Antibody-drug conjugate targeting EGFR and c-met

Abstract

Belongs to the field of biological medicine, and provides an EGFR (epidermal growth factor receptor) and c-Met targeting antibody drug conjugate which comprises a multispecific antibody, a connector and a cytotoxic drug which are connected with one another. Pharmaceutical compositions comprising the antibody drug conjugates and uses thereof are also provided. The antibody drug conjugate provided by the invention realizes excellent anti-tumor activity or / and relatively good safety. The provided antibody drug conjugate can be used for tumor treatment.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511682742.1 (22) Application Date 2025.10.22 (66) Domestic Priority Data 202411488108.X 2024.10.23 CN (62) Divisional Application Data 202511513272.6 2025.10.22 (71) Applicant: Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Address: No. 369, Yuzhou South Road, Lianyungang City, Jiangsu Province, 222062 (72) Inventors: Zhang Zhengping, Ma Yimin, Zhou Yaping, Yu Nianqin, Chen Hongmei, Shen Chen, Zhang Bing, Xu Tongjie (51) Int.Cl. A61K 47 / 68 (2017.01) A61K 38 / 08 (2019.01) A61P 35 / 00 (2006.01) (54) Invention Title: Antibody-Drug Conjugate Targeting EGFR and c-Met (57) Abstract: Belonging to the field of biomedicine, this invention provides an antibody-drug conjugate targeting EGFR and c-Met, wherein the antibody-drug conjugate comprises a linked multispecific antibody, a linker, and a cytotoxic drug. Pharmaceutical compositions comprising the antibody-drug conjugate and their uses are also provided. The provided antibody-drug conjugate achieves excellent antitumor activity and / or good safety. The provided antibody-drug conjugate can be used for the treatment of tumors. Claims 2 pages, Description 72 pages, Sequence Listing (electronic publication), Drawings 14 pages, CN 121570606 A 2026.02.27 CN 1 21 57 06 06 A 1. 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, wherein said multispecific antibody comprises (i) a first antigen-binding moiety binding to a first antigen; (ii) a second antigen-binding moiety binding to a second antigen; and (iii) a third antigen-binding moiety binding to the first antigen; wherein the first antigen is c-Met and the second antigen is EGFR, the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains and each independently comprises any one of the following: (1) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5. (1) CDR3 containing the amino acid sequence shown in SEQ ID NO: 7, 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) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; (4) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 12; (5) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 15; (6) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (7) A CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 19. CDR3 containing the amino acid sequence shown in SEQ ID NO: 22; or (8) CDR1 containing the amino acid sequence shown in SEQ ID NO: 23, CDR2 containing the amino acid sequence shown in SEQ ID NO: 24, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 24. 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 comprise any one of the following: (1) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is selected from S or T, preferably T; (2) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; or (8) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24. 3. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the antibody-drug conjugate according to claim 2.The antibody-drug conjugate, its stereoisomer, and a solvate of its pharmaceutically acceptable salt, 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. Claims 1 / 2 Page 2 CN 121570606 A 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 claim 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. 5. 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, wherein said multispecific antibody comprises (i) a first antigen-binding moiety binding to a first antigen; (ii) a second antigen-binding moiety binding to a second antigen; and (iii) a third antigen-binding moiety binding to the first antigen; wherein the first antigen is c-Met and the second antigen is EGFR, and the first and third antigen-binding moieties are each single variable domains and each independently comprises CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, or 39. 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 claim 5, wherein the first antigen-binding moiety and the third antigen-binding moiety each independently comprise an amino acid sequence as shown in SEQ ID NO: 35, 36, or 38.7. 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 claim 6, wherein the first antigen-binding moiety comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO: 36, and the third antigen-binding moiety comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO: 35. 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 claim 6, wherein the first antigen-binding moiety comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO: 36, and the third antigen-binding moiety comprises CDR1, CDR2, and CDR3 with a single variable domain as shown in SEQ ID NO: 38. 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 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. 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 any one of claims 1-9, 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: 41. Claims 2 / 2 Page 3 CN 121570606 A Antibody-drug conjugate targeting EGFR and c-Met

[0001] This application is filed on October 22, 2025, with Chinese application number 202511513272.6, and entitled "Invention".This is a divisional application of a patent application for an invention entitled "Antibody-Drug Conjugate Targeting EGFR and c-Met". Technical Field

[0002] This disclosure belongs to the field of biomedicine and relates to antibody-drug conjugates targeting EGFR and c-Met, which comprise 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 Art

[0003] Epidermal growth factor receptor (EGFR, also known as ErbB1 or HER1) is a 170 kDa type I transmembrane glycoprotein encoded by the proto-oncogene c-erbB1, belonging to the receptor tyrosine kinase (RTK) family. EGFR is a member of the human epidermal growth factor receptor (HER) family, which includes HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Increased expression or kinase activity of EGFR is associated with a range of human tumors, making EGFR an attractive target for tumor therapy.

[0004] c-Mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase, which is a heterodimer of approximately 190 kDa composed of a 50 kDa extracellular chain (α chain) and a 145 kDa transmembrane chain (β chain). The transmembrane chain (β chain) includes a SEMA homology region (SEMA), a PSI domain (plexin semaphorin-integrin, PSI), 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 and 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 activates HGF / c-Met signaling through paracrine mechanisms to exert its biological functions.

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

[0006] In camel-dwelling animals, in addition to the traditional four-chain structure IgG1, there are also naturally occurring heavy-chain-only antibodies (HcAbs) IgG2 and IgG3 that do not contain light chains. The single variable domain (VHH) in heavy-chain-only antibodies can specifically bind to antigens and has a high affinity for them. Based on its uniqueness, using the VHH domain as part of an antibody or antigen-binding fragment has significant advantages over conventional antibody fragments (such as scFv, Fab, etc.). For example, only a single domain is needed to specifically bind antigens with high affinity; it is easy to modify into multivalent and multispecific forms; the VHH domain is highly soluble and has no tendency to aggregate; the VHH molecule is small and thus has high tissue permeability; VHH does not need to pair with light chains; there is no light-heavy chain mismatch problem when forming bispecific or multispecific antibodies, etc.

[0007] Antibody-drug conjugates (ADCs) are a class of drugs that combine the high specificity of therapeutic antibodies and the high cytotoxic activity of cytotoxic drugs, wherein the therapeutic antibody part and the cytotoxic drug part are linked through an intermediate linker. In all cancer patients resistant to EGFR tyrosine kinase inhibitors, approximately 60% exhibited increased c-Met expression, c-Met amplification, or increased HGF, indicating that c-Met is activated as a compensatory pathway when EGFR is inhibited. While there are existing studies reporting on the development of antibody-drug conjugates targeting EGFR and c-Met for anti-tumor purposes, these are limited, and more available options are still needed. Summary of the Invention 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, and its pharmaceutically acceptable salt, comprising a multispecific antibody targeting EGFR and c-Met, wherein said multispecific antibody comprises (i) a first antigen-binding moiety binding to a first antigen; (ii) a second antigen-binding moiety binding to a second antigen; and (iii) a third antigen-binding moiety binding to the first antigen; wherein the first antigen is c-Met and the second antigen is EGFR, and the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains and each independently comprises any one of the following: (1) comprising SEQ ID NO:(1) CDR1 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; (2) CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T; (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; (4) CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 12; (5) CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 15, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 16, wherein X1 is S or T; (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.

