Antibody-drug conjugate targeting EGFR and c-met

Abstract

Belonging to the field of biomedicine, an antibody-drug conjugate targeting EGFR and c-Met is provided. The antibody-drug conjugate comprises a multispecific antibody, a linker, and a cytotoxic drug that are linked to one another. Also provided are a pharmaceutical composition comprising the antibody-drug conjugate and a use thereof. The provided antibody-drug conjugate achieves excellent antitumor activity and / or good safety. The provided antibody-drug conjugate can be used for tumor treatment.

Description

Antibody-drug conjugates targeting EGFR and c-Met Technical Field

[0001] This disclosure pertains to the field of biomedicine and relates to antibody-drug conjugates targeting EGFR and c-Met, comprising linked multispecific antibodies, adaptors, and cytotoxic drugs. This disclosure also relates to the use of said antibodies and antibody-drug conjugates in the preparation of medicaments for treating tumors. Background Technology

[0002] 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.

[0003] 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.

[0004] 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.

[0005] In camels, besides the traditional four-chain IgG1, naturally occurring heavy-chain-only antibodies (HcAbs) IgG2 and IgG3 also exist, lacking a light chain. 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 for high-affinity specific antigen binding, ease of modification into multivalent and multispecific forms, and V domain... H The 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.

[0006] Antibody-drug conjugates (ADCs) are a class of drugs that combine the high specificity of a therapeutic antibody with the high cytotoxic activity of a cytotoxic drug, with the therapeutic antibody portion and the cytotoxic drug portion linked by an intermediate linker. In all tumor patients resistant to EGFR tyrosine kinase inhibitors, approximately 60% exhibit increased c-Met expression, c-Met amplification, or increased HGF, indicating that c-Met is activated as a compensatory pathway in the presence of EGFR inhibition. While research has reported on the development of antibody-drug conjugates targeting EGFR and c-Met for antitumor purposes, the current pool is limited, and more available options are still needed.

[0007] Invention Summary

[0008] In one aspect, this disclosure provides an antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt, comprising a multispecific antibody targeting EGFR and c-Met, wherein said multispecific antibody comprises

[0009] (i) The first antigen-binding portion that binds to the first antigen;

[0010] (ii) the second antigen-binding portion that binds to the second antigen; and

[0011] (iii) The third antigen-binding portion that binds to the first antigen;

[0012] Wherein the first antigen is c-Met and the second antigen is EGFR, and both the first antigen-binding moiety and the third antigen-binding moiety are single variable domains and each independently contains any one of the following:

[0013] (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;

[0014] (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;

[0015] (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;

[0016] (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;

[0017] (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.

[0018] (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.

[0019] (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

[0020] (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.

[0021] In some embodiments, the first antigen-binding portion and the third antigen-binding portion each independently include any one of the following:

[0022] (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;

[0023] (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

[0024] (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.

[0025] In some embodiments, the first antigen-binding portion and the third antigen-binding portion each independently include any one of the following:

[0026] (2) 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; or

[0027] (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.

[0028] In some embodiments, the first antigen-binding portion and the third antigen-binding portion each independently include any one of the following:

[0029] (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

[0030] (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.

[0031] In some embodiments, the first antigen-binding portion and the third antigen-binding portion 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 portion and the third antigen-binding portion each independently comprise CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO:35, 36, or 38. The CDRs are defined, for example, according to Kabat, IMGT, Chothia, Contact, AbM, and / or CCG.

[0032] In some embodiments, the first antigen-binding portion and the third antigen-binding portion each independently comprise CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO:35 or 36. In other embodiments, the first antigen-binding portion and the third antigen-binding portion each independently comprise CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO:36 or 38. The CDRs are defined, for example, according to Kabat, IMGT, Chothia, Contact, AbM, and / or CCG.

[0033] In some specific embodiments, the first antigen-binding portion comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:36, and the third antigen-binding portion comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:35. In other specific embodiments, the first antigen-binding portion comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:36, and the third antigen-binding portion comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:38. The CDRs are defined, for example, according to Kabat, IMGT, Chothia, Contact, AbM, and / or CCG.

[0034] In some embodiments, the first antigen-binding portion 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 portion 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 portion comprises the amino acid sequence shown in SEQ ID NO:41, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:40. In some embodiments, the first antigen-binding portion 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 portion 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 portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:35.

[0035] In other embodiments, the first antigen-binding portion 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 portion 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 portion comprises the amino acid sequence shown in SEQ ID NO:41, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:42. In some embodiments, the first antigen-binding portion 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 portion 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 portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:38.

[0036] In some implementations, the first antigen-binding moiety and the third antigen-binding moiety bind different epitopes of c-Met.

[0037] The second antigen-binding region provides the ability to bind to EGFR. In some embodiments, the second antigen-binding region 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 portion 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 portion 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.

[0038] In some embodiments, the multispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.

[0039] In some specific embodiments, in the multispecific antibody, the first antigen-binding portion 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 portion 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 portion 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 comprising: 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; wherein X1 is S or T. In a further specific embodiment, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:41, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:40; and the heavy chain variable region of the second antigen-binding portion 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 portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:35; and the heavy chain variable region of the second antigen-binding portion 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.

[0040] In some other specific embodiments, in the multispecific antibody, the first antigen-binding portion 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 portion 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; and the second antigen-binding portion 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 comprising: 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. In a further specific embodiment, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:41, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:42; and the heavy chain variable region of the second antigen-binding portion 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 portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:38; and the heavy chain variable region of the second antigen-binding portion 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.

[0041] In these embodiments, one alternative configuration is that both the first and third antigen-binding moieties are single variable domains, the second antigen-binding moieties are Fab, the first antigen-binding moieties are fused at their C-terminus to the N-terminus of one Fc polypeptide in the Fc domain, the second antigen-binding moieties are fused at their C-terminus to the N-terminus of another Fc polypeptide in the Fc domain, and the third antigen-binding moieties are fused at their C-terminus to the N-terminus of the first antigen-binding moieties. In a further more specific embodiment, the multispecific antibody consists of three polypeptide chains: the first polypeptide chain contains the aforementioned first antigen-binding moieties, the third antigen-binding moieties, and one Fc polypeptide in the Fc domain; the second polypeptide chain contains the Fab heavy chain of the aforementioned second antigen-binding moieties and another Fc polypeptide in the Fc domain; and the third polypeptide chain is the Fab light chain of the aforementioned second antigen-binding moieties.

[0042] In some implementations, the multispecific antibody is trivalent.

[0043] In some embodiments, the antibody-drug conjugate is a product of the following formula Ia

[0044] The drug-connector shown in the diagram is connected to the multispecific antibody, and the drug-connector is connected to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia.

[0045] in,

[0046] R 1 and R 2 Each is independently selected from hydrogen atoms or deuterium atoms; preferably, the R 1 For hydrogen atoms, R 2 It is a deuterium atom.

[0047] This disclosure provides a method for preparing the antibody-drug conjugate of the present disclosure, its stereoisomers, its pharmaceutically acceptable salts, or solvates of said antibody-drug conjugate, its stereoisomers, and its pharmaceutically acceptable salts, comprising treating a multispecific antibody targeting EGFR and c-Met under reducing conditions, and then reacting the multispecific antibody targeting EGFR and c-Met with the linker-loaded product of the present disclosure.

[0048] On the other hand, this disclosure provides pharmaceutical compositions comprising the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, and pharmaceutically acceptable excipients.

[0049] On the other hand, this disclosure provides the use of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition thereof in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0050] On the other hand, this disclosure provides a method for treating tumors expressing c-Met and / or EGFR, comprising administering to the subject the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition thereof.

[0051] On the other hand, this disclosure provides the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition thereof, for use as a drug or for treatment.

[0052] The antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts disclosed herein target EGFR and c-Met, exhibiting good antitumor efficacy and / or safety. Attached Figure Description

[0053] Figures 1A-1G show the detection of different anti-human c-Met V antibodies by flow cytometry. H Binding curves of H-Fc chimeric antibody with target cells at different c-Met expression levels;

[0054] Figure 2 is a schematic diagram of the structure of an exemplary multispecific antibody of this disclosure;

[0055] Figures 3A-3C show the binding curves of anti-EGFR / anti-c-Met multispecific antibodies to cells expressing EGFR and c-Met, as detected by flow cytometry.

[0056] Figure 4 shows the inhibition of c-Met phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies detected by Western blotting. 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.

[0057] Figure 5 shows the inhibition of EGFR phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies using Western blotting. 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.

[0058] Figures 6A-6C show the curves of anti-EGFR / anti-c-Met multispecific antibodies competing with different concentrations of HGF ligands for c-Met, as detected by ELISA.

[0059] Figures 7A-7B show the inhibition of proliferation of tumor cells expressing EGFR and c-Met by anti-EGFR / anti-c-Met multispecific antibodies;

[0060] Figures 8A-8B illustrate the killing effect of anti-EGFR / anti-c-Met multispecific antibodies on tumor cells expressing EGFR and c-Met;

[0061] Figures 9A-9C show the internalization activity of anti-EGFR / anti-c-Met multispecific antibodies in cells expressing EGFR and c-Met, as detected by flow cytometry.

[0062] Figures 10A-10D show the binding curves of V42-DDDXd-6(1) to cells expressing EGFR and c-Met as detected by flow cytometry. Figure 10A shows HCC827 cells, Figure 10B shows NCI-H292 cells, Figure 10C shows HCA-7 cells, and Figure 10D shows NCI-H441 cells.

[0063] Figures 11A-11D show the internalization activity of V42-DDDXd-6(1) in cells expressing EGFR and c-Met as detected by flow cytometry, where Figure 11A is HCC827 cells, Figure 11B is NCI-H292 cells, Figure 11C is HCA-7 cells, and Figure 11D is NCI-H441 cells.

[0064] Figures 12A-12E show the killing effect of V42-DDDXd-6(1) on tumor cells expressing EGFR and c-Met, where Figure 12A is HCC827 cells, Figure 12B is MKN45 cells, Figure 12C is NCI-H292 cells, Figure 12D is BxPC3 cells, and Figure 12E is HCA-7 cells;

[0065] Figure 13 shows the bystander effect of V42-DDDXd-6(1) on EGFR and c-Met negative Jurkat cells in the presence of HCC827 cells;

[0066] Figure 14 shows the tumor volume curve of the xenograft tumor after administration of V42-DDDXd-6(1) in the NCI-H292 human lung cancer cell xenograft tumor model in nude mice.

[0067] Figure 15 shows the tumor volume curve of the xenograft tumor after administration of V42-DDDXd-6(1) in a nude mouse xenograft model of HCA-7 human colon cancer cells.

[0068] Invention Details

[0069] Definitions and Explanations

[0070] 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.

[0071] The term “optional” or “optionally” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0072] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxoation does not occur on the aromatic group. "Optionally substituted" means that it may or may not be substituted, unless otherwise specified; the type and number of substituents can be arbitrary to the extent chemically feasible.

[0073] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is substituted by two Rs, each R has an independent option.

[0074] The term "thiol" refers to the -SH group.

[0075] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration representing the center of a solid.

[0076] Unless otherwise specified, when a group has a connectable site, the connection position of that site with other groups can be indicated by an asterisk (*) or a hash (#). For example, the structure of "-(succinimide-3-yl-N)-" as used herein is shown in the following formula:

[0077] The asterisk (*) and the number (#) indicate that the carbon atom at position 3 and the nitrogen atom at position 1 of this structure are connected to other groups, respectively.

[0078] As used in this article, compounds formed by replacing atoms or groups of atoms in a parent compound molecule with other atoms or groups of atoms are called "derivatives" of the parent compound.

[0079] The compounds disclosed herein may exist in specific geometric or stereoisomer forms. This disclosure envisions all such compounds, including cis and trans isomers, levorotatory and dextrorotatory isomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this disclosure.

[0080] Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise because the single bonds of double bonds or cyclic carbon atoms cannot rotate freely.

[0081] Unless otherwise stated, the term "enantiomer" refers to stereoisomers that are mirror images of each other.

[0082] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0083] The compounds and intermediates disclosed herein may also exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton transfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerization. A specific example of a proton tautomer is the imidazole moiety, in which a proton can migrate between two ring nitrogens. Valence tautomers include interconversions via the recombination of some bonding electrons.

[0084] The term "bystander effect," as used herein, refers to an effect in which a cytotoxic drug coupled to an antibody via a cleavable or uncleavable linker is able to diffuse across the cell membrane after release from the antibody, thereby causing the killing of adjacent cells. The ability to diffuse across the cell membrane is related to the hydrophobicity of the cytotoxic drug or the combination of the cytotoxic drug and the linker. Such cytotoxic drugs can be, for example, ethatecan derivatives. The bystander effect may be desirable, particularly in tumors with heterogeneous target expression and in solid tumors where antibody penetration may be limited.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] The term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. The specific antigen-binding moiety can be Fab, scFv, or a single variable domain.

[0089] The terms “first,” “second,” or “third” used in this disclosure to refer to antigen-binding portions, antigens, Fc polypeptides, peptide linkers, polypeptide chains, etc., are used for ease of distinction when more than one type of portion is present. Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order or orientation.

[0090] The term "fusion" means that components (e.g., antigen-binding moieties, 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.

[0091] 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.

[0092] 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) located 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).

[0093] 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). V H The H domain is a small, stable, and efficient antigen recognition unit formed by a single domain.

[0094] 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 single variable domain antibodies typically have three CDRs (CDR1, CDR2, and CDR3).

