cells expressing EGFR and c-Met 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 Institute of Cell Biology), NCI-H1975 cells (medium EGFR expression, medium to low c-Met expression, source: Beina Biotechnology), and NCI-H1993 cells (medium EGFR expression, high c-Met expression, source: Beina Biotechnology) was analyzed by flow cytometry. The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0299] A431 cells, NCI-H1975 cells, and NCI-H1993 cells in logarithmic growth phase were used to adjust the viable cell density to 5 × 10⁶ cells / year using RPMI medium (Hyclon, catalog number SH30809.01) containing 2% fetal bovine serum. 6 ~ 1 × 10 7Cells were seeded at 50 μL / well in a 96-well U-shaped cell culture plate (Costar, catalog number 3799). Different concentrations of anti-EGFR / anti-c-Met multispecific antibodies were prepared using the above-mentioned culture medium, with a maximum concentration of 500 nM, using a 5-fold serial dilution for a total of 10 concentration gradients. 50 μL / well of each antibody concentration was added to the 96-well cell culture plate, mixed, and incubated at 4°C for 1 hour. Cells were washed with pre-chilled Running buffer (MACS, catalog number 130-091-221), and the supernatant was discarded. Pre-chilled fluorescently labeled goat anti-human IgG antibody (Jackson, catalog number 109-116-170) was added, 100 μL / well for resuspending, and incubated at 4°C for 30 minutes. After washing, 40 μL / well of pre-chilled Running buffer was added for resuspending, mixed, and the cells were collected using an iQue3 flow cytometer. Data were analyzed using software.

[0300] Table 7 and Figures 3A-3C The binding ability of the anti-EGFR / anti-c-Met multispecific antibody to A431, NCI-H1975, and NCI-H1993 cells was demonstrated. The results showed that the binding ability of V42 and V73 to the three cell lines was essentially equivalent to that of Amivantamab.

[0301] Table 7. Binding of anti-EGFR / anti-c-Met multispecific antibodies to EGFR and c-Met on the surface of tumor cells. 50 Value and maximum binding amount

[0302] The " / " indicates that the curve cannot be fitted to a numerical value.

[0303] Example 10: Phosphorylation of c-Met by HGF-stimulated anti-EGFR / anti-c-Met multispecific antibodies and downstream signaling pathways Road inhibition Western blotting was used to analyze the effects of anti-EGFR / anti-c-Met multispecific antibodies (including Amivantamab, V42, and V73) on HGF-induced c-Met phosphorylation and the inhibition of downstream signaling pathways. The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0304] The coding sequences of HGF-α (amino acid sequence as shown in SEQ ID NO: 61) and HGF-β (amino acid sequence as shown in SEQ ID NO: 62) were inserted into the pcDNA3.1(+) eukaryotic expression vector, expressed using the Expifectamine™ CHOTransfection Kit transient expression system (Thermo Fisher Scientific Inc., catalog No. A29129), and purified using a Ni Sepharose excel nickel column (GE, catalog No. 17-3712-01) to obtain HGF.

[0305] A549 cells (expressed in EGFR and c-Met, non-small cell lung cancer, source: Cell Center of Basic Medical Sciences, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) in logarithmic growth phase were collected, counted, and then the viable cell density was adjusted to 4 × 10⁶ cells / year using RPMI medium (Hyclon, catalog number SH30809.01) containing 10% fetal bovine serum. 5 Add 2 mL of RPMI medium per well to a 6-well cell culture plate and incubate at 37°C with 5% CO2 for 6 hours. Discard the medium and add 1 mL of RPMI medium per well for overnight starvation. Remove the overnight starved 6-well cell culture plate, discard the original medium, and add anti-EGFR / anti-c-Met multispecific antibody to a final concentration of 100 nM, or simultaneously add HGF and anti-EGFR / anti-c-Met multispecific antibody to a final concentration of 100 ng / mL. Incubate at 37°C with 5% CO2 for 15 minutes. Place the cell culture plate on ice, wash with pre-chilled PBS, and lyse with lysis buffer containing protease inhibitors and phosphatase inhibitors for 30 minutes. Collect the protein and quantify it using the BCA method. Western blotting is used to detect c-Met phosphorylation and downstream signaling pathways.