[0009] In some embodiments, the first antigen-binding portion and the third antigen-binding portion each independently comprise any one of the following: (2) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T; (3) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; or (8) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22 and an amino acid sequence shown in SEQ ID NO: 23.The CDR2 containing the amino acid sequence shown in SEQ ID NO: 24, and the CDR3 containing the amino acid sequence shown in SEQ ID NO: 24. Specification 2 / 72 pages 5 CN 121570606 A

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

[0011] In some embodiments, the first antigen-binding portion and the third antigen-binding portion each independently comprise any one of the following: (3) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; or (8) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24.

[0012] In some embodiments, the first antigen-binding portion and the third antigen-binding portion each independently comprise CDR1, CDR2, and CDR3 with 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.

[0013] 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.The first antigen-binding portion comprises CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 36, and the third antigen-binding portion comprises CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 35. In other specific embodiments, the first antigen-binding portion comprises CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 36, and the third antigen-binding portion comprises CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 38. The CDRs are defined, for example, according to Kabat, IMGT, Chothia, Contact, AbM, and / or CCG.

[0015] 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. (See specification page 3 / 72, 6 CN 121570606 A). 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.The amino acid sequence shown in SEQ ID NO: 35.

[0016] 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.

[0017] In some embodiments, the first antigen-binding portion and the third antigen-binding portion bind different epitopes of c-Met.

[0018] The second antigen-binding portion provides the ability to bind to EGFR. In some embodiments, the second antigen-binding portion 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 includes the amino acid sequence shown in SEQ ID NO: 69.The heavy chain variable region 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: 70, 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, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 70.

[0019] In some embodiments, the multispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.

[0020] In some specific embodiments, in the multispecific antibody, 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; 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; 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 comprising the amino acid sequence shown in SEQ ID NO: 63, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 64, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 65; the light chain variable region comprises: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 67, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 68. LCDR3 with the amino acid sequence shown in SEQ ID NO: 48; 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, the third antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 40 (page 4 / 72 of specification, CN 121570606 A); 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. Further...In a specific implementation, the first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 36, the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 35; and the heavy chain variable region of the second antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 69, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 70.

[0021] In some other specific embodiments, in the multispecific antibody, 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; 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; 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 comprising the amino acid sequence shown in SEQ ID NO: 63, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 64, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 65; the light chain variable region comprises: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 67, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 68. LCDR3 with the amino acid sequence shown in SEQ ID NO: 41. In a further specific embodiment, the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 41, 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, 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.

[0022] In these embodiments, an alternative configuration is that both the first and third antigen-binding portions are single variable domains, the second antigen-binding portion is Fab, and the first antigen-binding portion has a C-terminal domain of...The first antigen-binding moiety is fused to the N-terminus of one of the Fc peptides in the Fc domain, the second antigen-binding moiety is fused to the N-terminus of another Fc peptide in the Fc domain at the C-terminus of its Fab heavy chain or Fab light chain, and the third antigen-binding moiety is fused to the N-terminus of the first antigen-binding moiety at its C-terminus. In a more specific embodiment, the multispecific antibody consists of three polypeptide chains, wherein the first polypeptide chain contains the aforementioned first antigen-binding moiety, the third antigen-binding moiety, and one of the Fc peptides in the Fc domain; the second polypeptide chain contains the Fab heavy chain of the aforementioned second antigen-binding moiety and another Fc peptide in the Fc domain; and the third polypeptide chain is the Fab light chain of the aforementioned second antigen-binding moiety.

[0023] In some embodiments, the multispecific antibody is trivalent.

[0024] In some embodiments, the antibody-drug conjugate is formed by linking a drug-connector of the structure shown in Formula Ia to the multispecific antibody, wherein the drug-connector is linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia (page 5 / 72, CN 121570606 A); wherein R1 and R2 are independently selected from hydrogen atoms or deuterium atoms; preferably, R1 is a hydrogen atom and R2 is a deuterium atom.

[0025] This disclosure provides a method for preparing the antibody-drug conjugate of this disclosure, its stereoisomers, its pharmaceutically acceptable salts, or solvates of the 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 adapter-load of this disclosure.

[0026] 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 a pharmaceutically acceptable excipient.

[0027] 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.

[0028] On the other hand, this disclosure provides a method of 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.

[0029] On the other hand, this disclosure provides the antibody-drug conjugate, which can be used as a drug or for treatment, and its stereochemical composition.Isomers, pharmaceutically acceptable salts thereof, or solvates of the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or the pharmaceutical compositions thereof.

[0030] The antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts provided in this disclosure target EGFR and c-Met, exhibiting good antitumor efficacy and / or safety.

[0031] Figure 1A-1G shows the binding curves of different anti-human c-Met VHH-Fc chimeric antibodies with target cells expressing different levels of c-Met, as detected by flow cytometry; Figure 2 is a schematic diagram of the structure of an exemplary multispecific antibody of this disclosure; Figure 3A-3C shows the binding curves of anti-EGFR / anti-c-Met multispecific antibodies with cells expressing EGFR and c-Met, as detected by flow cytometry; 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, where the leftmost lane is the marker, BM is Amivantamab, negative is hIgG1, blank is no antibody, HGF "-" or "+" represents no HGF or HGF added, respectively, and p-cMet, p-Akt, p-Erk and p-EGFR are phosphorylated c-Met, phosphorylated Akt, phosphorylated Erk and phosphorylated EGFR, respectively; Figure 5 shows the binding curves of anti-EGFR / anti-c-Met multispecific antibodies with different levels of c-Met expression, as detected by Western blotting. The inhibition of EGFR phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies by blotting detection is shown in Figure 6A-6C. The leftmost lane represents the marker, BM represents Amivantamab, negative represents hIgG1, blank represents no antibody, and EGF "-" or "+" represents EGF-free or EGF-added, respectively. p-cMet, p-Akt, p-Erk, and p-EGFR represent phosphorylated c-Met, phosphorylated Akt, phosphorylated Erk, and phosphorylated EGFR, respectively. 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. Figures 7A-7B show the inhibition of the proliferation of tumor cells expressing EGFR and c-Met by anti-EGFR / anti-c-Met multispecific antibodies. (Instructions for use, page 6 / 72, 9 CN 121570606 A) Figures 8A-8B show the killing effect of anti-EGFR / anti-c-Met multispecific antibodies on tumor cells expressing EGFR and c-Met; 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.Figures 10A-10D show the binding curves of V42-DDDXd-6(1) to cells expressing EGFR and c-Met as detected by flow cytometry, where Figure 10A is HCC827 cells, Figure 10B is NCI-H292 cells, Figure 10C is HCA-7 cells, and Figure 10D is NCI-H441 cells; 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; 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; 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; 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; Figure 15 shows the tumor volume curve of the xenograft tumor after administration of V42-DDDXd-6(1) in the HCA-7 human colon cancer cell xenograft tumor model in nude mice.

[0032] Detailed Description of the Invention Definitions and Description 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 trade product or its active ingredient.

[0033] The terms “optional” or “optionally” mean that the event or condition described below may or may not occur, including both the occurrence and non-occurrence of said event or condition.

[0034] The term “substituted” means that any one or more hydrogen atoms on a particular atom are substituted by a substituent, provided that the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted, and oxo does not occur on the aromatic group. “Optionally substituted” means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically feasible basis.

[0035] 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.

[0036] The term “thiol” refers to the -SH group.

[0037] Unless otherwise specified, the absolute configuration of a stereocenter is indicated by a wedge solid line bond ( ) and a wedge dashed line bond ( ).

[0038] 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: Specification 7 / 72 page 10 CN 121570606 A, where * and # respectively indicate that the carbon atom at position 3 and the nitrogen atom at position 1 of the structure are connected with other groups.