[0095] Currently, there are many methods for defining CDRs. The Kabat definition, based on sequence variability, is the most commonly used; while the Chothia definition is based on the position of structural loops. The AbM definition is a compromise between the Kabat and Chothia definitions and is used by Oxford Molecular's AbM antibody modeling software. 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 CDRs for the same antibody variable region obtained based on different methods may differ; that is, the CDR sequences for the same antibody variable region defined by different methods may vary. Therefore, when referring to antibodies defined with a specific CDR sequence, the scope of antibodies also includes antibodies defined by CDR sequences of any other arbitrary definitions (e.g., one or more combinations of Kabat, IMGT, Chothia, Contact, AbM, CCG, etc.).

[0096] 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., the Fab heavy chain (VH-CH1, N-to-C-terminal) consisting of the VH variable region and the CH1 constant region of the heavy chain, and the Fab light chain (VL-CL, N-to-C-terminal) consisting of the VH variable region and the CL constant region 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.

[0097] The term "scFv" includes the VH and VL domains of an immunoglobulin, wherein these domains are present in 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.

[0098] 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.

[0099] 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.

[0100] The term "therapeutic effective amount" means 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 antibody-drug conjugate or pharmaceutical composition of the present disclosure 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] As used in this article, the term "EC" 50 "Effective concentration" refers to the concentration that elicits a 50% maximum response from the antibody or ADC. EC 50 It can be measured by ELISA or FACS analysis or any other method known in the art.

[0106] “K D "Refers to the equilibrium dissociation constant, which is the self-dissociation rate constant (k d ) relative binding rate constant (k a (that is, k) d / k a The ratio of the antibody to the ADC and expressed as molar concentration (M). DThe value can be determined using methods well-established in the art. K is used to determine antibodies or ADCs. 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.

[0107] 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 conservative substitutions as part of the sequence identity. Amino acid sequence alignment for identity can be performed in various ways within the scope of the art. Those skilled in the art can determine the appropriate parameters for the aligned sequences, including any algorithm required to achieve the maximum alignment across the full length of the compared sequences.

[0108] 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.

[0109] 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”.

[0110] 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.

[0111] In this document, unless the context clearly indicates otherwise, singular terms encompass plural referents, and vice versa.

[0112] Antibody-drug conjugates (ADCs)

[0113] This disclosure provides an antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt, comprising a multispecific antibody targeting EGFR and c-Met and a cytotoxic drug, wherein the multispecific antibody targeting EGFR and c-Met is conjugated to the cytotoxic drug.

[0114] Multispecific antibodies in ADCs

[0115] This disclosure provides an antibody-drug conjugate, its stereoisomer, a pharmaceutically acceptable salt thereof, or a solvation of said antibody-drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof, comprising a multispecific antibody, wherein the multispecific antibody comprises

[0116] (i) The first antigen-binding portion that binds to the first antigen;

[0117] (ii) the second antigen-binding portion that binds to the second antigen; and

[0118] (iii) The third antigen-binding portion that binds to the first antigen;

[0119] Wherein, the first antigen is c-Met and the second antigen is EGFR, and both the first antigen-binding portion and the third antigen-binding portion are single variable domains and each independently contains any one of the following:

[0120] (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;

[0121] (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;

[0122] (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;

[0123] (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;

[0124] (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.

[0125] (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.

[0126] (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

[0127] (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.

[0128] In this disclosure, both the first antigen-binding portion and the third antigen-binding portion are single variable domains that bind c-Met. In some embodiments, the first antigen-binding portion includes the complementarity-determining region described in any one of (1)-(8) above. In some embodiments, the third antigen-binding portion includes the complementarity-determining region described in any one of (1)-(8) above. In the multispecific antibody, the first antigen-binding portion and the third antigen-binding portion 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 portion and the third antigen-binding portion are the same. In other specific embodiments, the first antigen-binding portion and the third antigen-binding portion are different.

[0129] Table S1 exemplarily lists multispecific antibodies constructed using different combinations of the first antigen-binding portion and the third antigen-binding portion that define the CDR characteristics. In some embodiments, the first antigen-binding portion includes the complementarity-determining region described in (3) above, and the third antigen-binding portion includes the complementarity-determining region described in (2) above, i.e., the first antigen-binding portion 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 portion 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 portion 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 portion 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 portion 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 portion 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 portion includes the complementarity-determining region described in (3) above, and the third antigen-binding portion includes the complementarity-determining region described in (8) above, i.e., the first antigen-binding portion 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 portion 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 portion 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 portion comprises CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:24.

[0130] Table S1. Exemplary multispecific antibodies defining CDR characteristics of the first antigen-binding region and the third antigen-binding region. Note: In the table, (X)+(Y) represents the combination of the third antigen-binding part and the first antigen-binding part in a multispecific antibody. For example, (2)+(1) means that the third antigen-binding part of the multispecific antibody contains the complementary determination region described in (2) above, and the first antigen-binding part contains the complementary determination region described in (1) above.

[0131] Furthermore, in some embodiments, the first antigen-binding portion and the third antigen-binding portion 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 portion and the third antigen-binding portion 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.

[0132] In some embodiments, the first antigen-binding portion 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 portion 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 portion 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 portion 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.

[0133] In some embodiments, the third antigen-binding portion 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 portion 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 portion 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 portion 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.

[0134] In some embodiments, the first antigen-binding portion 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 portion 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 portion comprises the amino acid sequence shown in SEQ ID NO:41, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:40. In some more specific embodiments, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:27, 36 or 37, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:26, 33, 34 or 35.

[0135] In some embodiments, the first antigen-binding portion 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 portion 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 portion comprises the amino acid sequence shown in SEQ ID NO:41, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:42. In some more specific embodiments, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:27, 36 or 37, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:32, 38 or 39.

[0136] In one example, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:27, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0137] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:27, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0138] In one example, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0139] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0140] In one example, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:37, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0141] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:37, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:26, 32, 33, 34, 35, 38, or 39.

[0142] In one example, the first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:27, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:26.

[0143] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:27, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:26.

[0144] In one example, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:33.

[0145] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:33.

[0146] In one example, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:34.

[0147] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:34.

[0148] In one example, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:35.

[0149] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:35.

[0150] In one example, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:32.

[0151] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:32.

[0152] In one example, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:38.

[0153] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:38.

[0154] In one example, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:39.

[0155] In one example, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:39.

[0156] In one example, both the first antigen-binding portion and the third antigen-binding portion 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.

[0157] Table S2 provides exemplary examples of multispecific antibodies constructed using different combinations of the first and third antigen-binding portions of the full-length amino acid sequence.

[0158] Table S2. Exemplary multispecific antibodies that define the amino acid sequences of the first antigen-binding moiety and the third antigen-binding moiety. Note: In the table, (X)+(Y) represents the combination of the third antigen-binding part and the first antigen-binding part in a multispecific antibody. For example, "1B-1B6-V1+1B-1C7-V1 single variable domain" means that the third antigen-binding part of the multispecific antibody is the amino acid sequence of the 1B-1B6-V1 single variable domain, and the first antigen-binding part 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 S3.

[0159] Table S3. CDRs and full-length amino acid sequences of single variable domains (SEQ ID NO.)

[0160] In this disclosure, the first antigen-binding portion may be of camel origin or humanized. The third antigen-binding portion may be of camel origin or humanized. Humanization can reduce immunogenicity; in some embodiments, both the first and third antigen-binding portions shown are humanized. In some embodiments, both the first and third antigen-binding portions are of camel origin.

[0161] In the multispecific antibodies described in this disclosure, the second antigen-binding moiety provides the ability to target EGFR. The second antigen-binding moiety can be Fab, scFv, or ScFab (single-chain Fab). In some embodiments, the second antigen-binding moiety is an EGFR-binding Fab.

[0162] In some implementations, the second antigen-binding portion is murine, chimeric, or humanized.

[0163] In some embodiments, the second antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:63, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:64, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:65, 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. In some embodiments, the second antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:63, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:64, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:65, 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. In some embodiments, the second antigen-binding region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: 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 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 LCDR3 comprising the amino acid sequence shown in SEQ ID NO:68. In some embodiments, the second antigen-binding region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:63, HCDR2 of the amino acid sequence shown in SEQ ID NO:64, and HCDR3 of the amino acid sequence shown in SEQ ID NO:65; and the light chain variable region comprises LCDR1 of the amino acid sequence shown in SEQ ID NO:66, LCDR2 of the amino acid sequence shown in SEQ ID NO:67, and LCDR3 of the amino acid sequence shown in SEQ ID NO:68.

[0164] In one specific embodiment, the second antigen-binding region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:69, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:70. In another specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region shown in SEQ ID NO:69; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the variable region shown in SEQ ID NO:70. The CDRs are defined, for example, according to Kabat, IMGT, Chothia, Contact, AbM, and / or CCG.

[0165] In some embodiments, the second antigen-binding moiety 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 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 moiety 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 moiety is shown in SEQ ID NO:69, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:70.

[0166] The multispecific antibody described in this disclosure may be Fc domain-free, with the first antigen-binding portion, the second antigen-binding portion, and the third antigen-binding portion fused together via a suitable linker.

[0167] The multispecific antibodies described in this disclosure may have an Fc domain, which can extend the half-life and provide Fc domain-related effectors, among other functions.

[0168] Multispecific antibody configuration

[0169] The antigen-binding portions of the multispecific antibodies described in this disclosure can be fused together in various configurations. In some embodiments, the multispecific antibody further comprises (iv) an Fc domain consisting of two Fc polypeptides.

[0170] In some embodiments, the third antigen-binding moiety is fused to the first antigen-binding moiety, optionally via a peptide linker. More specifically, the third antigen-binding moiety is fused at its C-terminus to the N-terminus of the first antigen-binding moiety. Further, in some embodiments, the first antigen-binding moiety 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 moiety is Fab, with its C-terminus of either the Fab heavy chain or the Fab light chain fused to the N-terminus of another Fc peptide in the Fc domain.

[0171] In one specific embodiment, both the first and third antigen-binding moieties are single variable domains, the second antigen-binding moieties are Fab, the first antigen-binding moieties are fused at their C-terminus to the N-terminus of one Fc polypeptide in the Fc domain, the second antigen-binding moieties are fused at the C-terminus of their Fab heavy chain to the N-terminus of another Fc polypeptide in the Fc domain, and the third antigen-binding moieties are fused at their C-terminus to the N-terminus of the first antigen-binding moieties. This configuration is schematically depicted in Figure 2. In a further more specific embodiment, the multispecific antibody has three polypeptide chains: the first polypeptide chain contains the aforementioned first antigen-binding moieties, third antigen-binding moieties, and one Fc polypeptide in the Fc domain; the second polypeptide chain contains the Fab heavy chain of the aforementioned second antigen-binding moieties and another Fc polypeptide in the Fc domain; and the third polypeptide chain is the Fab light chain of the aforementioned second antigen-binding moieties.

[0172] In the above embodiments, the antigen-binding portions 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.

[0173] The third antigen-binding portion can be fused to the first antigen-binding portion directly or via a peptide linker. In one embodiment, the third antigen-binding portion is fused to the first antigen-binding portion via a peptide linker.

[0174] 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.

[0175] 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 moiety is fused to the first antigen-binding moiety via the peptide linker GGGGS (SEQ ID NO: 91) or (GGGGS)2.

[0176] When fused with Fc, fusion is typically via the hinge region. In one embodiment, the first antigen-binding moiety is fused to one of the Fc peptides in the Fc domain via a first hinge, and the second antigen-binding moiety is fused to another Fc peptide in 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).

[0177] Fc domain of multispecific antibodies

[0178] 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. The C-terminal lysine of the Fc polypeptide may be present or absent. In one embodiment, the multispecific antibody of this disclosure comprises one Fc domain.

[0179] 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.

[0180] 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.

[0181] In some implementations, the Fc includes modifications that promote heterodimerization.

[0182] The multispecific antibodies described in this disclosure comprise different antigen-binding moieties 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.

[0183] 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.

[0184] In a specific implementation, the 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).

[0185] 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.

[0186] 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.

[0187] 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.

[0188] In the multispecific antibodies described in this disclosure, 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:

[0189] 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

[0190] ii. (a) 435R or (b) 435R and 436F, occurring only on one of the Fc peptides.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] In some implementations, the Fc domain does not contain fucose.

[0195] In some embodiments, the multispecific antibodies described in 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.

[0196] In some embodiments, the multispecific antibody described in this disclosure is trivalent, meaning that the first antigen-binding portion, the second antigen-binding portion, and the third antigen-binding portion each provide monovalent binding to the corresponding antigen.

[0197] 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:71; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:71. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0198] 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:73; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:73. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0199] 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 ...0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:85. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0200] 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:77; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:77. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0201] 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:79; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:79; and a fourth polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:79. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0202] 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 ...0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:81. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0203] 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 ...0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:83. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0204] 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.

[0205] 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.

[0206] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein 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 a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0207] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein 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 a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0208] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein 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 a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0209] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein 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 a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0210] 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.

[0211] This disclosure provides exemplary trivalent multispecific antibodies.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] ADC

[0220] In some specific embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or solvates of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt provided in this disclosure, wherein the multispecific antibody comprises a first antigen-binding portion, a second antigen-binding portion, and a third antigen-binding portion, wherein the first antigen-binding portion comprises CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9; the second antigen-binding portion comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:63, HCDR2 of the amino acid sequence shown in SEQ ID NO:64, HCDR3 of the amino acid sequence shown in SEQ ID NO:65, LCDR1 of the amino acid sequence shown in SEQ ID NO:66, LCDR2 of the amino acid sequence shown in SEQ ID NO:67, and LCDR3 of the amino acid sequence shown in SEQ ID NO:68; and the third antigen-binding portion 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.

[0221] In some specific embodiments, the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts provided in this disclosure, wherein the multispecific antibody comprises a first antigen-binding moiety, a second antigen-binding moiety, and a third antigen-binding moiety, wherein the first antigen-binding moiety comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO:36; the second antigen-binding moiety comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:69, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:70; and the third antigen-binding moiety comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO:35. The CDRs are defined, for example, according to Kabat, IMGT, Chothia, Contact, AbM, and / or CCG.