[0306] Figure 4 The study demonstrated the inhibitory effects of anti-EGFR / anti-c-Met multispecific antibodies on c-Met phosphorylation and downstream signaling pathways. Results showed that V42 and V73 had inhibitory effects on HGF-stimulated c-Met phosphorylation and downstream signaling pathways similar to those of Amivantamab (…). Figure 4 It is equivalent to BM in the middle.

[0307] Example 11: EGFR phosphorylation and downstream signaling pathways stimulated by anti-EGFR / anti-c-Met multispecific antibodies. Road inhibition Western blotting was used to analyze the effect of anti-EGFR / anti-c-Met multispecific antibodies (including Amivantamab, V42, and V73) on EGF-induced EGFR phosphorylation and the inhibition of downstream signaling pathways. The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0308] Collect A549 cells in logarithmic growth phase, count them, and then adjust the viable cell density to 4 × 10⁹ cells using complete culture medium. 5 Add 2 mL / well of RPMI medium to each well of a 6-well cell culture plate and incubate at 37°C with 5% CO2 for 6 hours. Discard the complete medium and add 1 mL / well of RPMI medium (Hyclon, catalog number SH30809.01) for overnight starvation. Remove the overnight starved 6-well cell culture plate, discard the original medium, and add anti-EGFR / anti-c-Met multispecific antibody to a final concentration of 200 nM, or simultaneously add EGF (R&D, catalog number 236-EG) to a final concentration of 40 ng / mL and anti-EGFR / anti-c-Met multispecific antibody to a final concentration of 200 nM. Incubate at 37°C with 5% CO2 for 15 minutes. Place the cell culture plate on ice, wash with pre-chilled PBS, and lyse with lysis buffer containing protease inhibitors and phosphatase inhibitors for 30 minutes. Collect the protein and quantify using the BCA method. Western blotting is used to detect EGFR phosphorylation and downstream signaling pathways.

[0309] Figure 5 The study demonstrated the inhibitory effects of anti-EGFR / anti-c-Met multispecific antibodies on EGFR phosphorylation and downstream signaling pathways. Results showed that V42 and V73 had inhibitory effects on EGF-stimulated EGFR phosphorylation and downstream signaling pathways similar to those of Amivantamab (…). Figure 5 It is equivalent to BM in the middle.

[0310] Example 12: Anti-EGFR / anti-c-Met multispecific antibody competes with HGF ligand for c-Met binding activity. The activity of anti-EGFR / anti-c-Met multispecific antibodies (including Amivantamab, V42, and V73) competing with HGF ligands for c-Met binding was analyzed by ELISA. The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0311] Weigh 2.0 mg of biotin (Thermo, catalog number 20217) and add 590 µL of DMSO (Sigma, catalog number D2650), dissolve and mix well. Add biotin solution at a ratio of 2.7 µL of biotin solution per 100 µL of HGF protein (prepared according to Example 10, concentration 2 mg / mL) and mix. Incubate at room temperature for 40 min to prepare biotin-labeled HGF. Remove the c-Met-His-coated 96-well plate (Corning, catalog number 9018) that has been incubated overnight at 4°C. Discard the solution from the plate, wash three times with PBST, block with 3% BSA at room temperature for 2 hours, wash three times with PBST, add an equal volume of biotin-labeled HGF and anti-EGFR / anti-c-Met multispecific antibody mixture (final concentration of anti-EGFR / anti-c-Met multispecific antibody is 100 nM, final concentration of HGF protein is 50 ng / mL, 10 ng / mL, or 1 ng / mL), and incubate at room temperature for 2 hours. Wash three times with PBST, add HRP-Avidin antibody (Invitrogen, catalog number 18-4100-51), and incubate at room temperature for 1 hour. Wash the plate 5 times with PBST, add TMB (Thermo, 00-4201-56) and incubate at room temperature in the dark for 10 minutes. Add 1 M sulfuric acid stop solution and incubate at room temperature for 5 minutes. Use a microplate reader with 630 nm as the reference wavelength to read the absorbance value at a wavelength of 450 nm.