[0039] As used herein, a compound formed by replacing an atom or group of atoms in a parent compound molecule with other atoms or groups is called a “derivative” of the parent compound.

[0040] The compounds of this disclosure may exist in specific geometric isomer 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.

[0041] Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise from the fact that the single bonds of double bonds or cyclic carbon atoms cannot rotate freely.

[0042] Unless otherwise stated, the term "enantiomer" refers to stereoisomers that are mirror images of each other.

[0043] Unless otherwise stated, the term "diastereomer" refers to stereoisomers in which the molecules have two or more chiral centers and are not mirror images of each other.

[0044] The compounds and intermediates of this disclosure may also exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The term "tautomer" or "tautomer form" refers 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.

[0045] 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 capable of diffusing 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 cytotoxic drug or the cytotoxic drug and the linker.The combination of hydrophobicity is relevant. Such cytotoxic drugs can be, for example, ethatecan derivatives. Especially in tumors with heterogeneous target expression and solid tumors in which antibody penetration may be limited, the bystander effect may be desirable.

[0046] 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), and various antibody structures of single-chain antibodies, as long as they exhibit the desired antigen-binding activity.

[0047] The term “multispecific” means that an antibody is capable of specifically binding to a variety of different antigenic determinants, for example, capable of specifically binding to two or more different antigenic determinants. In this document, 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 may be expressed on the same or different cells. Different antigenic determinants may differ depending on the type of antigen (e.g., binding antigens EGFR and c-Met) or may be present on the same antigen. Antigenic determinants are special chemical groups with a certain composition and structure on the surface or other sites of antigen molecules, which can specifically bind to their corresponding antibodies or sensitized lymphocytes. An example of an antigenic determinant is c-Met, which has a variety of 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 can bind to EGFR and c-Met, for example.

[0048] The term "N-valent antibody" is intended to indicate that there are N antigen-binding sites in the antibody. For example, "bivalent antibody" or "antibody specification 8 / 72 page 11 CN 121570606 A 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 usually have two antigen-binding sites, Fab usually has a single antigen-binding site, and single variable domain and scFv usually have a single antigen-binding site.

[0049] The term “antigen-binding moiety” refers to a polypeptide molecule that specifically binds to an antigenic determinant. A specific antigen-binding moiety may be Fab, scFv, or a single variable domain.

[0050] The terms “first,” “second,” or “third” used in this disclosure with respect to antigen-binding moiety, antigen, Fc polypeptide, peptide linker, polypeptide chain, etc., are used for ease of distinction when more than one type of moiety is present. Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order or orientation.

[0051] The term “fusion” means that the components (e.g., antigen-binding moiety, Fc polypeptide, etc.) are directly or via one or more peptide linkers.Linked by peptide bonds. For example, some peptide linkers consist of 1 to 50 amino acids linked by peptide bonds, wherein the 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.

[0052] 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 region (also referred to as a heavy chain variable domain, VH, or VH domain) and a light chain variable region (also referred to as a light chain variable domain, VL, or VL domain), while heavy chain antibodies derived from animals such as camels or sharks have only a single 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 complementary determinant regions, referred to in the art and hereinafter as complementary determinant region 1 (or CDR1), complementary determinant region 2 (or CDR2), and complementary determinant region 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 endows the antibody with antigen specificity due to having an antigen binding site.

[0053] The term "single variable domain" refers to a variable domain that can specifically bind to an antigen epitope without pairing with other variable domains. A single variable domain usually has three CDRs (CDR1, CDR2, and CDR3) present on a single domain. In some cases, a single variable 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 consisting essentially of a single variable domain, so that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). Another example of a single variable domain is the "VHH domain" (or simply "VHH" or "VHH") of camels.

[0054] The term "VHH domain" is used to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies and the light chain variable domains present in conventional four-chain antibodies. The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (this differs from the VH or VL domains in conventional four-chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable, and efficient antigen recognition unit formed by a single domain.

[0055] The term "complementarity-determining region" (CDR) is also known as the "hypervariant region" (HVR). Natural four-chain antibodies typically contain six CDRs: three in the heavy chain variable region, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3); and three in the light chain variable region, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3). Heavy chain-only antibodies or single variable domain antibodies typically have three CDRs (CDR1, CDR2, and CDR3).

[0056] Currently, there are many methods for defining CDRs. Among them, the Kabat definition is based on sequence variability and is the most commonly used; while the Chothia definition is based on the position of the structural loop. The AbM definition is a compromise between the Kabat and Chothia definitions and is used by the AbM antibody modeling software of Oxford Molecular. The definition of “contact” is described on page 9 / 72 of CN 121570606 A. The basis of CDR is the analysis of the available crystal structure of the complex. In addition, there is the CCG definition. However, it should be noted that the boundaries of CDRs for the same antibody variable region obtained by different methods of delineation may differ, that is, the CDR sequences for the same antibody variable region defined by different methods may be different. Therefore, when referring to antibodies defined by a specific CDR sequence, the scope of said antibody also covers antibodies defined by CDR sequences of other arbitrary definitions (e.g., one or more of the definitions of Kabat, IMGT, Chothia, Contact, AbM, CCG, etc.).

[0057] 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 immunoglobulins. In this document, Fab refers to a Fab molecule in its natural form or modified form, namely, a Fab heavy chain (VH-CH1, N-to-C-terminal direction) comprising a heavy chain variable region VH and a constant region CH1, and a Fab light chain (VL-CL, N-to-C-terminal direction) comprising a light chain variable region and a constant region CL. A modified Fab may, for example, be 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 may be a Fab with amino acid substitutions introduced into the CL domain.

[0058] 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.

[0059] The terms “Fc domain,” “Fc,” or “Fc region” are used herein to define the C-terminal region of the immunoglobulin heavy chain, whichContains at least a portion of the constant region. This term includes the native sequence Fc and variant Fc. The C-terminal lysine (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 of the “Fc polypeptides” 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.

[0060] The term “treatment” means administering the compounds or pharmaceutical compositions described herein 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; (iv) reducing any direct or indirect pathological consequences of the disease or disease state.

[0061] The term “therapeutic effective amount” means (i) the amount of the compounds of this disclosure used to treat or prevent a particular disease, condition, or disorder; (ii) reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) 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 this disclosure constituting a “therapeutic effective amount” may 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 may also be routinely determined by those skilled in the art based on their own knowledge and the content of this disclosure.

[0062] The term “pharmaceuticalally 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.

[0063] The term “excipient” refers to any component other than the active ingredient (e.g., the antibody of this disclosure). ExcipientsThe choice will largely depend on factors such as the specific method of application, the efficacy of the excipient for solubility and stability, and the nature of the dosage form.

[0064] The term “isolated” means a target compound that has been isolated from its natural environment, such as an antibody or its antigenic conjugate fragment, VHH, or nucleic acid.

[0065] The terms “Xn” and “Xaa” are equivalent and refer to an unspecified amino acid, the range of which is specified by subsequent definitions in the relevant description.

[0066] As used herein, the term “EC50” refers to the effective concentration that elicits a 50% maximum response to the antibody or ADC. EC50 can be measured by ELISA or FACS analysis or any other method known in the art.

[0067] “KD” refers to the equilibrium dissociation constant, which is derived from the ratio of the dissociation rate constant (kd) to the binding rate constant (ka) (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody or ADC can be determined using methods well established in the art. A preferred method for determining the KD of an antibody or ADC is by using surface plasmon resonance (SPR) technology, preferably using a biosensor system such as the Biacore surface plasmon resonance system.