[0222] In some specific embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided in this disclosure, wherein the multispecific antibody comprises a first antigen-binding moiety, a second antigen-binding moiety, and a third antigen-binding moiety, wherein the first antigen-binding moiety 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; the second antigen-binding moiety 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; The heavy chain variable region of the amino acid sequence shown in NO:69 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:70, and a light chain variable region of the 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; and the third antigen-binding portion comprises an amino acid sequence identical to that shown in SEQ ID NO:69. The amino acid sequence shown in NO:35 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0223] In some specific embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided in this disclosure, wherein the multispecific antibody comprises a first antigen-binding moiety, a second antigen-binding moiety, and a third antigen-binding moiety, wherein the first antigen-binding moiety comprises the amino acid sequence shown in SEQ ID NO:36, the second antigen-binding moiety comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:69 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:70, and the third antigen-binding moiety comprises the amino acid sequence shown in SEQ ID NO:35.

[0224] In some specific embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided in this disclosure, wherein 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 ... fourth polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, or 100% identity with the amino acid sequence shown in SEQ ID NO:85. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0225] In some specific embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt provided in this disclosure, wherein the multispecific antibody is composed of three polypeptide chains, wherein 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 a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0226] In some specific embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or solvates of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt provided in this disclosure, wherein the multispecific antibody is composed 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.

[0227] In some embodiments, the multispecific antibody can be modified, for example, by inserting, deleting, or substituting one or more amino acids. In this document, the modified multispecific antibody retains its activity of specifically binding to EGFR (e.g., human EGFR and / or monkey EGFR) and c-Met (e.g., human c-Met and / or monkey c-Met). In some embodiments, the C-terminal lysine residue of the heavy chain constant region of the multispecific antibody may be present or absent.

[0228] In the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts provided in this disclosure, the cytotoxic drug is conjugated to the multispecific antibody via a linker. In some specific embodiments, in the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts provided in this disclosure, each cytotoxic drug is conjugated to the multispecific antibody via a linker. The linkers of this disclosure can be linked to the multispecific antibody by any method known in the art. In some preferred embodiments, the linker is linked to the multispecific antibody via a thiol group and / or an amino group. In some more preferred embodiments, the linker is linked to the multispecific antibody via a thiol group.

[0229] In some embodiments, the connector may be a cleavable connector or a non-cleavable connector. In some embodiments, the connector is a cleavable connector, such as one that is degraded at low pH (including hydrazone bonds, carbonate bonds, etc.), proteolytic (including peptide bonds), or degraded at high glutathione concentrations (including disulfide bonds). The cleavable connector can cleave within the target cell, thereby releasing the cytotoxic drug. In other embodiments, the connector is a non-cleavable connector, such as one containing maleiminohexanoyl.

[0230] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt provided by the present invention, wherein a multispecific antibody is conjugated to one or more cytotoxic drugs, such as alkaloids, antimetabolites, antitumor antibiotics, alkylating agents, and platinum-based drugs. In some embodiments, the cytotoxic drug is a DNA-acting cytotoxic drug. In some embodiments, the DNA-acting cytotoxic drug includes, but is not limited to, calicutamide derivatives, duocarmycin derivatives, pyrrolobenzodiazepine (PBD), topoisomerase I inhibitors, and topoisomerase II inhibitors.

[0231] In some embodiments, the cytotoxic drug is a topoisomerase I inhibitor. In some embodiments, the cytotoxic drug is a camptothecin-based topoisomerase I inhibitor. In some embodiments, the cytotoxic drug is SN-38, an SN-38 derivative, ethanotecan, or an ethanotecan derivative. In some specific embodiments, the cytotoxic drug is an ethanotecan derivative. In some specific embodiments, the cytotoxic drug is an ethanotecan derivative with deuteration modification.

[0232] In some implementations, the cytotoxic drug is linked to the adapter via a functional group, and the antibody-drug conjugate is hydrolyzed within tumor cells to form a free cytotoxic drug, thereby exerting an anti-tumor effect.

[0233] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided in this disclosure, wherein the antibody-drug conjugate is a product of the following formula Ia

[0234] The drug-connector shown in the diagram is connected to the multispecific antibody, and the drug-connector is connected to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia.

[0235] Among them, R 1 and R 2 The atoms are independently selected from hydrogen or deuterium atoms. In some embodiments, the drug-connector is attached to the disulfide bond site of the multispecific antibody at the position indicated by * in the structure shown in Formula Ia, preferably via a thioether bond.

[0236] In some implementations, the R 1 For hydrogen atoms, R 2 It is a deuterium atom.

[0237] In some embodiments, the antibody-drug conjugate is a product of formula Ia-1

[0238] The drug-connector of the structure shown is formed by linking the multispecific antibody to the structure shown in Formula Ia-1, with the drug-connector attached to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia. In some embodiments, the drug-connector is attached to the disulfide bond site of the multispecific antibody at the position indicated by * in the structure shown in Formula Ia, preferably via a thioether bond.

[0239] The number of cytotoxic drugs linked to the multispecific antibody in the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or solvates of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt provided in this disclosure can vary, such that the antibody-drug conjugate (ADC) can be heterogeneous, that is, the antibody-drug conjugate includes multispecific antibodies linked to different numbers of cytotoxic drugs, for example, one molecule of multispecific antibody linked to 0 (i.e., without cytotoxic drugs), 1, 2, 3, 4, 5, or 6 or more molecules of cytotoxic drugs.

[0240] By controlling the proportions of multispecific antibodies linked to different numbers of cytotoxic drugs, antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of said antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts with different drug-antibody ratios (DARs) can be generated. It should be understood that the DAR is the average number of cytotoxic drugs linked to each multispecific antibody in the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the solvate of said antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt. For example, "DAR 6" refers to an antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt, comprising a heterogeneous mixture in which each multispecific antibody is linked to the same or different numbers of cytotoxic drugs (e.g., each multispecific antibody is linked to 0, 1, 2, 3, 4, 5, and / or 6 cytotoxic drugs), but the average number of cytotoxic drugs linked to each multispecific antibody is 6.

[0241] In some of the above embodiments, the DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6. In some of the above embodiments, the DAR of the antibody-drug conjugate is 5-6. In some of the above embodiments, the DAR of the antibody-drug conjugate is 5.5-6. In some of the above embodiments, the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0242] The antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts provided in this disclosure may also be represented by the structure shown in Formula II:

[0243] The multispecific antibody is as described in the multispecific antibody of this disclosure;

[0244] R 1 and R 2 Each is independently selected from either hydrogen or deuterium atoms. In some embodiments, the R... 1 For hydrogen atoms, R 2 It is a deuterium atom.

[0245] In some embodiments, the 3-position of -(succinimide-3-yl-N)- in Formula II is linked to the multispecific antibody via a thioether bond. n has the same meaning as DAR, representing the average number of cytotoxic agents linked to each multispecific antibody. In some embodiments, n is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6. In some embodiments, n is 5-6. In some embodiments, n is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0246] In some embodiments, the antibody-drug conjugate can be represented by the structure shown in Formula II-1:

[0247] Wherein, the multispecific antibody is as described in the multispecific antibody of this disclosure, and n is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6. In some embodiments, n is 3.5-5. In some embodiments, n is 5-6. In some embodiments, n is 5.5-6. In some embodiments, n is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0248] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided herein, wherein the antibody-drug conjugate is formed by linking a drug-connector of the structure shown in Formula Ia-1 to a multispecific antibody, the drug-connector being linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia-1, the multispecific antibody comprising a first antigen-binding portion, a second antigen-binding portion, and a third antigen-binding portion, wherein the first antigen-binding portion comprises CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9; the second antigen-binding portion comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:63, HCDR2 of the amino acid sequence shown in SEQ ID NO:64, HCDR3 of the amino acid sequence shown in SEQ ID NO:65, LCDR1 of the amino acid sequence shown in SEQ ID NO:66, LCDR2 of the amino acid sequence shown in SEQ ID NO:67, and SEQ ID NO:68. LCDR3 of the amino acid sequence shown in NO:68; and the third antigen-binding portion 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 some such embodiments, the DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6; preferably, the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0249] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided herein, wherein the antibody-drug conjugate is formed by linking a drug-linker of the structure shown in Formula Ia-1 to a multispecific antibody, the drug-linker being linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia-1, the multispecific antibody comprising a first antigen-binding portion, a second antigen-binding portion, and a third antigen-binding portion, wherein the first antigen-binding portion comprises CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO:36; the second antigen-binding portion comprises HCDR1, HCDR2, and HCDR3 of a heavy chain variable region as shown in SEQ ID NO:69, and LCDR1, LCDR2, and LCDR3 of a light chain variable region as shown in SEQ ID NO:70; and the third antigen-binding portion comprises an amino acid sequence as shown in SEQ ID NO:36. CDR1, CDR2, and CDR3, representing the single variable domains shown in NO:35, are defined, for example, according to Kabat, IMGT, Chothia, Contact, AbM, and / or CCG. In some such embodiments, the DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6; preferably, the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0250] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided herein, wherein the antibody-drug conjugate is formed by linking a drug-connector of the structure shown in Formula Ia-1 to a multispecific antibody, the drug-connector being linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia-1, the multispecific antibody comprising a first antigen-binding portion, a second antigen-binding portion, and a third antigen-binding portion, wherein the first antigen-binding portion 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; the second antigen-binding portion 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; The heavy chain variable region of the amino acid sequence shown in NO:69 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:70, and a light chain variable region of the 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; and the third antigen-binding portion comprises an amino acid sequence identical to that shown in SEQ ID NO:69. The amino acid sequence shown in NO:35 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity. In some such embodiments, the DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6; preferably, the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0251] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided herein, wherein the antibody-drug conjugate is formed by linking a drug-connector of the structure shown in Formula Ia-1 to a multispecific antibody, the drug-connector being linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia-1, the multispecific antibody comprising a first antigen-binding portion, a second antigen-binding portion, and a third antigen-binding portion, wherein the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, the second antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:69 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:70, and the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:35. In some such embodiments, the DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6; preferably, the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0252] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided herein, wherein the antibody-drug conjugate is formed by linking a drug-linker of the structure shown in Formula Ia-1 to a multispecific antibody, the drug-linker being linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia-1, the multispecific antibody being composed of 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, and another polypeptide chain contains an amino acid sequence having ... The amino acid sequence shown in NO:85 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:87, and a polypeptide chain containing 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. In some such embodiments, the DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6; preferably, the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0253] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided herein, wherein the antibody-drug conjugate is formed by linking a drug-connector of the structure shown in Formula Ia-1 to a multispecific antibody, the drug-connector being linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia-1, the multispecific antibody being composed of three polypeptide chains, wherein 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 a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87. In some such embodiments, the DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6; preferably, the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0254] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt provided herein, wherein the antibody-drug conjugate is formed by linking a drug-connector of the structure shown in Formula Ia-1 to a multispecific antibody, the drug-connector being linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia-1, the multispecific antibody being composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains being as shown in SEQ ID NO:77, SEQ ID NO:85, and SEQ ID NO:87, respectively. In some such embodiments, the DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6; preferably, the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0255] In some embodiments, the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts provided in this disclosure, wherein the DAR of the antibody-drug conjugates is 5-6, 5.5-6, or 6, and the mass fraction of the antibody-drug conjugates to which each multispecific antibody is linked to 6 cytotoxic drugs is not less than 40%, not less than 45%, not less than 50%, not less than 65%, not less than 70%, not less than 75%, not less than 80%, not less than 85%, not less than 90%, not less than 95%, or not less than 99%.

[0256] In some embodiments, the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of said antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts provided in this disclosure exhibit one or more combinations of the following properties:

[0257] (1) Combined with human c-Met, preferably with K 6E-10M or smaller. D Value combined with human c-Met;

[0258] (2) Combined with human EGFR;

[0259] (3) Combined with monkey c-Met;

[0260] (4) Combined with monkey EGFR;

[0261] (5) Internalization occurs in cells expressing c-Met and / or EGFR;

[0262] (6) It exhibits cytotoxic activity against tumor cells expressing c-Met and / or EGFR; and

[0263] (7) It has the bystander effect.

[0264] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt has the properties (1) to (7).

[0265] The antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of said antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts provided in this disclosure achieve excellent antitumor efficacy and / or safety. In some embodiments, the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of said antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts exhibit good killing activity against various tumor cells expressing EGFR and / or c-Met. In some embodiments, the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of said antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts have good in vivo antitumor activity. In some embodiments, the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of said antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts have excellent safety. In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or solvates of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt are not prone to aggregation.

[0266] Pharmaceutical Composition

[0267] This disclosure provides pharmaceutical compositions comprising the antibody-drug conjugate of this disclosure, its stereoisomer, a pharmaceutically acceptable salt thereof, or a solvation of the antibody-drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0268] use

[0269] This disclosure provides for the use of the antibody-drug conjugates of this disclosure, their stereoisomers, pharmaceutically acceptable salts thereof, or solvates of said antibody-drug conjugates, their stereoisomers, or pharmaceutically acceptable salts thereof. This disclosure also provides for the use of the pharmaceutical compositions of this disclosure.