[0312] Figures 6A-6C The results showed that the anti-EGFR / anti-c-Met multispecific antibodies competitively bind to c-Met with HGF ligands. The results also indicated that the c-Met binding activities of V42 and V73 with HGF ligands were essentially equivalent to those of Amivantamab.

[0313] Example 13: Inhibitory activity of anti-EGFR / anti-c-Met multispecific antibodies against tumor cell proliferation The inhibitory activity of anti-EGFR / anti-c-Met multispecific antibodies (including Amivantamab, V42, and V73) on tumor cell proliferation was analyzed using an enzyme-linked immunosorbent assay (ELISA) reader. The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0314] Log-phase NCI-H292 cells (expressed in EGFR, low in c-Met, source: Beina Biotechnology) and KP4 cells (expressed in EGFR, low in c-Met, source: Kebai Biotechnology) were collected and their viable cell density was adjusted to 1.5 × 10⁶ cells / year using RPMI medium (Hyclon, catalog number SH30809.01) containing 2% fetal bovine serum. 4 ~ 2 × 10 4Cell culture medium was prepared using RPMI medium at a concentration of 100 μL / well in a 96-well plate (Costar, catalog number 3599). Anti-EGFR / anti-c-Met multispecific antibodies were prepared using RPMI medium, with a maximum concentration of 2500 nM for NCI-H292 cells (5-fold serial dilutions, 10 concentration gradients in total) and a maximum concentration of 555 nM for KP4 cells (3-fold serial dilutions, 8 concentration gradients in total). Different concentrations of antibody were added to the aforementioned 96-well cell culture plates at 100 μL / well and incubated at 37°C with 5% CO2. For NCI-H292, 50 μL of HGF was added to the medium to a final concentration of 1 ng / mL, and the culture was carried out for 144 hours; for KP4, the culture was carried out for 120 hours. CCK8 working solution was added to the cell culture plates at 20 μL / well and incubated at 37°C with 5% CO2 for 2 hours. The absorbance was read at 450 nm using a microplate reader with 630 nm as the reference wavelength.

[0315] Figures 7A-7B Table 8 shows the inhibitory activity of anti-EGFR / anti-c-Met multispecific antibodies against tumor cell proliferation. The results showed that in NCI-H292 and KP4 cells, V73 and V42 exhibited superior inhibitory activity against tumor cell proliferation compared to Amivantamab.

[0316] Table 8. IC50 of each antibody on the inhibition of NCI-H292 and KP4 cell proliferation 50 value