[0068] The term “identity” is also called consistency. The “percentage of identity (%)” of an amino acid sequence refers to the percentage of amino acid residues in the sequence to be aligned that are identical to the amino acid sequence shown herein when the sequence to be aligned is compared with the specific amino acid sequence shown herein and, if necessary, vacancies are introduced to achieve the maximum percentage of sequence identity, without regard to any conservative substitutions as part of the sequence identity. Amino acid sequence alignment for identity can be performed in a variety of ways within the art. Those skilled in the art can determine suitable parameters for the alignment of sequences, including any algorithm required to obtain the maximum alignment across the full length of the comparison sequence.

[0069] 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" or "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 the disease or condition.

[0070] As used herein, "about" means the range of error acceptable to a particular value as determined by a person skilled in the art.Within this scope, it depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can, according to art practice, mean within 1 or more standard deviations. Alternatively, “about” can 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 this disclosure, unless otherwise stated, “about” should be understood to mean within an acceptable error range for that particular value. In this document, unless otherwise stated, the value of a step parameter or condition is by default modified by “about”.

[0071] The terms “comprise,” “containing,” and “comprising” and their equivalents (e.g., contain, contains, containing, include, includes, and including) should 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.

[0072] In this document, unless the context clearly specifies otherwise, singular terms cover plural references, and vice versa.

[0073] Antibody-Drug Conjugate (ADC) 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.

[0074] Multispecific antibodies in ADCs 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, specification 11 / 72 pages 14 CN 121570606 A wherein the multispecific antibody comprises (i) a first antigen-binding moiety binding a first antigen; (ii) a second antigen-binding moiety binding a second antigen; and (iii) a third antigen-binding moiety binding a first antigen; wherein the first antigen is c-Met and the second antigen is EGFR, the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains and each independently comprises any one of the following: (1) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; (2) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 4. CDR2 of the amino acid sequence shown in Figure 5, and containing SEQ ID(2) CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is S or T; (3) CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9; (4) CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 12; (5) CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 15; (6) CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (7) CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. CDR1 of the amino acid sequence shown in SEQ ID NO: 20 includes CDR2 of the amino acid sequence shown in SEQ ID NO: 21 and CDR3 of the amino acid sequence shown in SEQ ID NO: 21; or (8) CDR1 of the amino acid sequence shown in SEQ ID NO: 22 includes CDR2 of the amino acid sequence shown in SEQ ID NO: 23 and CDR3 of the amino acid sequence shown in SEQ ID NO: 24.

[0075] 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.

[0076] 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 feature. 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, that is, the first antigen-binding portion includes: SEQ ID NO: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 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. 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: 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 comprises the complementarity-determining region described in (3) above, and the third antigen-binding portion comprises the complementarity-determining region described in (8) above, that is, 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. More specifically, in one 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, and CDR3 shown in SEQ ID NO: 9.CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24.

[0077] Table S1. Exemplary multispecific antibodies defining the CDR characteristics of the first antigen-binding portion and the third antigen-binding portion

[0078] Note: In the table, (X)+(Y) indicates the combination of the third antigen-binding portion and the first antigen-binding portion in the multispecific antibody. For example, (2)+(1) indicates that the third antigen-binding portion of the multispecific antibody contains the complementary determination region described in (2) above, while the first antigen-binding portion contains the complementary determination region described in (1) above.

[0079] Further, 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.

[0080] 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, or 39. In some specific embodiments, the first antigen-binding portion comprises SEQ ID NO: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 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.

[0081] 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.

[0082] 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 SEQ ID NO: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.

[0083] 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.

[0084] As an 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.

[0085] As an 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.

[0086] 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.

[0087] 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.

[0088] In one example, the first antigen-binding portion comprises SEQ ID NO: 36, SEQ ID NO: 36, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 ...The third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 37, and the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 26, 32, 33, 34, 35, 38, or 39.

[0089] In one example, the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 37, and the first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 26, 32, 33, 34, 35, 38, or 39.

[0090] 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.

[0091] In one example, the third antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 27, and the first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 26.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] In one example, the third antigen-binding portion comprises SEQ ID NO:The first antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 36, and the second antigen-binding portion contains the amino acid sequence shown in SEQ ID NO: 38.

[0101] In one example, 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: 38.

[0102] In one example, 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: 39.

[0103] In one example, 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.

[0104] 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.

[0105] Table S2 exemplarily lists multispecific antibodies constructed by different combinations of the first antigen-binding portion and the third antigen-binding portion with full-length amino acid sequence characteristics.

[0106] Table S2. Exemplary multispecific antibody specification page 15 / 72 18 CN 121570606 A for defining the amino acid sequences of the first antigen-binding part and the third antigen-binding part

[0107] Note: In the table, (X)+(Y) represents the combination of the third antigen-binding part and the first antigen-binding part in the 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.

[0108] Table S3. CDR and full-length amino acid sequence of a single variable domain (SEQ ID NO.) Specification 16 / 72 pages 19 CN 121570606 A

[0109] 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 embodimentsIn the above, both the first and third antigen-binding portions are humanized. In some embodiments, both the first and third antigen-binding portions are from camels.

[0110] In the multispecific antibody of this disclosure, the second antigen-binding portion provides the ability to target EGFR. The second antigen-binding portion may be Fab, scFv, or ScFab (single-chain Fab). In some embodiments, the second antigen-binding portion is a Fab that binds to EGFR.

[0111] In some embodiments, the second antigen-binding portion is murine, chimeric, or humanized.

[0112] In some embodiments, 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. In some embodiments, 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. In some 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: 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. 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: 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.

[0113] In one specific embodiment, 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. In one specific embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region shown in SEQ ID NO: 69 (page 17 / 72 of specification, 20 CN 121570606 A); 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.

[0114] In some embodiments, 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. In some embodiments, the heavy chain variable region of the second antigen-binding portion 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.

[0115] The multispecific antibody described in this disclosure may be without an Fc domain, and the first antigen-binding moiety, the second antigen-binding moiety, and the third antigen-binding moiety are fused together by a suitable linker.

[0116] The multispecific antibody described in this disclosure may be with an Fc domain, which can prolong the half-life, provide Fc domain-related effectors, and other functions.

[0117] Multispecific antibody configuration The antigen-binding moieties in the multispecific antibody described in this disclosure can be fused together in various configurations. In someIn some embodiments, the multispecific antibody further comprises (iv) an Fc domain consisting of two Fc polypeptides.

[0118] In some embodiments, the third antigen-binding portion is fused to the first antigen-binding portion, optionally via a peptide linker. In some more specific embodiments, the third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion. Further, in some embodiments, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one of the Fc polypeptides of the Fc domain, the second antigen-binding portion is Fab, and the second antigen-binding portion is fused at its C-terminus of its Fab heavy chain or Fab light chain to the N-terminus of the other Fc polypeptide of the Fc domain.

[0119] In one specific embodiment, both the first antigen-binding portion and the third antigen-binding portion are single variable domains, the second antigen-binding portion is Fab, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one of the Fc polypeptides of the Fc domain, the second antigen-binding portion is fused at its C-terminus to the N-terminus of its Fab heavy chain to the N-terminus of the other Fc polypeptide of the Fc domain, and the third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion. Such a construction is schematically depicted in Figure 2. In this embodiment, and more specifically, the multispecific antibody has three polypeptide chains, wherein the first polypeptide chain contains the aforementioned first antigen-binding moiety, the third antigen-binding moiety, and one of the Fc polypeptides of the Fc domain; the second polypeptide chain contains the Fab heavy chain of the aforementioned second antigen-binding moiety and another Fc polypeptide of the Fc domain; and the third polypeptide chain is the Fab light chain of the aforementioned second antigen-binding moiety.