[0270] In one aspect, this disclosure provides the use of the antibody-drug conjugate of this disclosure, its stereoisomers, its pharmaceutically acceptable salts, or solvates of said antibody-drug conjugates, their stereoisomers, or their pharmaceutically acceptable salts in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0271] In one aspect, this disclosure provides the use of the pharmaceutical compositions of this disclosure in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0272] In one aspect, this disclosure provides the use of the antibody-drug conjugates of this disclosure, their stereoisomers, their pharmaceutically acceptable salts, or solvates of said antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, and one or more other therapeutic agents in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0273] In one aspect, this disclosure provides the use of the pharmaceutical compositions of this disclosure, as well as one or more additional therapeutic agents, in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0274] In one aspect, this disclosure provides a method of treating a disease expressing c-Met and / or EGFR, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvation of said antibody-drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof. In some embodiments, the antibody-drug conjugate of the present disclosure, its stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvation of said antibody-drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof is administered to the subject in a therapeutically effective amount.

[0275] In one aspect, this disclosure provides a method of treating a disease expressing c-Met and / or EGFR, the method comprising administering a pharmaceutical composition of the present disclosure to a subject. In some embodiments, the pharmaceutical composition of the present disclosure is administered to the subject in a therapeutically effective amount.

[0276] In one aspect, this disclosure provides a method of treating a disease expressing c-Met and / or EGFR, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvation of said antibody-drug conjugate, its stereoisomer, a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents. In some embodiments, the antibody-drug conjugate of the present disclosure, its stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvation of said antibody-drug conjugate, its stereoisomer, a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents are administered to the subject in a therapeutically effective amount.

[0277] In one aspect, this disclosure provides a method of treating a disease expressing c-Met and / or EGFR, the method comprising administering to a subject a pharmaceutical composition of the present disclosure and one or more additional therapeutic agents. In some embodiments, the pharmaceutical composition of the present disclosure and one or more additional therapeutic agents are administered to the subject in a therapeutically effective amount.

[0278] In some embodiments, the disease expressing c-Met and / or EGFR is a tumor. In some embodiments, the additional therapeutic agent may be a tumor therapeutic agent known in the art.

[0279] In some embodiments, the method includes contacting tumor cells with the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition, thereby killing tumor cells or inhibiting tumor cell growth.

[0280] In some embodiments, the method includes contacting tumor cells with the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition, and simultaneously or sequentially contacting the tumor cells with one or more additional therapeutic agents, thereby killing the tumor cells or inhibiting tumor cell growth.

[0281] In some embodiments, administration to a subject of the disclosed antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure can kill tumor cells or inhibit tumor cell growth. In some embodiments, administration to a subject of a therapeutically effective amount of the disclosed antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure can kill tumor cells or inhibit tumor cell growth.

[0282] In some embodiments, administering to a subject the antibody-drug conjugate of this disclosure, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, and one or more additional therapeutic agents can kill tumor cells or inhibit tumor cell growth. In some embodiments, administering to a subject a therapeutically effective amount of the antibody-drug conjugate of this disclosure, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, and one or more additional therapeutic agents can kill tumor cells or inhibit tumor cell growth. In some embodiments, administering to a subject the pharmaceutical composition of this disclosure and one or more additional therapeutic agents can kill tumor cells or inhibit tumor cell growth. In some embodiments, administering to a subject a therapeutically effective amount of the pharmaceutical composition of this disclosure and one or more additional therapeutic agents can kill tumor cells or inhibit tumor cell growth.

[0283] In some embodiments, the tumor is a c-Met and / or EGFR-positive tumor. In some embodiments, the tumor is epithelial carcinoma, squamous cell carcinoma, glioblastoma, 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, and / or thymic cancer. In some embodiments, the lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC).

[0284] Reagent test kit

[0285] This disclosure provides a kit comprising the antibody-drug conjugate of this disclosure, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a pharmaceutical composition of this disclosure.

[0286] This disclosure describes kits comprising the antibody-drug conjugate, its stereoisomer, a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, its stereoisomer, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof. The kits can be used to implement the use or other uses of the antibody-drug conjugate, its stereoisomer, a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, its stereoisomer, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof provided in this disclosure. In some embodiments, the kit may include the antibody-drug conjugate, its stereoisomer, a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, its stereoisomer, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof; optionally, the kit may also include instructions for use. The kit may also include other materials required from a commercial and user perspective, such as other buffers, diluents, needles, syringes, etc.

[0287] Connector - Payload

[0288] In some respects, this disclosure provides a linker-payload with the structure shown in Formula I:

[0289] Among them, R 1 and R 2 They are each independently selected from hydrogen atoms or deuterium atoms.

[0290] In some implementations, the R 1 For hydrogen atoms, R 2 It is a deuterium atom.

[0291] In one specific embodiment, the present invention provides a joint-load capacity with the structure shown in Formula I-1:

[0292] Preparation method

[0293] This disclosure provides a method for preparing the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt, comprising treating a multispecific antibody under reducing conditions, followed by reacting the multispecific antibody with a linker-loador selected from the structure shown in Formula I, wherein R 1 and R 2 Each is independently selected from either hydrogen or deuterium atoms. In some embodiments, the R... 1 For hydrogen atoms, R 2 It is a deuterium atom.

[0294] In some embodiments, the method includes treating a multispecific antibody under reducing conditions, followed by reacting the multispecific antibody with a linker-loador selected from the structure shown in Formula I-1.

[0295] In some implementations, the reduction condition is in the presence of TCEP. Detailed Implementation

[0296] This disclosure also provides the following specific implementation schemes, but the scope of protection of this disclosure is not limited thereto:

[0297] Implementation Scheme 1. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt, comprising a multispecific antibody, wherein said multispecific antibody comprises (i) a first antigen-binding portion that binds to a first antigen.

[0298] (ii) the second antigen-binding portion that binds to the second antigen; and

[0299] (iii) The third antigen-binding portion that binds to the first antigen;

[0300] Wherein, the first antigen is c-Met and the second antigen is EGFR, and both the first antigen-binding portion and the third antigen-binding portion are single variable domains and each independently contains any one of the following:

[0301] (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;

[0302] (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;

[0303] (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;

[0304] (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;

[0305] (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.

[0306] (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.

[0307] (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

[0308] (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.

[0309] Implementation Scheme 2. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 1, wherein the first antigen-binding moiety and the third antigen-binding moiety each independently comprise any one of the following:

[0310] (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;

[0311] (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

[0312] (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.

[0313] Implementation Scheme 3. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 2, wherein the first antigen-binding portion 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 portion 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.

[0314] Implementation Scheme 4. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 2, wherein the first antigen-binding portion 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 portion 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.

[0315] Implementation Scheme 5. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt, comprising a multispecific antibody, wherein said multispecific antibody comprises

[0316] (i) The first antigen-binding portion that binds to the first antigen;

[0317] (ii) the second antigen-binding portion that binds to the second antigen; and

[0318] (iii) The third antigen-binding portion that binds to the first antigen;

[0319] Wherein, the first antigen is c-Met and the second antigen is EGFR, and both the first antigen-binding portion and the third antigen-binding portion 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.

[0320] Implementation Scheme 6. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 5, wherein the first antigen-binding moiety and the third antigen-binding moiety each independently comprise CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO:35, 36, or 38.

[0321] Implementation Scheme 7. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 6, wherein the first antigen-binding portion comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO:36, and the third antigen-binding portion comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO:35.

[0322] Implementation Scheme 8. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 6, wherein the first antigen-binding portion comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO:36, and the third antigen-binding portion comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO:38.

[0323] Implementation Scheme 9. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-8, wherein the first antigen-binding portion and the third antigen-binding portion 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.

[0324] Implementation Scheme 10. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-9, wherein the first antigen-binding portion 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.

[0325] Implementation Scheme 11. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to Implementation Scheme 10, wherein the first antigen-binding portion 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 portion 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.

[0326] Implementation Scheme 12. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-11, wherein the third antigen-binding portion 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.

[0327] Implementation Scheme 13. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to Implementation Scheme 12, wherein the third antigen-binding moiety 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 moiety 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.

[0328] Implementation Scheme 14. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-11, wherein the third antigen-binding portion 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.

[0329] Implementation Scheme 15. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to Implementation Scheme 14, wherein the third antigen-binding moiety 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 moiety 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.

[0330] Implementation Scheme 16. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-8, wherein the first antigen-binding moiety and the third antigen-binding moiety are selected from any one of the following:

[0331] (1) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:35;

[0332] (2) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:27, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:26;

[0333] (3) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:33;

[0334] (4) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:34;

[0335] (5) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:32;

[0336] (6) The third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:38; or

[0337] (7) The third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:39.

[0338] Implementation Scheme 17. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-16, wherein the single variable domain is of camelid origin or humanized.

[0339] Implementation Scheme 18. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-17, wherein the second antigen-binding portion is Fab, scFv, or scFab.

[0340] Implementation Scheme 19. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-18, wherein the second antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:63, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:64, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:65, 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.

[0341] Implementation Scheme 20. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-18, wherein the second antigen-binding portion comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:69, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:70.

[0342] Implementation Scheme 21. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-20, wherein the second antigen-binding portion 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 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.

[0343] Implementation Scheme 22. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 21, wherein the heavy chain variable region of the second antigen-binding moiety 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.

[0344] Implementation Scheme 23. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-22, wherein the second antigen-binding portion is murine, chimeric, or humanized.

[0345] Implementation Scheme 24. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-23, wherein the third antigen-binding portion and the first antigen-binding portion are fused to each other, optionally fused to each other via a peptide linker.

[0346] Implementation Scheme 25. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 24, wherein the third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion.

[0347] Implementation Scheme 26. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-25, wherein the multispecific antibody further comprises (iv) an Fc domain consisting of two Fc polypeptides.

[0348] Implementation Scheme 27. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 26, wherein the first antigen-binding moiety 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 moiety is Fab and the second antigen-binding moiety is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain.

[0349] Implementation Scheme 28. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to Implementation Scheme 26 or 27, wherein the Fc domain is an IgG Fc domain, preferably an IgG1 Fc domain.

[0350] Implementation Scheme 29. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 28, wherein the IgG Fc domain is a human IgG Fc domain, preferably a human IgG1 Fc domain.

[0351] Implementation Scheme 30. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt 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.

[0352] Implementation Scheme 31. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 30, wherein, according to EU designation, one of the Fc peptides of the Fc domain comprises amino acid substitutions 354C and 366Y / W, and the other Fc peptide comprises amino acid substitutions 349C, 366S, 368A, and 407T / V.

[0353] Implementation Scheme 32. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt 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.

[0354] Implementation Scheme 33. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Implementation Scheme 32, wherein, according to the EU designation, the Fc domain comprises an amino acid substitution (a) 435R or (b) 435R and 436F occurring only in one of the Fc polypeptides.

[0355] Implementation Scheme 34. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt 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.

[0356] Implementation Scheme 35. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 26-29, wherein, according to the EU designation, one of the Fc peptides of the Fc domain comprises amino acid substitutions: 349C, 366S, 368A, 407V, and 435R, and the other Fc peptide comprises amino acid substitutions: 354C and 366W.

[0357] Implementation Scheme 36. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-35, wherein the multispecific antibody is trivalent.

[0358] Implementation Scheme 37. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt, according to any one of Implementation Schemes 1-36, wherein the multispecific antibody comprises three polypeptide chains, wherein:

[0359] (1) One polypeptide chain 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:77; another polypeptide chain 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:85; and a third polypeptide chain 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:77. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0360] (2) One polypeptide chain 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:71; another polypeptide chain 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:85; and a third polypeptide chain 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:71. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0361] (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: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:73; and a fourth polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:73. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0362] (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:75. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0363] (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: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:79; and a fourth polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:79. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0364] (6) One polypeptide chain 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:81; another polypeptide chain 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:85; and a third polypeptide chain 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:85; and a fourth polypeptide chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:81. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; or

[0365] (7) One polypeptide chain 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:83; another polypeptide chain 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:85; and a third polypeptide chain 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:83; and a fourth polypeptide chain 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:85. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0366] Implementation Scheme 38. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt, comprising a multispecific antibody, wherein said multispecific antibody is composed of three polypeptide chains, wherein:

[0367] (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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87;

[0368] (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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0369] (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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0370] (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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0371] (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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0372] (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 a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87; or

[0373] (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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87.

[0374] Implementation Scheme 39. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-38, wherein the multispecific antibody is unfucosylated.

[0375] Implementation Scheme 40. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-39, wherein said antibody-drug conjugate is a product of the following formula Ia

[0376] The drug-connector shown in the diagram is connected to the multispecific antibody, and the drug-connector is connected to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia.

[0377] in,

[0378] R 1 and R 2 They are each independently selected from hydrogen atoms or deuterium atoms.

[0379] Implementation Scheme 41. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to Implementation Scheme 40, wherein the R 1 For hydrogen atoms, R 2 It is a deuterium atom.

[0380] Implementation Scheme 42. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-41, wherein the DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6.

[0381] Implementation Scheme 43. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-41, wherein the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0382] Implementation Scheme 44. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 1-43, wherein the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt exhibits one or more combinations of the following properties:

[0383] (1) Combined with human c-Met, preferably with K 6E-10M or smaller. D Value combined with human c-Met;

[0384] (2) Combined with human EGFR;

[0385] (3) Combined with monkey c-Met;

[0386] (4) Combined with monkey EGFR;

[0387] (5) Internalization occurs in cells expressing c-Met and / or EGFR;

[0388] (6) It exhibits cytotoxic activity against tumor cells expressing c-Met and / or EGFR; and

[0389] (7) It has the bystander effect.

[0390] Implementation Scheme 45. A pharmaceutical composition comprising, as described in any one of Implementation Schemes 1-44, an antibody-drug conjugate thereof, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of said antibody-drug conjugate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; optionally, said pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0391] Implementation Scheme 46. A method for preparing the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of Implementation Schemes 40-44, comprising treating the multispecific antibody under reducing conditions, followed by reacting the multispecific antibody with a linker-loador selected from the structure shown in Formula I, wherein the structure shown in Formula I is as follows:

[0392] R 1 and R 2 They are each independently selected from hydrogen atoms or deuterium atoms.