[0317] Example 14: ADCC effect of anti-EGFR / anti-c-Met multispecific antibody on tumor cells This study investigated the antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-EGFR / anti-c-Met multispecific antibodies (including Amivantamab, V42, and V73) on tumor cells using human PBMCs (peripheral blood mononuclear cells). The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0318] Logarithmically growing NCI-H292 and KP4 cells were harvested and their viable cell density was adjusted to 3 × 10⁶ cells / year using RPMI medium (Hyclon, catalog number SH30809.01) containing 2% fetal bovine serum. 5 PBMCs were selected as target cells at a density of [number] cells / mL. Anti-EGFR / anti-c-Met multispecific antibodies were prepared in RPMI medium to achieve concentrations ranging from 8 nM to 200 nM, with 5-fold serial dilutions for a total of 8-10 concentration gradients. After resuscitation, PBMCs were counted, and the viable cell density was adjusted to 1.5 × 10⁶ cells / mL using RPMI medium. 6Cells were selected at a density of 50 μL / mL as effector cells. The following groups were established: drug administration group (50 μL target cells + 100 μL effector cells + 50 μL antibody), target cell group (50 μL target cells + 150 μL culture medium), effector cell group (100 μL effector cells + 100 μL culture medium), target cell + effector cell group (50 μL target cells + 100 μL effector cells + 50 μL culture medium), blank control group (200 μL culture medium), lysis buffer control group (200 μL culture medium + 20 μL lysis buffer), and target cell maximum release group (50 μL target cells + 150 μL culture medium + 20 μL lysis buffer). All cells were added to 96-well plates (Costar, catalog number 3599) to achieve an effector-to-target ratio of 10:1. Cells were incubated at 37°C with 5% CO2 for 24 hours. Detection was performed using the CytoTox96® non-radioactive cytotoxicity assay kit (Promega, catalog number G1780). Finally, the absorbance value at a wavelength of 490 nm was measured using an ELISA reader.

[0319]

[0320] Figures 8A-8B Table 9 shows the ADCC effect of anti-EGFR / anti-c-Met multispecific antibodies on tumor cells.

[0321] Table 9. Effects of various antibodies on the lysis EC of NCI-H292 and KP4 cells 50 Value and maximum pyrolysis rate

[0322] Example 15: Endocytotic activity of anti-EGFR / anti-c-Met multispecific antibodies against tumor cells The endocytic activity of anti-EGFR / anti-c-Met multispecific antibodies (including Amivantamab, V42, and V73) on A431, NCI-H1975, and NCI-H441 cells (expressed in both EGFR and c-Met, source: Beina Biotechnology) was analyzed by flow cytometry. The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0323] A431 cells, NCI-H1975 cells, and NCI-H441 cells in the logarithmic growth phase were harvested and their viable cell density was adjusted to 2 × 10⁻⁶ cells using complete culture medium. 6Cells were seeded at 20 μL / well in a 96-well V-type cell culture plate (Costar, catalog number 3894). Anti-EGFR / anti-c-Met multispecific antibody complexes were prepared according to the Antibody Internalization Human Reagent (Sartorius, catalog number 90564) manufacturer's instructions, achieving a final concentration of 100–167 nM for the anti-EGFR / anti-c-Met multispecific antibody, and incubated at 37°C for 15 minutes. Subsequent 5-fold dilutions were performed for a total of 10 concentration gradients. 20 µL / well of the complex solution was added to each cell culture plate, mixed thoroughly, and incubated at 37°C with 5% CO2 for 2 hours. Fluorescence changes were detected using the RL1 channel of an iQue3 flow cytometer, and data analysis was performed using software.

[0324] Figures 9A-9C Table 10 shows the endocytic activity of the anti-EGFR / anti-c-Met multispecific antibody against A431 cells, NCI-H1975 cells, and NCI-H441 cells.

[0325] Table 10. Endocytotic activity of anti-EGFR / anti-c-Met multispecific antibodies against A431, NCI-H1975, and NCI-H441 cells.

[0326] Example 16: Subcutaneous migration of anti-EGFR / anti-c-Met multispecific antibodies in U-87 MG human glioma nude mice Pharmacodynamic evaluation in tumor-planting models SPF-grade female BALB / c-nu nude mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were subcutaneously inoculated with U-87 MG cells (ATCC HTB-14™) until the tumors grew to 100-150 mm. 3 Then, the mice were divided into a model group (physiological saline), a V42 group, and a V73 group, with 8 mice in each group.

[0327] Day 0 (D0) was the day of the first administration. The drug (0.25 mg / kg or 0.75 mg / kg) was administered intravenously (iv) twice weekly for a total of 6 administrations, with an injection volume of 0.1 mL / 10 g mouse body weight. Tumors were measured twice weekly using calipers. Efficacy was evaluated based on the tumor growth inhibition rate (TGI).