[0120] In the above embodiments, the antigen-binding moieties of the multispecific antibody can be operatively linked, such as 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.

[0121] The third antigen-binding moiety can be fused to the first antigen-binding moiety directly or via a peptide linker. In one embodiment, the third antigen-binding moiety is fused to the first antigen-binding moiety via a peptide linker.

[0122] Each peptide linker can be independently adopted from any suitable one, for example, charged and / or flexible linker peptides can be used. In a specific embodiment, the peptide linker consists of 1 to 50 amino acids linked by peptide bonds, wherein the 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. Therefore, an exemplary peptide linker may be a poly(peptide linker).Glycine (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.

[0123] In some embodiments, the peptide linker may be a peptide linker composed of glycine and serine. In some embodiments, the number of amino acids contained in the peptide linker may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20. In some embodiments, the peptide linker is a peptide linker containing GGGGS units. 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.

[0124] When fused with Fc, fusion is typically via a hinge region. In one embodiment, the first antigen-binding moiety is fused to one of the Fc polypeptides in the Fc domain via a first hinge, and the second antigen-binding moiety is fused to another Fc polypeptide in the Fc domain via a second hinge. In some embodiments, the first hinge and the second hinge are capable of forming covalent bonds, such as disulfide bonds, with each other. The first hinge and / or the second hinge may contain amino acids from the hinge region of human IgG, which contains a natural hinge region or a variant thereof. In some embodiments, the first hinge and / or the second hinge contains amino acids from the hinge region of human IgG1. In some embodiments, the first hinge and / or the second hinge contains 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).

[0125] Fc Domain of Multispecific Antibodies The Fc domain of a multispecific antibody consists of a pair of polypeptide chains containing heavy chain domains of immunoglobulin molecules. ExampleFor example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each Fc polypeptide contains CH2 and CH3 of the constant region of the IgG heavy chain. 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 an Fc domain.

[0126] 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.

[0127] In some embodiments, the Fc domain contains modifications, such as amino acid substitutions. The modifications may be, for example, modifications that promote heterodimerization or modifications that alter the binding ability to protein A.

[0128] In some embodiments, the Fc contains modifications that promote heterodimerization.

[0129] The multispecific antibodies described in this disclosure comprise different antigen-binding portions 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 produce several possible combinations of the two polypeptides. To improve the yield and purity of the multispecific antibody in recombinant production, it would be 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 of the Fc domain.

[0130] The site of the most extensive protein-protein interaction between the two Fc polypeptides of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is in the CH3 domain of the Fc domain. Specification 19 / 72 pages 22 CN 121570606 A

[0131] In a specific embodiment, the modification is a so-called "knob-into-hole" modification, which comprises a "knob" modification in one of the two Fc polypeptides of the Fc domain and a "pot" modification in the other of the two Fc polypeptides of the Fc domain. Typically, the method involves introducing a protrusion ("knob") at the interface of one Fc polypeptide and a corresponding depression ("pot") at the interface of the other Fc polypeptide, such that the protrusion can be positioned in 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, etc.). A complementary depression with 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, etc.).

[0132] Therefore, in a specific embodiment, in the CH3 domain of one Fc polypeptide of the multispecific antibody, amino acid residues are substituted with amino acid residues having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the Fc polypeptide that can be positioned in the CH3 domain of another Fc polypeptide. In the CH3 domain of the other Fc polypeptide, amino acid residues are substituted with amino acid residues having a smaller side chain volume, thereby creating a depression in the CH3 domain of the Fc polypeptide.

[0133] In some embodiments, according to EU designations, one Fc polypeptide of the Fc domain contains amino acid substitutions 354C and / or 366Y / W, and the other Fc polypeptide contains amino acid substitutions 349C, 366S, 368A and / or 407T / V. In some specific embodiments, according to EU designations, one Fc polypeptide of the Fc domain contains amino acid substitutions 354C and 366Y / W, and the other Fc polypeptide contains amino acid substitutions 349C, 366S, 368A and 407T / V. In some more specific embodiments, according to EU designations, one of the Fc polypeptides in the Fc domain comprises amino acid substitutions 354C and 366W, and the other Fc polypeptide comprises amino acid substitutions 349C, 366S, 368A, and 407V. In the above embodiments, the Fc may be the Fc of human IgG1. In one specific embodiment, according to EU designations, 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.

[0134] In some embodiments, the Fc domain comprises modifications that reduce or eliminate the binding of the CH3 region of one Fc polypeptide in the Fc domain to protein A (from Staphylococcus aureus). In some embodiments, the Fc domain comprises amino acid substitutions that reduce or eliminate the binding of the CH3 region of one Fc polypeptide in the Fc domain to protein A. In some embodiments, according to the EU designation, the Fc domain comprises amino acid substitutions (a) 435R or (b) 435R and 436F, which occur only on one Fc polypeptide and not on the other. In some embodiments, according to the EU designation, the Fc domain comprises amino acid substitutions (a) 435R or (b) 435R and 436F occurring only on one of the Fc polypeptides. In some specific embodiments, according to the EU designation, the Fc domain comprises amino acid substitutions H435R and Y436F occurring only on one of the Fc polypeptides. In some specific embodiments, according to the EU designation, the Fc domain comprises amino acid substitution H435R occurring only on one of the Fc polypeptides. In the above embodiments, the Fc is IgG1 Fc, particularly human IgG1 Fc.

[0135] 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 one Fc polypeptide in 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 one Fc polypeptide in the Fc domain to protein A. For example, in one specific embodiment, according to EU designation, the Fc domain includes the following amino acid substitutions: i. 349C, 366S, 368A, 407T / V, 354C, and 366Y / W; wherein amino acid substitutions 354C and 366Y / W are on the same Fc polypeptide and are not on the same Fc polypeptide as the other amino acid substitutions in (i); and ii. (a) 435R or (b) 435R and 436F, occurring only on one of the Fc polypeptides.

[0136] 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 particular embodiment, the Fc is IgG1 Fc, especially human IgG1 Fc. Specification 20 / 72 pages 23 CN 121570606 A

[0137] 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, especially human IgG1 Fc.

[0138] In the context of this disclosure, amino acid substitution is expressed 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. Thus, for example, "H435R" or "435R" means that the amino acid H or the original amino acid at position 435 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.

[0139] In some embodiments, the Fc domain does not contain fucose.

[0140] In some embodiments, the multispecific antibody of this disclosure is unfucosylated. Defucosylation can enhance the interaction between the multispecific antibody and FcγRIIIa, thereby enhancing the ADCC activity of the antibody. The method of generating multispecific antibodies with little or no fucose at the glycosylation site of the Fc domain without changing the amino acid sequence...Methods 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.

[0141] In some embodiments, the multispecific antibody described herein is trivalent, that is, the first antigen-binding portion, the second antigen-binding portion, and the third antigen-binding portion each provide monovalent binding to the corresponding antigen.

[0142] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 71; another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 87. The amino acid sequence of the same identity.

[0143] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 73; another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 87. The amino acid sequence is identical to that shown in SEQ ID NO: 75.

[0144] In some embodiments, the multispecific antibody consists of three polypeptide chains, one of which contains amino acids having a content of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, and 94% of the amino acid sequence shown in SEQ ID NO: 75.One polypeptide chain contains an amino acid sequence having at least 85%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 87.

[0145] 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: 85; another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 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: 85; and a fourth 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. The amino acid sequence shown in 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.

[0146] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 79; another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 87.The sequence 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.

[0147] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 81; another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 87. The amino acid sequence of the same identity.