[0393] Implementation Scheme 47. The method according to Implementation Scheme 46, wherein the R 1 For hydrogen atoms, R 2 It is a deuterium atom.

[0394] Implementation Scheme 48. Use of any antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition of Implementation Scheme 45 in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0395] Implementation Scheme 49. Use of an antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition of Implementation Scheme 45, and one or more additional therapeutic agents in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0396] Implementation Scheme 50. The use according to Implementation Scheme 48 or 49, wherein the disease expressing c-Met and / or EGFR is a tumor.

[0397] Implementation Scheme 51. The use according to Implementation Scheme 49 or 50, wherein the additional therapeutic agent is a tumor therapeutic agent.

[0398] Implementation Scheme 52. The use according to Implementation Scheme 50 or 51, wherein the tumor is a c-Met and / or EGFR positive tumor; preferably, the tumor is epithelial carcinoma, squamous cell carcinoma, glioblastoma, 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, and / or thymic cancer.

[0399] Implementation Scheme 53. A method for treating a disease expressing c-Met and / or EGFR, comprising administering to a subject an antibody-drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of said antibody-drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof as described in Implementation Scheme 45.

[0400] Implementation Scheme 54. The method according to Implementation Scheme 53, wherein the disease expressing c-Met and / or EGFR is a tumor.

[0401] Implementation Scheme 55. The method according to Implementation Scheme 54, the method comprising contacting tumor cells with any one of the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or the pharmaceutical composition of Implementation Scheme 45, thereby killing tumor cells or inhibiting tumor cell growth.

[0402] Implementation Scheme 56. The method according to any one of Implementation Schemes 53-55, wherein the method further comprises administering one or more additional therapeutic agents to the subject.

[0403] Implementation Scheme 57. The method according to Implementation Scheme 56, wherein the additional therapeutic agent is a tumor therapeutic agent.

[0404] Implementation Scheme 58. The method according to any one of Implementation Schemes 54-57, wherein the tumor is a c-Met and / or EGFR positive tumor; preferably, the tumor is epithelial carcinoma, squamous cell carcinoma, glioblastoma, 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, and / or thymic cancer.

[0405] Implementation Scheme 59. A kit comprising the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition of Implementation Scheme 45; optionally, the kit further comprising instructions for use.

[0406] 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. The RAA22 / B09-57 used in the examples was prepared according to conventional antibody methods, first by vector construction, then transfection into eukaryotic cells, followed by purification and expression. The sequences are derived from SEQ ID NO:59 (anti-EGFR-HC), SEQ ID NO:61 (anti-EGFR-LC), SEQ ID NO:60 (anti-c-Met-HC), and SEQ ID NO:62 (anti-c-Met-LC) in patent disclosure US20230183358A1.

[0407] Example 1: Anti-human c-Met V H Construction of H phage display library

[0408] 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 used for the initial subcutaneous multi-site immunization of Bactrian camels. Subsequently, booster immunizations were performed 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.

[0409] The amplified V HThe 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 No. pMECS) to construct a recombinant vector. This vector was then electroporated into *E. coli* TG1 (Lucigen, catalog number No. 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 No. 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), 2.5M 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.

[0410] Example 2: Anti-human c-Met V H H screening

[0411] 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.

[0412] 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, V of 1B-1B6. H The amino acid sequence of H is shown in SEQ ID NO:26, 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, V of 4&5B-2F01. HThe 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.

[0413] Example 3: Anti-human c-Met V H Preparation of H-Fc chimeric antibodies

[0414] 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 Expifectamine was used. TM The CHO Transfection Kit transient expression system (Thermo Fisher Scientific Inc., catalog number A29129) expresses these V... 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.

[0415] 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).

[0416] Example 4: Anti-human V H Affinity of H-Fc chimeric antibody to human and cynomolgus monkey c-Met

[0417] 4.1 Determination of the affinity of the antibody for human and cynomolgus monkey c-Met using surface plasmon resonance (SPR) technique

[0418] 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. H H-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 monkey 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, and bound at a flow rate of 50 μL / min for 200 s. Dissociation was then allowed to proceed after 600–1400 s. Data analysis was performed using software, and the binding rate constant k was calculated by fitting the data to 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.

[0419] Table 1-1.V H Affinity of H-Fc chimeric antibody to human and cynomolgus monkey c-Met

[0420] Table 1-2.V H Affinity of H-Fc chimeric antibody to human and cynomolgus monkey c-Met

[0421] 4.2 Flow cytometry determination of antibody-cell binding

[0422] 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 human lung adenocarcinoma cells with high c-Met expression; MKN45 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60488) are human gastric cancer cells with moderate c-Met expression; KP4 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60541) are human pancreatic cancer cells with low c-Met expression; NCI-H1975 cells (Beina Biotechnology, catalog number BNCC100690) are human non-small cell lung adenocarcinoma cells with low to moderate c-Met expression; and NCI-H292 cells (Beina Biotechnology, catalog number BNCC100671) are human epidermal lung cancer cells 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 for 2×10 5 Target cells were incubated on ice for 1 hour, then washed and incubated with PE-labeled anti-human IgG Fc antibody (Jackson Immuno Research, catalog number No. 109-116-170) for 0.5 hours on ice. After washing, the cells were analyzed by flow cytometry (Thermo Fisher Scientific Inc., Attune NXT). The results are shown in Figures 1A-1G, Tables 2-1 and 2-2. Cells 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 affinity to target cells with different c-Met expression levels. Cells 4&5B-2F01-Fc and 4&5C-12B04-Fc were superior to the control 1016-069.

[0423] Table 2-1. Anti-human c-Met V H H-Fc chimeric antibody binding to target cells

[0424] Table 2-2. Anti-human c-Met V H H-Fc chimeric antibody binding to target cells

[0425] Example 5: Anti-human c-Met V H Epitope Differences Among Different Clones

[0426] Epitope competition analysis was performed using a biomolecular interaction system (Fortebio, catalog number Octet RED96). An Anti-Penta-HIS (HIS1K) sensor (Fortebio, catalog number No. 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 binding 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.

[0427] 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.

[0428] The data in Table 3-3 shows that 4&5B-2F01-Fc and 4&5C-12B04-Fc are in full competition with 1016-069.

[0429] Table 3-1. Epitope Difference Analysis

[0430] Table 3-2 Epitope Difference Analysis

[0431] Table 3-3. Epitope Difference Analysis

[0432] Example 6: Anti-human c-Met V H Construction, expression, purification, and affinity detection of H-Fc humanized antibodies

[0433] 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 Expifectamine was used. TM The CHO Transfection Kit transient expression system (Thermo Fisher Scientific Inc., catalog number A29129) expresses these V... 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 V... H H represents 12B04-V1 and 12B04-V2, respectively, with amino acid sequences shown in SEQ ID NO:38 and 39, respectively.

[0434] 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 number 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 form 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.

[0435] The affinity data of humanized antibodies (1B-1B6-V1, 1B-1B6-V2, 1B-1B6-V3, 1B-1C7-V1 and 1B-1C7-V2) and chimeric antibodies (1B-1B6-Fc and 1B-1C7-Fc) to human c-Met protein are shown in Table 4.

[0436] Table 4. Anti-human c-Met V H Binding affinity of H-Fc chimeric antibodies and humanized antibodies to human c-Met-His

[0437] Tests showed that 1B-1B6-Fc and its humanized antibody, as well as 1B-1C7-Fc and its humanized antibody, can specifically bind to c-Met-His protein.

[0438] Example 7: Construction, expression, and purification of anti-EGFR / anti-c-Met multispecific antibodies

[0439] 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), and 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).

[0440] Anti-EGFR / anti-c-Met multispecific antibodies were constructed according to the configuration shown in Figure 2, and named V12, V41, V35, V42, V67, V73, and V74, respectively. The second antigen-binding portion 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) HThe H-Fc contains two tandemly linked 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 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. The high-yield expression system (catalog number: MIR 6270) delivers the above expression vector 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 (named CHO FUT8) with the FUT8 gene knocked out. - / - In the cells, the transfected cell density was 4 × 10⁻⁶. 6 Cells / mL. Cells were cultured continuously until day 10 post-transfection, then centrifuged to collect the supernatant. [The following is a separate, unrelated sentence:] ...using... Protein purification was performed using the 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), molecular weight analysis (TOF MS), and sequence analysis confirmed the acquisition of the aforementioned multispecific antibody with the expected structure and sequence.

[0441] Table 5. Composition and origin of the antigen-binding domain for c-Met in anti-EGFR / anti-c-Met multispecific antibodies

[0442] Example 8: Affinity of anti-EGFR / anti-c-Met multispecific antibody to human c-Met

[0443] 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.

[0444] Table 6. Binding affinity of anti-EGFR / anti-c-Met multispecific antibodies to human c-Met-His

[0445] Example 9: Binding of anti-EGFR / anti-c-Met multispecific antibodies to tumor cells expressing EGFR and c-Met

[0446] 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).

[0447] A431 cells, NCI-H1975 cells, and NCI-H1993 cells in the 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, serially diluted 5-fold 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.

[0448] Table 7 and Figures 3A-3C show the binding ability of the anti-EGFR / anti-c-Met multispecific antibodies to A431, NCI-H1975, and NCI-H1993 cells. The results show that the binding ability of V42 and V73 to the three cell lines is basically equivalent to that of Amivantamab.

[0449] 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 The " / " indicates that the curve cannot be fitted to a numerical value.

[0450] Example 10: Inhibition of HGF-stimulated c-Met phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies

[0451] 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).

[0452] 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, respectively, and were analyzed using Expifectamine. TMHGF was expressed using the CHO Transfection 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.

[0453] 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 / 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 medium and add 1 mL / well of RPMI medium for overnight starvation. Remove the overnight starved 6-well cell culture plate, discard the original medium, and add 100 nM of anti-EGFR / anti-c-Met multispecific antibody, or simultaneously add 100 ng / mL of HGF and 100 nM of anti-EGFR / anti-c-Met multispecific antibody. 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.

[0454] Figure 4 shows the inhibitory effects of anti-EGFR / anti-c-Met multispecific antibodies on c-Met phosphorylation and downstream signaling pathways. The results show that the inhibitory effects of V42 and V73 on HGF-stimulated c-Met phosphorylation and downstream signaling pathways are comparable to those of Amivantamab (BM in Figure 4).

[0455] Example 11: Inhibition of EGF-stimulated EGFR phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies

[0456] 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).

[0457] A549 cells in the logarithmic growth phase were collected, counted, and then the viable cell density was adjusted to 4 × 10⁹ cells using complete culture medium. 5Add 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.

[0458] Figure 5 shows the inhibitory effects of anti-EGFR / anti-c-Met multispecific antibodies on EGFR phosphorylation and downstream signaling pathways. The results show that the inhibitory effects of V42 and V73 on EGF-stimulated EGFR phosphorylation and downstream signaling pathways are comparable to those of Amivantamab (BM in Figure 5).

[0459] Example 12: Anti-EGFR / anti-c-Met multispecific antibody competes with HGF ligand for c-Met binding activity.

[0460] 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).

[0461] Weigh 2.0 mg of biotin (Thermo, catalog number 20217) and add it to 590 μL of DMSO (Sigma, catalog number D2650), then dissolve and mix well. Add biotin solution at a ratio of 2.7 μL of biotin solution to every 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 1M sulfuric acid stop solution and incubate at room temperature for 5 minutes. Use a microplate reader with 630nm as the reference wavelength to read the absorbance value at a wavelength of 450nm.

[0462] Figures 6A-6C show the activity of anti-EGFR / anti-c-Met multispecific antibodies in competitive binding to c-Met with HGF ligands. The results show that the activity of V42 and V73 in competitive binding to c-Met with HGF ligands is basically equivalent to that of Amivantamab.

[0463] Example 13: Inhibitory activity of anti-EGFR / anti-c-Met multispecific antibodies against tumor cell proliferation

[0464] 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).

[0465] Log-phase NCI-H292 (EGFR-expressed, c-Met-low-expressed, source: Beina Biotechnology) and KP4 (EGFR-expressed, c-Met-low-expressed, source: Kebai Biotechnology) cells were collected and their viable cell density was adjusted to 1.5 × 10⁻⁶ 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 plates were incubated for 144 hours; for KP4, the plates were incubated 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.

[0466] Figures 7A-7B and Table 8 show 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.

[0467] Table 8. IC50 of each antibody on the inhibition of NCI-H292 and KP4 cell proliferation 50 value

[0468] Example 14: ADCC effect of anti-EGFR / anti-c-Met multispecific antibody on tumor cells

[0469] 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).

[0470] Logarithmic growth phase 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. The non-radioactive cytotoxicity assay kit (Promega, catalog number G1780) was used for detection. Finally, the absorbance value at a wavelength of 490 nm was measured using a microplate reader.

[0471] Figures 8A-8B and Table 9 show the ADCC effect of anti-EGFR / anti-c-Met multispecific antibodies on tumor cells.

[0472] Table 9. Effects of each antibody on the lysis EC of NCI-H292 and KP4 cells 50 Value and maximum pyrolysis rate

[0473] Example 15: Internalization activity of anti-EGFR / anti-c-Met multispecific antibodies in tumor cells

[0474] The internalization activity of anti-EGFR / anti-c-Met multispecific antibodies (including Amivantamab, V42, and V73) in 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).

[0475] A431 cells, NCI-H1975 cells, and NCI-H441 cells in the logarithmic growth phase were collected 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 with Antibody Internalization Human Reagent (Sartorius, catalog number 90564) were prepared according to the 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.