[0328] The formulas for calculating the test indicators are as follows: Tumor volume (mm) 3 ) = 1 / 2 × (a × b 2 ), where a represents the long diameter of the tumor and b represents the short diameter of the tumor.

[0329] Relative tumor proliferation 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; T0 and C0 are the tumor volumes of the treatment group and the model group at the beginning of the experiment respectively.

[0330] If the tumor is smaller than the tumor volume at the beginning of the experiment, that is, when T < T0 or C < C0, it is defined as partial tumor regression (PR). When tumor regression occurs, TGI(%) = 100 - (T - T0) / T0×100%.

[0331] The results of the detection indexes are shown in Table 11. In the subcutaneous xenograft tumor model of U - 87 MG human glioma nude mice, both V42 and V73 have significant tumor - inhibiting effects.

[0332] Table 11. Effects of anti - EGFR / anti - c - Met multispecific antibody on subcutaneous xenograft tumors of U - 87 MG human glioma nude mice

[0333] The P value is compared with the model group.

[0334] The sequence information of the present disclosure is summarized in Table S4 below.

[0335]

[0336] For purposes of description and disclosure, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided solely because their publications predate the filing date of this disclosure. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. Furthermore, in any country, any reference to these publications herein does not constitute an endorsement that such publications are part of the general knowledge in the art.

[0337] Although this disclosure has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this disclosure are all within the scope of protection claimed by this disclosure.

Claims

1. A multispecific antibody comprising (i) a first antigen-binding module that binds to a first antigen; (ii) a second antigen-binding module that binds to a second antigen; and (iii) a third antigen-binding module that binds to the first antigen; wherein, The first antigen is c-Met and the second antigen is EGFR. Both the first antigen-binding module and the third antigen-binding module are single variable domains and each independently comprises any one of the following: (1) a CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is selected from S or T, preferably T; (2) a CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; (3) a CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; (4) a CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and a CDR3 containing the amino acid sequence shown in SEQ ID NO:

12. (5) CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 15; (6) CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (7) CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 21; or (8) CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

24.

2. The multispecific antibody according to claim 1, wherein, The first antigen-binding module and the third antigen-binding module each independently include any one of the following: (1) CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is selected from S or T, preferably T; (2) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; or (8) CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

24.

3. The multispecific antibody according to claim 2, wherein, The first antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; and the third antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is selected from S or T; preferably, X1 is T.

4. The multispecific antibody according to claim 2, wherein, The first antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; and the third antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

24.

5. A multispecific antibody comprising (i) a first antigen-binding module that binds to a first antigen; (ii) a second antigen-binding module that binds to a second antigen; and (iii) a third antigen-binding module that binds to the first antigen; wherein, The first antigen is c-Met and the second antigen is EGFR. Both the first antigen-binding module and the third antigen-binding module are single variable domains and each independently contains CDR1, CDR2 and CDR3 of the single variable domain as shown in SEQ ID NO: 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38 or 39.

6. The multispecific antibody according to claim 5, wherein, The first antigen-binding module and the third antigen-binding module each independently contain CDR1, CDR2 and CDR3 with a single variable domain as shown in SEQ ID NO: 35, 36 or 38.

7. The multispecific antibody according to claim 6, wherein, The first antigen-binding module comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO: 36, and the third antigen-binding module comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:

35.

8. The multispecific antibody according to claim 6, wherein, The first antigen-binding module comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO: 36, and the third antigen-binding module comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:

38.

9. The multispecific antibody according to any one of claims 1-8, wherein, The first antigen-binding module and the third antigen-binding module each independently comprise 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 shown in SEQ ID NO: 40, 41, 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, or 42.

10. The multispecific antibody according to any one of claims 1-9, 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 shown in SEQ ID NO: 41.

Images