[0148] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 83; another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 87. The amino acid sequence of the same identity.

[0149] In some embodiments, the multispecific antibody consists of three polypeptide chains, wherein 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 a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0150] In some embodiments, the multispecific antibody consists of three polypeptide chains, wherein one polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 71.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.

[0151] In some embodiments, the multispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 75, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0152] In some embodiments, the multispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 77, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0153] 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.

[0154] 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.

[0155] In some embodiments, the multispecific antibody comprises three polypeptide chains, wherein 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 a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0156] This disclosure provides exemplary trivalent multispecific antibodies.

[0157] As one example, the 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.

[0158] As one example, the multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in SEQ ID NO: 73, SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86, respectively.As shown in SEQ ID NO: 85 and SEQ ID NO: 87. 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.

[0159] 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.

[0160] 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.

[0161] In one example, the 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.

[0162] In one example, the 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.

[0163] In one example, the 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.

[0164] 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...In this 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; 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.

[0165] 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 moiety, a second antigen-binding moiety, and a third antigen-binding moiety, wherein the first antigen-binding moiety comprises CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 36; the second antigen-binding moiety 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 moiety comprises CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 35. The CDR is defined, for example, according to Kabat, IMGT, Chothia, Contact, AbM, and / or CCG.

[0166] 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 having an amino acid sequence of at least 85%, 86%, and 87% as shown in SEQ ID NO: 36.The second antigen-binding portion comprises an amino acid sequence having at least 85%, 86%, 87%, 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, and an amino acid sequence having at 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 having at least 85%, 86%, 87%, 88%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 35. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.

[0167] 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.

[0168] 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 is 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 the other 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.The amino acid sequence, and a polypeptide chain 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: 87.

[0169] 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 comprises the amino acid sequence shown in SEQ ID NO: 77, another polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 87.

[0170] In some specific embodiments, the multispecific antibody in 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 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.

[0171] In some embodiments, the multispecific antibody can be modified, for example, by including the insertion, deletion, or substitution of 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.

[0172] 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.

[0173] In some embodiments, the adapter is a cleavable adapter or a non-cleavable adapter. In some embodiments, the adapter is a cleavable adapter, for example, it may be a low pH-dependent degradation type (including hydrazone bonds, carbonate bonds, etc.), a proteolytic type (including peptide bonds), or a high glutathione concentration-dependent degradation type (including disulfide bonds), etc. The cleavable adapter can break within the target cell, thereby releasing the cytotoxic drug. In other embodiments, the adapter is a non-cleavable adapter, for example, it may be maleiminohexanoyl, etc.

[0174] In some embodiments, in the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt provided by the present invention, a multispecific antibody is conjugated to one or more cytotoxic drugs, the cytotoxic drugs being, for example, alkaloids, antimetabolites, antitumor antibiotics, alkylating agents, and platinum-based drugs, etc. In some embodiments, the cytotoxic drug is a DNA-acting cytotoxic drug. In some embodiments, the DNA-targeting cytotoxic drug includes, but is not limited to, calcitrazine, duocarmycin, pyrrolobenzodiazepine (PBD), topoisomerase I inhibitors, and topoisomerase II inhibitors.

[0175] 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, ethatecan, or an ethatecan derivative. In some specific embodiments, the cytotoxic drug is an ethatecan derivative. In some specific embodiments, the cytotoxic drug is an ethatecan derivative and is deuterated.

[0176] In some embodiments, the cytotoxic drug is linked to a linker via a functional group, and the antibody-drug conjugate is hydrolyzed within tumor cells to form a free cytotoxic drug, thereby exerting an antitumor effect.

[0177] 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 with the structure shown in the specification shown on page 25 / 72 of CN 121570606 A Ia to the multispecific antibody, and the drug-connector is linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia;Wherein, R1 and R2 are independently selected from hydrogen atoms 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.

[0178] In some embodiments, R1 is a hydrogen atom and R2 is a deuterium atom.

[0179] In some embodiments, the antibody-drug conjugate is formed by linking the drug-connector of the structure shown in Formula Ia-1 to the multispecific antibody, wherein the drug-connector is attached to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia-1. 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.

[0180] In the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt provided in this disclosure, the number of cytotoxic drugs linked to the multispecific antibody 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.

[0181] By controlling the proportion of the multispecific antibodies linked to different numbers of cytotoxic drugs, antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvate of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt with different drug-antibody ratios (DAR) can be generated. It should be understood that the DAR is the average number of cytotoxic drugs linked to each multispecific antibody in 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. For example, "DAR of 6" means that such 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 comprises 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.

[0182] 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 as stated on page 26 / 72 of the specification, CN 121570606 A. 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.

[0183] 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 may also be represented by the structure shown in Formula II: II, wherein the multispecific antibody is as described in the multispecific antibody of this disclosure; R1 and R2 are independently selected from hydrogen atoms or deuterium atoms. In some embodiments, R1 is a hydrogen atom and R2 is a deuterium atom.

[0184] 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.

[0185] In some embodiments, the antibody-drug conjugate can be represented by the structure shown in Formula II-1: II-1, 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.

[0186] 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, and the multispecific antibody comprising the first...The device comprises an 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 (page 27 / 72 of specification, 30 CN 121570606 A), 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. 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.

[0187] 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, 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: CDR1, CDR2, and CDR3 of the single variable structural domain shown in Figure 35.The CDR is 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.

[0188] 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 amino acid sequence shown in SEQ ID NO: 69 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: 70, 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: 35; 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 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, or 4.5-6.4, 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.

[0189] 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 comprising an 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.

[0190] 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 an amino acid sequence having at 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 the other polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, or 89% identity with the amino acid sequence shown in SEQ ID NO: 85.An amino acid sequence having 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.

[0191] 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.

[0192] 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, 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: 85, respectively.As shown in Figure 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.

[0193] In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or solvation of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt provided in this disclosure, wherein the DAR of the antibody-drug conjugate described in the specification (pages 29 / 72, CN 121570606 A) is 5-6, 5.5-6, or 6, and the mass fraction of the antibody-drug conjugate with each multispecific antibody 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%.

[0194] 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 exhibits one or more combinations of the following properties: (1) binding to human c-Met, preferably binding to human c-Met with a KD value of 6E-10 M or less; (2) binding to human EGFR; (3) binding to monkey c-Met; (4) binding to monkey EGFR; (5) internalization in cells expressing c-Met and / or EGFR; (6) cytotoxic activity against tumor cells expressing c-Met and / or EGFR; and (7) having a bystander effect.

[0195] 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 said property (1) to property (7).

[0196] 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 achieve excellent antitumor efficacy and / or safety. 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 exhibit good killing activity against a variety of tumor cells expressing EGFR and / or c-Met. In some embodiments, the antibody-drug conjugates,Its stereoisomers, its pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts exhibit good in vivo antitumor activity. 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 have excellent safety. 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 are not prone to aggregation.

[0197] Pharmaceutical Compositions This disclosure provides pharmaceutical compositions comprising the antibody-drug conjugates, their stereoisomers, their pharmaceutically acceptable salts, or solvates of the antibody-drug conjugates, their stereoisomers, and their pharmaceutically acceptable salts of the present disclosure. In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0198] Uses This disclosure provides for the use of the antibody-drug conjugates of the present disclosure, their stereoisomers, their pharmaceutically acceptable salts, or solvates of said antibody-drug conjugates, their stereoisomers, or their pharmaceutically acceptable salts. This disclosure also provides for the use of the pharmaceutical compositions of the present disclosure.