[0476] Figures 9A-9C and Table 10 show the internalization activity of the anti-EGFR / anti-c-Met multispecific antibody in A431, NCI-H1975, and NCI-H441 cells.

[0477] Table 10. Internalization activity of anti-EGFR / anti-c-Met multispecific antibodies in A431, NCI-H1975, and NCI-H441 cells.

[0478] Example 16: Pharmacodynamic evaluation of anti-EGFR / anti-c-Met multispecific antibody in a U-87MG human glioma subcutaneous xenograft model in nude mice

[0479] SPF-grade female BALB / c-nu nude mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were subcutaneously inoculated with U-87MG cells (ATCC HTB-14). TM Once the tumor grows to 100-150mm 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.

[0480] 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).

[0481] The formulas for calculating the test indicators are as follows:

[0482] 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.

[0483] The relative tumor proliferation rate T / C(%) = (T - T0) / (C - C0) × 100%,

[0484] 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.

[0485] If the tumor is smaller than the tumor volume at the start 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%.

[0486] The results of the detection indexes are shown in Table 11. In the U-87MG human glioma nude mouse subcutaneous transplantation tumor model, both V42 and V73 have significant tumor inhibitory effects.

[0487] Table 11. Effects of anti-EGFR / anti-c-Met multispecific antibody on U-87MG human glioma nude mouse subcutaneous transplantation tumor The P value is compared with the model group.

[0488] Example 17: Preparation of Linker-payload MC-GGFG-deuterated DXd (MC-GGFG-DDDXd)

[0489] Step 1 Synthesis of Intermediate A

[0490] Under nitrogen protection, 80 g of ethyl diazoacetate was added to a 3 L single-neck flask, and 800 mL of dichloromethane and 800 mL of 1% deuterated acetic acid solution (8 g of deuterated acetic acid dissolved in 800 mL of deuterium water) were added. The single-neck flask was placed under light-shielding conditions and stirred at room temperature for 75 hours. The organic phase was separated and collected by liquid separation. The aqueous phase was extracted with dichloromethane twice (200 mL × 2), and the organic phases were combined. The organic phase was washed with 200 mL of deuterium water, and the obtained organic phase was dried with anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to dryness at 20 °C to obtain 49.25 g of Intermediate A; the CAS of this Intermediate A is 1356471-71-0.

[0491] Step 2 Synthesis of Intermediate B

[0492] Weigh 50 g of N-fluorenemethoxycarbonyl-glycyl-glycine into a 2 L round-bottom flask, add 750 mL of tetrahydrofuran and 150 mL of glacial acetic acid, stir at 40 °C for 20 minutes, add 100 g of lead tetraacetate, raise the temperature to 80 °C and continue the reaction for 3 hours. Cool to room temperature, filter, and wash the filter cake with 250 mL of ethyl acetate. Concentrate the filtrate to dryness, add 330 mL of dichloromethane and 670 mL of ethyl acetate to dissolve and obtain the organic phase. Wash the organic phase three times with 30% potassium bicarbonate aqueous solution (500 mL × 3), then dry with anhydrous sodium sulfate and filter. Concentrate the filtrate to dryness under reduced pressure, add 100 mL of dichloromethane to dissolve, then add 100 mL of n-hexane, stir at room temperature until a solid precipitates, then add 300 mL of a mixed solution of n-hexane and dichloromethane (n-hexane:dichloromethane = 1:1), and stir overnight. The filter cake was filtered and dried in a vacuum oven at 40°C for 4 hours to obtain 37.3 g of intermediate B. LC-MS (ESI) m / z: 391.09 [M+Na] + The CAS number for intermediate B is 1599440-06-8.

[0493] Step 3 Synthesis of intermediate C

[0494] Weigh 78g of intermediate B and add it to a 3000mL single-necked flask. Add 800mL of dichloromethane and 45g of intermediate A. Cool the 3000mL single-necked flask to 0℃ in an ice-water bath. Dissolve 16g of lithium tert-butoxide in 400mL of dichloromethane to prepare a lithium tert-butoxide solution. Add the lithium tert-butoxide solution to the 3000mL single-necked flask and react at 0℃ for 3 hours. Transfer to room temperature and add 800mL of water with stirring. Separate the liquid and collect the organic phase. Extract the aqueous phase with 400mL of dichloromethane and combine the organic phases. Wash the organic phase once with 800mL of saturated brine, dry to anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and perform silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain 61g of intermediate C. LC-MS (ESI) m / z: 437.34 [M+Na] + The CAS number for intermediate C is 2760715-83-9.

[0495] Step 4: Synthesis of Compound D

[0496] 31.2 g of intermediate C was added to 270 mL of deuterated methanol and 70 mL of heavy water, stirred in an ice bath, followed by the addition of 5.5 g of NaOH, and stirred overnight at room temperature. The reaction mixture was then extracted with 300 mL of ethyl acetate and 300 mL of water. The pH of the aqueous layer was adjusted to 2-3 with 20 mL of glacial acetic acid, resulting in the precipitation of a solid. Filtering yielded 20.3 g of compound D. 1H-NMR (500MHz, DMSO-d6) δ8.70(t,J=6.6Hz,1H),7.89(d,J=7.5Hz,2H),7.72(d,J=7.5Hz,2H),7.57(t,J=6.0Hz,1H),7.42(t,J=7.5Hz,2H ),7.34(t,J=7.5Hz,2H),4.61(d,J=6.6Hz,2H),4.30(d,J=7.1Hz,2H),4.23(1H,m),3.64(d,J=6.0Hz,2H); LC-MS(ESI)m / z:409.08[M+Na] + .

[0497] Step 5: Synthesis of Compound E

[0498] Weigh 2.0 g of ethatecan mesylate dihydrate and 1.63 g of compound D into a 100 mL round-bottom flask. Add 40 mL of N,N-dimethylformamide and stir. Cool to 0 °C. Add 2.0 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and 1.82 g of N,N-diisopropylethylamine sequentially, and react at 0 °C for 3 h. Pour the reaction solution into 120 mL of ice water and stir for 1 h. Filter and dissolve the filter cake in dichloromethane. Perform silica gel column chromatography (100 g of 100-200 mesh silica gel, dichloromethane:methanol = 30:1, 2 L) to obtain 2.6 g of compound E. 1 H-NMR (500MHz, DMSO-d6), δ8.78(t,J=6.6Hz,1H),8.47(d,J=9.0Hz,1H),7.86(d,J=7.5Hz,2H),7.73(d,J=11.0 Hz,1H),7.68(d,J=7.5Hz,2H),7.53(t,J=6.0Hz,1H),7.39(t,J=7.5Hz,2H),7.30(m,2H),7.29(s,1H),6.50(br s,1H),5.56(m,1H),5.39(m,2H),5.14(m,2H),4.64(m,2H),4.25(d,J=6.8Hz,2H),4.19(m,1H),3.62(d,J=6.0H z,2H),3.15(m,2H),2.35(s,3H),2.17(m,2H),1.83(m,2H),0.85(t,J=7.3Hz,3H); LC-MS(ESI)m / z:804.84[M+H] + .

[0499] Step 6: Preparation of compound F

[0500] Weigh 0.38 g of 1,8-diazabicyclo[5.4.0]undec-7-ene and add it to a 100 mL round-bottom flask. Then add 20 mL of tetrahydrofuran to the round-bottom flask and stir. Cool to 0 °C. Weigh 2.0 g of compound E and prepare a solution with 20 mL of tetrahydrofuran. Slowly add the prepared solution of compound E to the 100 mL round-bottom flask and allow it to warm naturally to room temperature. React for 3 h. Filter under nitrogen protection to obtain 1.45 g of compound F. 1 H-NMR (500MHz, DMSO-d6) δ8.76(m,1H),7.72(m,1H),7.28(s,1H),5.48(m,3H),5.16(m,2H),4.61(m,2H),3.40( m,2H),3.20(m,2H),2.35(s,3H),2.17(m,2H),1.83(m,2H),0.86(t,J=7.1Hz,3H); LC-MS(ESI)m / z:582.39[M+H] + .

[0501] Step 7: Preparation of compound H

[0502] Weigh 1.00 g of compound F and 0.97 g of compound G into a 100 mL round-bottom flask, and add 10 mL of N,N-dimethylformamide. Cool to -20 °C, add 0.34 g of 1-hydroxybenzotriazole and 0.49 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and react at -20 °C for 3 h. Add 20 mL of dichloromethane and 20 mL of water to the reaction mixture, stir for 0.5 h, let stand, separate the layers, and collect the organic phase. Dry the organic phase with anhydrous sodium sulfate and filter. Concentrate the filtrate to dryness under reduced pressure. Analyze by silica gel column chromatography (dichloromethane:methanol = 15:1) to obtain 400 mg of compound H, MS m / z: 1037.08 [M+H]. + . 1H-NMR (500MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.49(d,J=8.5Hz,1H),8.2 9(t,J=5.5Hz,1H),8.12(d,J=8.0Hz,1H),8.06(t,J=5.5Hz,1H),8.00(t,J=5 .5Hz,1H),7.74(d,J=10.5Hz,1H),7.30(s,1H),7.27-7.12(m,5H),6.98(s,2 H),6.51(brs,1H),5.61-5.58(m,1H),5.45-5.37(m,2H),5.22-5.13(m,2H), 4.64(d,J=6.5Hz,2H),4.49-4.45(m,1H),3.76-3.57(m,6H),3.37-3.32(m, 2H),3.24-3.09(m,2H),3.02(dd,J=4.5Hz,14.0Hz,1H),2.77(dd,J=9.5Hz,1 3.5Hz,1H),2.36(s,3H),2.23-2.14(m,2H),2.09(t,J=7.5Hz,2H),1.91-1.7 9(m,2H),1.49-1.42(m,4H),1.20-1.14(m,2H),0.87(t,J=7.5Hz,3H); HR-MS m / z: 1036.4194 [M+H] + .

[0503] Example 18: Fabrication of an ADC targeting EGFR and c-Met

[0504] Example 18-1: Preparation of ADC V42-DDDXd-6(1) targeting EGFR and c-Met

[0505] Reagents:

[0506] The antibody was V42, an anti-EGFR / anti-c-Met multispecific antibody, and the linker payload was MC-GGFG-DDDXd synthesized in Example 17.

[0507] Experimental procedure:

[0508] 1. Antibody Reduction: Adjust the antibody concentration to approximately 8-10 g / L using 20 mM histidine buffer (containing 1.43 mg / mL L-histidine, 2.27 mg / mL L-histidine monohydrate, 5% sucrose, pH 6.0), and then adjust the pH to approximately 6.0 using 0.3 M disodium hydrogen phosphate aqueous solution. Temperature the above solution to 30°C, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution to make the molar ratio of TCEP·HCl to antibody 6.0:1. React at 30°C with stirring at 50-60 rpm for 2 hours in the dark to obtain reaction solution 1.

[0509] 2. Antibody-linker-payload coupling: Add DMSO to reaction solution 1 to make the final concentration 5% (v / v), then add DMSO dissolved in Linker-payload at a concentration of 10 g / L, so that the molar ratio of Linker-payload to antibody is 9.25:1. React in the dark for 2 hours at 22℃ and stirring at 50-60 rpm to obtain reaction solution 2.

[0510] 3. Conjugation termination and purification: After conjugation, reaction solution 2 was replaced with 20mM histidine buffer (20g / kg polysorbate 80(II), 0.753g / kg histidine, 3.1762g / kg histidine hydrochloride, pH 5.8) using a 30kDa ultrafiltration membrane to obtain antibody-drug conjugate V42-DDDXd-6(1).

[0511] Referring to the method of Example 19, the DAR value of V42-DDDXd-6(1) was measured to be 6.0.

[0512] The structure of V42-DDDXd-6(1) is as follows:

[0513] Example 18-2: Preparation of ADC V42-DDDXd-6(2) targeting EGFR and c-Met

[0514] Reagents:

[0515] The antibody was V42, an anti-EGFR / anti-c-Met multispecific antibody, and the linker payload was MC-GGFG-DDDXd synthesized in Example 17.

[0516] Experimental procedure:

[0517] 1. Antibody Reduction: Adjust the antibody concentration to approximately 18-20 g / L using 20 mM histidine buffer (containing 1.43 mg / mL L-histidine, 2.27 mg / mL L-histidine monohydrate, 5% sucrose, pH 6.0), and then adjust the pH to approximately 6.5 using 0.3 M disodium hydrogen phosphate aqueous solution. Temperature the above solution to 30°C, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution to make the molar ratio of TCEP·HCl to antibody 6.0:1. React at 30°C with stirring at 50-60 rpm for 2 hours in the dark to obtain reaction solution 1.

[0518] 2. Antibody-linker-payload coupling: Add DMSO to reaction solution 1 to make the final concentration 5% (v / v), then add DMSO dissolved in Linker-payload at a concentration of 10 g / L, so that the molar ratio of Linker-payload to antibody is 9.25:1. React in the dark for 2 hours at 22℃ and stirring at 50-60 rpm to obtain reaction solution 2.

[0519] 3. Conjugation termination and purification: After conjugation, reaction solution 2 was replaced with 20mM histidine buffer (pH 5.8) using a 30kDa ultrafiltration membrane to obtain antibody-drug conjugate V42-DDDXd-6(2).

[0520] Referring to the method of Example 19, the DAR value of V42-DDDXd-6(2) was measured to be 5.9.

[0521] The structure of V42-DDDXd-6(2) is as follows:

[0522] Example 18-3: Preparation of AZD9592, an ADC benchmark targeting EGFR and c-Met

[0523] Reagents:

[0524] Antibody: The antibody is RAA22 / B09-57; the linker payload is Mal-PEG8-amide-Val-Ala-(4-NH2)-Exatecan (AZ0133, manufacturer: MCE, catalog number: HY-145399).

[0525] Experimental procedure:

[0526] 1. Antibody reduction: The antibody was replaced with histidine buffer (1.43 mg / mL L-histidine, 2.27 mg / mL L-histidine monohydrate) at pH 6.0, and the antibody concentration was adjusted to approximately 10 mg / mL with histidine buffer at pH 6.0. The antibody solution was added to a light-proof glass bottle, and 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution was added to make the antibody to TCEP molar ratio 1:10. The solution was incubated at 37°C in the dark for 1 hour with gentle stirring to reduce the disulfide bonds between the antibody chains, resulting in reaction solution 1.

[0527] 2. Coupling of antibody and adapter-loador: Add adapter-loador solution with a concentration of 10 mg / mL dissolved in DMSO to reaction solution 1, so that the molar ratio of antibody to adapter-loador is 1:12. Incubate at 22°C in the dark for 1 hour, stirring gently to link the antibody and adapter-loador, to obtain reaction solution 2.

[0528] 3. Conjugation termination and purification: reaction solution 2 was ultrafiltered with pH 6.0 histidine buffer to obtain antibody-drug conjugate AZD9592.

[0529] Referring to the method of Example 19, the DAR value of AZD9592 was measured to be 6.0.

[0530] The structure of AZD9592 is as follows:

[0531] Example 19: Determination of DAR value of antibody-drug conjugates

[0532] The DAR value of ADC was determined by hydrophobic interaction chromatography (HIC). The neutral high-salt mobile phase was used to enhance the hydrophobicity of protein molecules, thereby enabling them to bind with hydrophobic bonds in the chromatographic column. Then, the elution was carried out by gradually decreasing the salt concentration and gradually increasing the proportion of isopropanol, with less hydrophobic substances eluted first and more hydrophobic substances eluted later.

[0533] ADC components targeting EGFR and c-Met were separated using a non-porous polystyrene / divinylbenzene (PS / DVB) packing material with bonded butyl groups. The chromatographic column specifications were Sepax HIC-Butyl, 4.6 × 100 mm, 5 μm, with a column temperature of 25℃. Mobile phase A was 25 mmol / L phosphate buffer-2 mol / L ammonium sulfate, pH 7.0 (3.55 g of anhydrous disodium hydrogen phosphate and 264.28 g of ammonium sulfate were weighed, added to approximately 800 mL of ultrapure water, stirred until fully dissolved, adjusted to pH 7.0 ± 0.1 with phosphoric acid, and brought to a final volume of 1 L. After mixing, the solution was filtered through a 0.22 μm filter membrane). Mobile phase B was 25 mmol / L phosphate buffer, pH 7.0 (3.55 g of anhydrous disodium hydrogen phosphate was weighed, added to approximately 800 mL of ultrapure water, stirred until fully dissolved, adjusted to pH 7.0 ± 0.1 with phosphoric acid, and brought to a final volume of 1 L. After mixing, the solution was filtered through a 0.22 μm filter). Mobile phase C was 100% isopropanol. The ADC sample was diluted 1-fold with the initial proportioned mobile phase to prepare the test solution. The injection volume was adjusted according to the sample concentration, and 50 μg of protein was injected. Detection was performed at a wavelength of 280 nm. The flow rate was 0.5 mL / min, and gradient elution was performed for 30 min. The elution program is shown in Table 12.

[0534] Data processing employed area normalization for quantitative analysis. The peak area percentages of ADCs containing 0, 1, 2, 3, 4, 5, 6, 7, and 8 cytotoxic drugs were calculated, and the DAR value was also calculated. The formula is: DAR value = (peak area percentage of ADCs containing 0 cytotoxic drugs × 0 + peak area percentage of ADCs containing 1 cytotoxic drug × 1 + peak area percentage of ADCs containing 2 cytotoxic drugs × 2 + peak area percentage of ADCs containing 3 cytotoxic drugs × 3 + peak area percentage of ADCs containing 4 cytotoxic drugs × 4 + peak area percentage of ADCs containing 5 cytotoxic drugs × 5 + peak area percentage of ADCs containing 6 cytotoxic drugs × 6 + peak area percentage of ADCs containing 7 cytotoxic drugs × 7 + peak area percentage of ADCs containing 8 cytotoxic drugs × 8) / 100%.

[0535] Table 12. Gradient elution parameters

[0536] Example 20: Aggregate Validation of Antibody-Drug Conjugates

[0537] Gel chromatography was used to separate the components of an ADC sample targeting EGFR and c-Met. A neutral pH buffer containing 10% isopropanol was used as the mobile phase for elution, and the components were eluted sequentially in descending order of molecular weight. The column used was an ACQUITY UPLC Protein BEH SEC Column. A 1.7 μm, 4.6 × 300 mm gel chromatographic column was used at a column temperature of 25 °C. The mobile phase was 50 mmol / L phosphate buffer-200 mmol / L sodium chloride-10% isopropanol, pH 7.0 (12.53 g of disodium hydrogen phosphate dodecahydrate, 2.33 g of sodium dihydrogen phosphate dihydrate, and 11.69 g of sodium chloride were weighed, added to approximately 800 mL of ultrapure water, stirred until fully dissolved, and then ultrapure water was added to 1000 mL. After mixing, 900 mL of the solution was taken and 100 mL of isopropanol was added. The solution was mixed and filtered through a 0.22 μm filter membrane). 20 μg of the ADC sample was accurately injected into the liquid chromatograph and detected at a wavelength of 280 nm. The flow rate was 0.3 mL / min, and isocratic elution was performed for 15 min. Data processing was performed, and the results were quantitatively analyzed using the area normalization method. The peak area percentages of aggregates, immunoglobulin monomers, and low molecular weight impurities were calculated separately. The peak preceding the main peak represents aggregates, the main peak represents immunoglobulin monomers, and the peak following the main peak represents low molecular weight impurities. The detection results are shown in Table 13.

[0538] Table 13. Monomer, aggregate, and low molecular weight impurity content of ADCs targeting EGFR and c-Met

[0539] Example 21: Affinity of ADCs targeting EGFR and c-Met with human EGFR and human c-Met

[0540] The affinity of the ADC for human EGFR-His or human c-Met-His protein was detected using a biomolecular interaction analysis system (Cytiva, Biacore 8K). An amino-conjugated anti-hIgG (Fc) antibody (Cytiva, catalog No. BR-1008-39) was added to the CM5 sensor chip. The antibody was diluted to 4 μg / mL using running buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4, 0.05% surfactant P-20 (w / v), pH 7.4), and captured through the experimental channel at a flow rate of 30 μL / min. Human EGFR-His or human c-Met-His was diluted with run buffer to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM. Binding was performed at a flow rate of 50 μL / min for 200 s, followed by dissociation at a time of 600 s. Data was acquired and analyzed in real-time using software, and 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 KD (M) value. The test results are shown in Table 14.

[0541] Table 14. Affinity of Antibody-Drug Conjugates

[0542] Example 22: Cell binding activity of ADCs targeting EGFR and c-Met

[0543] Flow cytometry was used to detect the cell binding activity of ADCs targeting EGFR and c-Met. Human non-small cell lung cancer cells HCC827 (high EGFR expression, low to moderate c-Met expression, Yaji Biotechnology, catalog number: YS122C), NCI-H441, NCI-H292, and human colon adenocarcinoma cells HCA-7 (low to moderate EGFR expression, low to moderate c-Met expression, Nanjing Kebai, catalog number: CBP60035) were diluted to 2×10⁻⁶ cells / mL. 6 Add 50 μL / well of FACS buffer (Miltenyi Biotec, catalog number 130-091-221) to each well of a 96-well plate, and add 50 μL / well of serially diluted ADCs targeting EGFR and c-Met (V42-DDDXd-6(1) with a final concentration of 115.4 nM as the starting concentration, and AZD9592 with a final concentration of 100 nM as the starting concentration, 5-fold serial dilution, for a total of 8 concentrations). Incubate at 4℃ for 1 h, then centrifuge at 1000 rpm for 5 min. Discard the supernatant, wash 3 times with pre-cooled FACS buffer, and add 100 μL / well of goat anti-human IgG Fcγ-PE secondary antibody (Jackson immunoresearch, catalog number 109-116-170) diluted 1:200 (v / v). Incubate at 4℃ for 20 min. Wash three times with pre-chilled FACS buffer, resuspend in 50 μL of FACS buffer, and then analyze the fluorescence signal using flow cytometry (Sartorius, iQUE3). The binding activity of the ADC targeting EGFR and c-Met to EGFR and c-Met on the cell surface was represented by the mean fluorescence intensity (MFI). Data analysis was performed using GraphPad Prism 5, and the results are shown in Figures 10A-10D. The calculated EC50 values ​​were... 50 The results are shown in Table 15 below. The results show that V42-DDDXd-6(1) can effectively bind to cells with different levels of EGFR and c-Met expression, and the binding activity is better than that of AZD9592.

[0544] Table 15. Binding of ADCs targeting EGFR and c-Met to cells.

[0545] Example 23: Internalization activity of ADCs targeting EGFR and c-Met

[0546] Flow cytometry was used to detect the internalization of ADCs targeting EGFR and c-Met in cells with different EGFR and c-Met expression levels. The cell concentrations of HCC827, NCI-H292, HCA-7, and NCI-H441 cells were adjusted to 2 × 10⁻⁶. 6 50 μL / well was seeded into 96-well plates and 50 μL / well was added to each well of FACS buffer (Miltenyi Biotec, catalog number 130-091-221) serially diluted with EGFR and c-Met ADCs (for HCC827 and HCA-7 cells, the starting concentrations were V42-DDDXd-6(1) at 115.4 nM and AZD9592 at 100 nM, with 5-fold serial dilutions for a total of 8 concentrations; for NCI-H292 and NCI-H441 cells, the starting concentrations were V42-DDDXd-6(1) at 23.08 nM and AZD9592 at 20 nM, with 5-fold serial dilutions for a total of 7 concentrations). The plates were incubated at 4°C for 1 h. After incubation, the 96-well cell culture plates were removed, centrifuged at 400 g for 5 min at 4°C, and the supernatant was discarded. Add 100 μL / well of pHrodo (Thermo, P35358)-labeled mouse anti-human IgG antibody (Jackson, 109-005-190) diluted 1:200 (v / v), mix well, and incubate at 4°C. After 30 min, wash the plate, add 40 μL of cell culture medium to each well, mix well, and incubate at 37°C for 2 h. Place the plate in a flow cytometer (Sartorius, iQUE3) and measure the fluorescence reading of the RL1 channel. Analyze the data; the results are shown in Figures 11A-11D. Calculate the EC50. 50 As shown in Table 16 below. The results indicate that V42-DDDXd-6(1) can be effectively internalized in cells with different expression levels of EGFR and c-Met.

[0547] Table 16. Internalization activity of ADCs targeting EGFR and c-Met

[0548] Example 24: Killing of tumor cells by ADCs targeting EGFR and c-Met

[0549] To detect the killing effect of EGFR and c-Met-targeting ADCs on EGFR and c-Met-positive tumor cells, the killing activity was tested using natural human non-small cell lung cancer cells HCC827, NCI-H292, human gastric cancer cells MKN45 (EGFR low-to-medium expression, c-Met high expression, Nanjing Kebai Biotechnology, catalog number: CBP60488), human colon adenocarcinoma cells HCA-7, and human pancreatic cancer cells BxPC3 (EGFR high expression, c-Me low-to-medium expression, Pronosai, catalog number: CL-0042).

[0550] Cells in the logarithmic growth phase were harvested, and the cell densities of HCC827, MKN45, NCI-H292, and BxPC3 cells were adjusted to 2 × 10⁻⁶. 4 HCA-7 cells were reduced to a density of 4 × 10⁶ cells / mL. 4 The concentration of ADCs targeting EGFR and c-Met was increased to 100 μL / well in 96-well plates and cultured at 37°C and 5% CO2 for 4-6 h. ADCs targeting EGFR and c-Met were prepared using the complete culture medium of the corresponding cells. For HCC827 cells, V42-DDDXd-6(1) and AZD9592 were started at 25 nM and serially diluted 4-fold to obtain 10 concentrations. For MKN45, NCI-H292, and BxPC3 cells, V42-DDDXd-6(1) and AZD9592 were started at 100 nM and serially diluted 4-fold to obtain 10 or 11 concentrations. For HCA-7 cells, V42-DDDXd-6(1) and AZD9592 were started at 25 nM and serially diluted 4-fold to obtain 9 concentrations. Remove adherent cells from the culture plate. For the experimental group, add 50 μL / well of diluted ADC targeting EGFR and c-Met, while for the control group, add 50 μL / well of the corresponding complete culture medium. Continue culturing for 96 h, 120 h, or 144 h, then use the CellCounting-Lite 2.0 Luminescent Cell Viability Assay kit (Vazyme, catalog number: DD1101-03) for detection. Specifically: remove a 96-well plate, add 75 μL of CellCounting-Lite detection solution (Vazyme, catalog number: DD1101-03) to each well, vortex to mix, incubate at room temperature in the dark for 10 min, then aspirate 170 μL from each well to an opaque white plate, remove air bubbles, and read the chemiluminescence value using a microplate reader (PE, Envision 2105) to calculate the cell killing rate.

[0551] The data was analyzed, and the results are shown in Figures 12A-12E. The calculated EC 50The results are shown in Table 17 below. The results indicate that V42-DDDXd-6(1) has a better killing effect on tumor cells than AZD9592.