[0199] In one aspect, this disclosure provides for the use of the antibody-drug conjugates of the present disclosure, their stereoisomers, their 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. Specification 30 / 72 pages 33 CN 121570606 A

[0200] In one aspect, this disclosure provides for the use of the pharmaceutical compositions of the present disclosure in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0201] In one aspect, this disclosure provides the use of the antibody-drug conjugate of the present disclosure, its stereoisomers, its pharmaceutically acceptable salts, or solvates of said antibody-drug conjugate, its stereoisomers, its pharmaceutically acceptable salts, and one or more additional therapeutic agents in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0202] In one aspect, this disclosure provides the use of the pharmaceutical compositions of the present disclosure and one or more additional therapeutic agents in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0203] In one aspect, this disclosure provides a method of treating diseases expressing c-Met and / or EGFR, the method comprising administering to a subject the antibody-drug conjugate of the present disclosure, its stereoisomers, its pharmaceutically acceptable salts, or said antibody-drug conjugate, its stereoisomers, its pharmaceutically acceptable salts, or said antibody-drug conjugate, its stereoisomers, its pharmaceutically acceptable salts, and one or more additional therapeutic agents.Antibody-drug conjugates, their stereoisomers, and solvates of their pharmaceutically acceptable salts. In some embodiments, the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt is administered to a subject in a therapeutically effective amount.

[0204] 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 this disclosure to a subject. In some embodiments, the pharmaceutical composition of this disclosure is administered to a subject in a therapeutically effective amount.

[0205] 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, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, and one or more additional therapeutic agents. In some embodiments, a subject is administered 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, and one or more additional therapeutic agents.

[0206] In one aspect, the present disclosure provides a method of treating a disease expressing c-Met and / or EGFR, the method comprising administering a subject a pharmaceutical composition of the present disclosure and one or more additional therapeutic agents. In some embodiments, a subject is administered a therapeutically effective amount of the disclosed pharmaceutical composition and one or more additional therapeutic agents.

[0207] 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.

[0208] 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 to kill tumor cells or inhibit tumor cell growth.

[0209] 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 to kill tumor cells or inhibit tumor cell growth.

[0210] In some embodiments, administering to a subject 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 thereof.The drug, or the pharmaceutical composition of this disclosure, can kill tumor cells or inhibit tumor cell growth. In some embodiments, administering a therapeutically effective amount of 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 the pharmaceutical composition of this disclosure to a subject can kill tumor cells or inhibit tumor cell growth. Specification 31 / 72 pages 34 CN 121570606 A

[0211] In some embodiments, administering a subject to an 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, and one or more other therapeutic agents can kill tumor cells or inhibit tumor cell growth. In some embodiments, administering a therapeutically effective amount of the antibody-drug conjugate of the present disclosure, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of said antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, and one or more other therapeutic agents to a subject can kill tumor cells or inhibit tumor cell growth. In some embodiments, administering a pharmaceutical composition of the present disclosure and one or more other therapeutic agents to a subject can kill tumor cells or inhibit tumor cell growth. In some embodiments, administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure and one or more other therapeutic agents to a subject can kill tumor cells or inhibit tumor cell growth.

[0212] 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, gastric 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).

[0213] Kit This disclosure provides a kit comprising the antibody-drug conjugate of the present disclosure, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a pharmaceutical composition of the present disclosure.

[0214] This disclosure describes kits 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 thereof. The kits can be used to implement the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition provided herein.The reagent kit may include, in some embodiments, 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 a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the pharmaceutical composition provided herein; optionally, the reagent kit may also include instructions for use. The reagent kit may also include other materials required from a commercial and user perspective, such as other buffers, diluents, needles, syringes, etc.

[0215] Linker-payload In some aspects, the present disclosure provides a linker-payload with the structure shown in Formula I: I, wherein R1 and R2 are independently selected from hydrogen atoms or deuterium atoms, respectively.

[0216] In some embodiments, R1 is a hydrogen atom and R2 is a deuterium atom.

[0217] In one specific embodiment, the present invention provides a linker-loader with the structure shown in Formula I-1: Specification 32 / 72 pages 35 CN 121570606 AI-1.

[0218] Preparation Method The present 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, and then reacting the multispecific antibody with a linker-loader selected from the structure shown in Formula I, wherein R1 and R2 are independently selected from hydrogen or deuterium atoms. In some embodiments, R1 is a hydrogen atom and R2 is a deuterium atom.

[0219] In some embodiments, the method comprises treating a multispecific antibody under reducing conditions, and then reacting the multispecific antibody with a linker-loader selected from the structure shown in Formula I-1.

[0220] In some embodiments, the reducing conditions are in the presence of TCEP. Detailed Embodiments

[0221] The present disclosure also provides the following specific embodiments, but the scope of protection of the present disclosure is not limited thereto: Embodiment 1. 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, wherein the multispecific antibody comprises (i) a first antigen-binding portion binding to a first antigen; (ii) a second antigen-binding portion binding to a second antigen; and (iii) a third antigen-binding portion binding 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 comprises any one of the following: (1) comprising SEQ IDCDR1 containing the amino acid sequence shown in NO: 4, comprising 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, comprising 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, comprising 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, comprising 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, comprising CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and comprising SEQ ID NO: 6. (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.

[0222] Embodiment 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 Embodiment 1, wherein the first antigen-binding portion and the third antigen-binding portion each independently comprise any one of the following: (1) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is selected from S or T, preferably T; (2) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, comprising SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, wherein X1 is selected from S or T, preferably T;CDR2 containing the amino acid sequence shown in 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.

[0223] Embodiment 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 Embodiment 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.

[0224] Embodiment 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 Embodiment 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.

[0225] Embodiment 5. 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 comprises (i) a first antigen-binding moiety binding to a first antigen; (ii) a second antigen-binding moiety binding to a second antigen; and (iii) a third antigen-binding moiety binding to the first antigen; wherein the first antigen is c-Met and the second antigen is EGFR, and the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains and each independently contains an amino acid sequence as shown in SEQ ID NO:CDR1, CDR2, and CDR3 of a single variable domain as shown in SEQ ID NO: 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, or 39.

[0226] Embodiment 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 Embodiment 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.

[0227] Embodiment 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 Embodiment 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.

[0228] Embodiment 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 Embodiment 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.

[0229] Embodiment 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 Embodiments 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.

[0230] Embodiment 10. An antibody-drug conjugate, its stereoisomer, according to any one of Embodiments 1-9,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: 41.

[0231] Embodiment 11. 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 Embodiment 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.

[0232] Embodiment 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 Embodiments 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.

[0233] Embodiment 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 Embodiment 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 amino acid sequence.

[0234] Embodiment 14. An antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, according to any one of Embodiments 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.

[0235] Embodiment 15. 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 Embodiment 14, 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: 32, 38, or 39; 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: 38.

[0236] Embodiment 16. 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 Embodiments 1-8, 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 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; (2) 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; (3) 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: 36.(3) 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 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: 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.

[0237] Embodiment 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 Embodiments 1-16, wherein the single variable domain is of camelid origin or humanized.

[0238] Embodiment 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 embodiments 1-17, wherein the second antigen-binding portion is Fab, scFv, or scFab.

[0239] Embodiment 19. 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 Embodiments 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.

[0240] Embodiment 20. 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 Embodiments 1-18, wherein the second antigen-binding moiety comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 69, and an amino acid sequence as shown in SEQ ID NO: 69.The LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in NO: 70.

[0241] Embodiment 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 Embodiments 1-20, wherein the second antigen-binding portion comprises a heavy chain variable region having at least 85%, 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.