[0552] Table 17. Killing effect of ADCs targeting EGFR and c-Met on tumor cells

[0553] Example 25: Bystander effect of EGFR and c-Met-targeting ADCs on tumor cells

[0554] To detect the bystander effect of ADCs targeting EGFR and c-Met, HCC827 cells were used as positive cells and Jurkat cells as negative cells, and the detection was performed using a FACS-based method. HCC827 and Jurkat cells in logarithmic growth phase were harvested, and the viable cell density of HCC827 cells was adjusted to 2 × 10⁻⁶. 4 Jurkat viable cell density was adjusted to 1 × 10⁶ cells / mL and 1 mL was added to each well of a 6-well plate. The plates were then incubated overnight at 37°C with 5% CO₂. ADC was pre-diluted to 1.6 nM (final concentration) and 2 mL was added to each well. 4 Cells were added at a density of 1 mL / well to the corresponding cell plate. After 5 days of incubation, cells from the 6-well plate were removed, digested with trypsin, and counted. Cells from different wells were collected, and the cell density was adjusted to 1 × 10⁶ cells / mL using FACS buffer. 6 Cells / mL, 100 μL / well added to 96-well V plates. Add Fixable Viability Stain 780 (BD, catalog number: 565388) to the 96-well plates, incubate at room temperature for 15 min, wash cells twice, then add EGFR direct-labeled antibody (abcam, catalog number: 130738) and incubate at 4°C for 40 min. After incubation, wash cells twice, resuspend in FACS buffer, and perform flow cytometry (Invitrogen, model: [model number missing]). NxT) detection signal.

[0555] The data were analyzed and the results are shown in Figure 13. The results show that V42-DDDXd-6(1) has a bystander effect.

[0556] Example 26: Pharmacodynamic evaluation of EGFR and c-Met-targeting ADCs in NCI-H292 human lung cancer cell xenograft tumor model in nude mice

[0557] SPF-grade female BALB / c nude mice (source: Experimental Animal Center of Hangzhou Medical College) were subcutaneously inoculated with NCI-H292 human lung cancer cells in the right axilla, 5 × 10⁻⁶ cells per cell line. 6 One per tumor. Tumors are collected when their average volume reaches 100 mm². 3At approximately 10:00 AM, the animals were divided into 6 groups of 5 animals each. The specific grouping and administration regimens are shown in Table 18.

[0558] Table 18. Grouping and Dosing Regimens

[0559] Day 0 was the day of grouping, and the mouse was administered the drug once via tail vein on day 1. Tumor volume was measured twice a week, and the mice were weighed and the data were recorded. The general performance of the mice was observed and recorded daily. After the experiment, the tumors were removed, weighed, and photographed.

[0560] The detection indicators and calculation formulas are as follows:

[0561] Tumor volume, TV (mm) 3 )=1 / 2×(a×b 2 ); where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0562] Relative to tumor volume, RTV = TV t / TV0; where TV0 is the tumor volume on day 0, TV t This represents the tumor volume at each measurement.

[0563] Relative tumor proliferation rate, T / C (%) = T RTV / C RTV ×100%; where, T RTV For the treatment group, RTV; C RTV The solvent control group is RTV.

[0564] Tumor growth inhibition rate, TGI(%) = (1-TW / TW0)×100%; where TW is the tumor weight in the treatment group and TW0 is the tumor weight in the solvent control group.

[0565] Weight change rate, WCR (%) = (Wt) t -Wt0) / Wt0×100%; where Wt0 is the mouse body weight on day 0, Wt t The mouse's weight at each measurement.

[0566] The results of each test index on day 18 are shown in Table 19 and Figure 14 below. There was no obvious toxicity in any group. Among them, V42-DDDXd-6(1) showed inhibitory activity against mouse NCI-H292 human lung cancer xenograft tumors at both low and high doses. At the same dose of cytotoxic drugs, V42-DDDXd-6(1) was more effective than AZD9592.

[0567] Table 19. Effects of ADCs targeting EGFR and c-Met on various parameters in the NCI-H292 human lung cancer cell subcutaneous xenograft model in nude mice. Note: Compared with the solvent control group (group 1),* P<0.05; N / A indicates not applicable.

[0568] Example 27: Pharmacodynamic evaluation of EGFR and c-Met-targeting ADCs in a nude mouse xenograft model of HCA-7 human colon cancer cells.

[0569] HCA-7 human colon cancer cells were subcutaneously injected into the right axilla of SPF-grade female BALB / c nude mice (source: Experimental Animal Center of Hangzhou Medical College), 2 × 10⁻⁶ cells per mouse. 6 One per tumor. Tumors are expected to reach an average volume of 150 mm². 3 At approximately 10:00 AM, the animals were divided into 6 groups of 5 animals each. The specific grouping and administration regimens are shown in Table 20.

[0570] Table 20. Mouse grouping and drug dosage

[0571] Day 0 was designated as the day of grouping. The mice were administered the drug via tail vein on day 1 of each group, twice a week for a total of four administrations. Tumor volume and mouse weight were measured twice weekly and recorded. General mouse behavior was observed and recorded daily. At the end of the experiment, the tumors were removed, weighed, and photographed.

[0572] The detection indicators and calculation formulas are as follows:

[0573] Tumor volume, TV (mm) 3 )=1 / 2×(a×b 2 ); where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0574] Relative to tumor volume, RTV = TV t / TV0; where TV0 is the tumor volume on day 0, TV t This represents the tumor volume at each measurement.

[0575] Relative tumor proliferation rate, T / C (%) = T RTV / C RTV ×100%; where, T RTV For the treatment group, RTV; C RTV The solvent control group is RTV.

[0576] Tumor growth inhibition rate, TGI(%) = (1-TW / TW0)×100%; where TW is the tumor weight in the treatment group and TW0 is the tumor weight in the solvent control group.

[0577] Weight change rate, WCR (%) = (Wt) t -Wt0) / Wt0×100%; where Wt0 is the mouse body weight on day 0, Wt t The mouse's weight at each measurement.

[0578] The results of each test indicator on day 21 are shown in Table 21 and Figure 15 below. There was no obvious toxicity in any group. Among them, the high dose of V42-DDDXd-6(2) had inhibitory activity on mouse HCA-7 human colon cancer xenograft tumors.

[0579] Table 21. Effects of ADC-EGFR cMet on various indicators in a nude mouse subcutaneous xenograft model of HCA-7 human colon cancer cells. Note: Compared with the solvent control group. * P<0.05; N / A indicates not applicable.

[0580] The sequence information disclosed herein is summarized in Table S4 below.

[0581] Table S4. Sequence Information

[0582] 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.

[0583] 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. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt, comprising a multispecific antibody, wherein, The multispecific antibody includes (i) The first antigen-binding portion that binds to the first antigen; (ii) The second antigen-binding portion that binds to the second antigen; and (iii) The third antigen-binding portion 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 portion and the third antigen-binding portion 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: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; (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) 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.

2. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to claim 1, wherein, The first antigen-binding portion and the third antigen-binding portion 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; Preferably, The first antigen-binding portion 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 portion 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; or The first antigen-binding portion 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 portion 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.

3. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt, comprising a multispecific antibody, wherein, The multispecific antibody includes (i) The first antigen-binding portion that binds to the first antigen; (ii) The second antigen-binding portion that binds to the second antigen; and (iii) The third antigen-binding portion that binds to the first antigen; Wherein, the first antigen is c-Met and the second antigen is EGFR, and the first antigen-binding portion and the third antigen-binding portion are both single variable domains and each independently contains the single variable domain CDR1, CDR2 and CDR3 as shown in SEQ ID NO:35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38 or 39; Preferably, the first antigen-binding portion and the third antigen-binding portion each independently contain CDR1, CDR2 and CDR3 of a single variable domain as shown in SEQ ID NO:35, 36 or 38; More preferably, the first antigen-binding portion comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:36, and the third antigen-binding portion comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:35; or, the first antigen-binding portion comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:36, and the third antigen-binding portion comprises CDR1, CDR2, and CDR3 with single variable domains as shown in SEQ ID NO:

38.

4. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-3, wherein, The first antigen-binding portion and the third antigen-binding portion 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. Preferably, the first antigen-binding portion 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; more preferably, the first antigen-binding portion 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; even more preferably, the first antigen-binding portion 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; The amino acid sequence shown in NO:36 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. And / or, The third antigen-binding portion 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; more preferably, the third antigen-binding portion 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; more preferably, the third antigen-binding portion 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; The amino acid sequence shown in NO:35 has an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%; or, The third antigen-binding portion 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; more preferably, the third antigen-binding portion 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; more preferably, the third antigen-binding portion 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; The amino acid sequence shown in NO:38 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

5. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-4, wherein, The first antigen-binding portion and the third antigen-binding portion are selected from any one of the following: (1) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:35; (2) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:27, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:26; (3) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:33; (4) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:34; (5) The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:32; (6) The third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO:38; or (7) The third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:36, and the first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO:

39.

6. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-5, wherein, The single variable domain is from camel-like animals or of human origin; and / or The second antigen-binding moiety is Fab, scFv, or scFab; and / or The second antigen-binding portion is murine, chimeric, or humanized.

7. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-6, wherein, The second antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO:63, HCDR2 containing the amino acid sequence shown in SEQ ID NO:64, HCDR3 containing the amino acid sequence shown in SEQ ID NO:65, 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; and / or The second antigen-binding region comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:69, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:

70. Preferably, the second antigen-binding portion 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; Preferably, the heavy chain variable region of the second antigen-binding portion 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.

8. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-7, wherein, The third antigen-binding portion and the first antigen-binding portion are fused together, optionally via a peptide linker; Preferably, the third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion; Preferably, the multispecific antibody is trivalent.

9. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-8, wherein the multispecific antibody further comprises... (iv) An Fc domain consisting of two Fc polypeptides; Preferably, the first antigen-binding moiety 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 moiety is Fab and the second antigen-binding moiety is fused at its C-terminus to the N-terminus of another Fc peptide in the Fc domain. Preferably, the Fc domain is an IgG Fc domain, and more preferably an IgG1 Fc domain; Preferably, the IgG Fc domain is a human IgG Fc domain, and more preferably a human IgG1 Fc domain.

10. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to claim 9, wherein, The Fc domain contains amino acid substitutions that promote the association of two Fc polypeptides within the Fc domain. Preferably, according to the EU designation, one of the Fc polypeptides in 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; And / or, the Fc domain contains amino acid substitutions that reduce or eliminate the binding of the CH3 region of an Fc polypeptide in the Fc domain to protein A; Preferably, according to the EU designation, the Fc domain comprises amino acid substitutions occurring only in one of the Fc polypeptides: (a) 435R or (b) 435R and 436F; more preferably, according to the EU designation, one of the Fc polypeptides in 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; or, more preferably, according to the EU designation, one of the Fc polypeptides in 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.

11. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-10, wherein the multispecific antibody comprises three polypeptide chains, wherein: (1) One polypeptide chain 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:77; another polypeptide chain 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:85; and a third polypeptide chain 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:

77. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. (2) One polypeptide chain 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:71; another polypeptide chain 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:85; and a third polypeptide chain 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:

71. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. (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: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:73; and a fourth polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

73. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. (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:

75. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. (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: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:79; and a fourth polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

79. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. (6) One polypeptide chain 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:81; another polypeptide chain 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:85; and a third polypeptide chain 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:85; and a fourth polypeptide chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

81. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; or (7) One polypeptide chain 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:83; another polypeptide chain 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:85; and a third polypeptide chain 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:83; and a fourth polypeptide chain 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:

85. The amino acid sequence shown in NO:87 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. Preferably, the multispecific antibody is unfucosylated.

12. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvation of said antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt, comprising a multispecific antibody, wherein, The multispecific antibody consists 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 yet 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 yet 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 yet 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 yet 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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:

87. (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 a third 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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:87; Preferably, the multispecific antibody is unfucosylated.

13. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-12, wherein, The antibody-drug conjugate is formulated as follows: Ia The drug-connector shown in the diagram is connected to the multispecific antibody, and the drug-connector is connected to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia. in, R 1 and R 2 Each is independently selected from hydrogen atoms or deuterium atoms; preferably, the R 1 For hydrogen atoms, R 2 It is a deuterium atom.

14. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-13, wherein, The DAR of the antibody-drug conjugate is 1-10, 2-8, 2-6, 4-7, 5-6, or 5.5-6; Preferably, the DAR of the antibody-drug conjugate is 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.

15. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-14, wherein, The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt exhibits one or more of the following properties: (1) Combined with human c-Met, preferably with K 6E-10M or smaller. D Value combined with human c-Met; (2) Combined with human EGFR; (3) Combined with monkey c-Met; (4) Combined with monkey EGFR; (5) Internalization occurs in cells expressing c-Met and / or EGFR; (6) It exhibits cytotoxic activity against tumor cells expressing c-Met and / or EGFR; and (7) It has the bystander effect.

16. A pharmaceutical composition comprising the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt, as described in any one of claims 1-15; optionally, the pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

17. A method for preparing the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt according to any one of claims 1-15, comprising treating the multispecific antibody under reducing conditions, followed by reacting the multispecific antibody with a linker-loador selected from the structure shown in Formula I. in, The structure shown in Equation I is as follows: R 1 and R 2 Each is independently selected from hydrogen atoms or deuterium atoms; preferably, the R 1 For hydrogen atoms, R 2 It is a deuterium atom.

18. A method of treating a disease expressing c-Met and / or EGFR, comprising administering to a subject the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition of claim 16. Preferably, the disease expressing c-Met and / or EGFR is a tumor; More preferably, the tumor is a c-Met and / or EGFR positive tumor; more preferably, the tumor is epithelial carcinoma, squamous cell carcinoma, glioblastoma, 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, and / or thymic cancer.

19. The method according to claim 18, wherein, The method further includes administering one or more additional therapeutic agents to the subject; preferably, the additional therapeutic agent is a tumor therapeutic agent.

20. A kit comprising the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition of claim 16; optionally, the kit further comprising instructions for use.

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