[0242] Embodiment 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 Embodiment 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.

[0243] Embodiment 23. 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 any one of Embodiments 1-22, wherein the second antigen-binding moiety is murine, chimeric, or humanized.

[0244] Embodiment 24. 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 according to any one of embodiments 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.

[0245] Embodiment 25. 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 according to embodiment 24, wherein the third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion.

[0246] Embodiment 26. An antibody-drug conjugate, its stereoisomer, ...The multispecific antibody further comprises (iv) an Fc domain consisting of two Fc polypeptides.

[0247] Embodiment 27. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt, or the solvation of the antibody-drug conjugate, its stereoisomer, and its pharmaceutically acceptable salt according to Embodiment 26, wherein the first antigen-binding moiety is fused at its C-terminus to the N-terminus of one of the Fc polypeptides of the Fc domain, and the second antigen-binding moiety is Fab and the second antigen-binding moiety is fused at the C-terminus of its Fab heavy chain to the N-terminus of the other Fc polypeptide of the Fc domain.

[0248] Embodiment 28. 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 Embodiment 26 or 27, wherein the Fc domain is an IgG Fc domain, preferably an IgG1 Fc domain.

[0249] Embodiment 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 Embodiment 28, wherein the IgG Fc domain is a human IgG Fc domain, preferably a human IgG1 Fc domain.

[0250] Embodiment 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 embodiments 26-29, wherein the Fc domain comprises an amino acid substitution that promotes association between two Fc polypeptides of the Fc domain.

[0251] Embodiment 31. 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 embodiment 30, wherein, according to EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions 354C and 366Y / W, and the other Fc polypeptide comprises amino acid substitutions 349C, 366S, 368A, and 407T / V. Instructions for Use, Pages 37 / 72, 40 CN 121570606 A

[0252] Embodiment 32. 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 embodiments 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.

[0253] 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 EU designation, the Fc domain comprises an amino acid substitution occurring only in one of the Fc polypeptides: (a) 435R or (b) 435R and 436F.

[0254] Implementation Scheme 34. 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 any one of Implementation Schemes 26-29, wherein, according to EU designation, one of the Fc polypeptides in the Fc domain comprises an amino acid substitution: 349C, 366S, 368A, 407V, 435R, and 436F, and the other Fc polypeptide comprises an amino acid substitution: 354C and 366W.

[0255] Embodiment 35. 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 according to any one of Embodiments 26-29, wherein, according to EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: 349C, 366S, 368A, 407V, and 435R, and the other Fc polypeptide comprises amino acid substitutions: 354C and 366W.

[0256] Embodiment 36. 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 according to any one of Embodiments 1-35, wherein the multispecific antibody is trivalent.

[0257] Embodiment 37. 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 Embodiments 1-36, 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: 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; The amino acid sequence shown in 87 has at least 85%(1) an amino acid sequence having at 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; (2) 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: 85; 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: 85; 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: 85; The amino acid sequence shown in SEQ ID NO: 87 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: 73; (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: 85, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 85, and yet ... The amino acid sequence shown in SEQ ID NO: 75 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: 75; (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: 85, and another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain(5) A polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 87; and another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 79, and yet another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85 ... The amino acid sequence shown in SEQ ID NO: 87 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: 81; (6) 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: 85, another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85, and yet ... The amino acid sequence shown in SEQ ID NO: 87 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: 83; or (7) one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identity with the amino acid sequence shown in SEQ ID NO: 85.An amino acid sequence with 98%, 99%, or 100% identity, 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.

[0258] Embodiment 38. 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 three polypeptide chains, wherein: (1) one polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 77, another polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 87; (2) one polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 71, another polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 87; (3) one polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 73, another polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 85, and a third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 87; (4) one polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: (5) One polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 79, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 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 another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87; or (7) One polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 83, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 85, and another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 87.

[0259] Embodiment 39. An antibody-drug conjugate according to any one of Embodiments 1-38, its stereoisomer, Specification 39 / 72 pages 42 CN 121570606 AThe multispecific antibody is a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt thereof, wherein the multispecific antibody is unfucosylated.

[0260] Embodiment 40. The antibody-drug conjugate, its stereoisomer, its pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt thereof, according to any one of Embodiments 1-39, wherein the antibody-drug conjugate is formed by linking a drug-connector of the structure shown in Formula Ia to the multispecific antibody, wherein the drug-connector is linked to the multispecific antibody at the position indicated by * in the structure shown in Formula Ia; wherein R1 and R2 are independently selected from hydrogen atoms or deuterium atoms, respectively.

[0261] Embodiment 41. 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 Embodiment 40, wherein R1 is a hydrogen atom and R2 is a deuterium atom.

[0262] Embodiment 42. 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 any one of Embodiments 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.

[0263] Embodiment 43. 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 embodiments 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.

[0264] Embodiment 44. 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 Embodiments 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: (1) binding to human c-Met, preferably binding to human c-Met with a KD value of 6E-10 M or less; (2) binding to human EGFR; (3) binding to monkey c-Met; (4) binding to monkey EGFR; (5) internalization in cells expressing c-Met and / or EGFR; (6) [missing information - likely related to surface]Tumor cells reaching c-Met and / or EGFR have cytotoxic activity; and (7) have a bystander effect.

[0265] Embodiment 45. 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 embodiments 1-44; optionally, the pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0266] Embodiment 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 as described in any one of Embodiments 40-44, comprising treating the multispecific antibody under reducing conditions, and then reacting the multispecific antibody with a linker-loader selected from the structure shown in Formula I, wherein the structure shown in Formula I is as follows: Specification 40 / 72 pages 43 CN 121570606 AI, R1 and R2 are each independently selected from hydrogen atoms or deuterium atoms.

[0267] Embodiment 47. The method according to Embodiment 46, wherein R1 is a hydrogen atom and R2 is a deuterium atom.

[0268] Embodiment 48. 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 of Embodiment 45 in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR, according to any one of Embodiments 1-44.

[0269] Embodiment 49. 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 of Embodiment 45, and one or more additional therapeutic agents in the preparation of a medicament for treating diseases expressing c-Met and / or EGFR.

[0270] Embodiment 50. Use according to Embodiment 48 or 49, wherein the disease expressing c-Met and / or EGFR is a tumor.

[0271] Embodiment 51. The use according to Embodiment 49 or 50, wherein the additional therapeutic agent is an antitumor therapeutic agent.

[0272] Embodiment 52. The use according to Embodiment 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, or lung cancer.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.

[0273] Embodiment 53. A method for treating a disease expressing c-Met and / or EGFR, comprising administering to a subject 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 Embodiment 45.

[0274] Embodiment 54. The method according to Embodiment 53, wherein the disease expressing c-Met and / or EGFR is a tumor.

[0275] Embodiment 55. The method according to Embodiment 54, wherein the method comprises contacting tumor cells with an 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 of Embodiment 45, thereby killing tumor cells or inhibiting tumor cell growth.

[0276] Embodiment 56. The method according to any one of Embodiments 53-55, wherein the method further comprises administering one or more additional therapeutic agents to a subject.

[0277] Embodiment 57. The method according to Embodiment 56, wherein the additional therapeutic agent is an antitumor therapeutic agent. Instruction manual, pages 41 / 72, CN 121570606 A

[0278] Embodiment 58. The method according to any one of embodiments 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.

[0279] Embodiment 59. A kit comprising the 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 Embodiment 45; optionally, the kit further comprising instructions for use.

[0280] For clarity, the present disclosure is further illustrated by examples, but the examples are not intended to limit the scope of the 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.

[0281] Example 1: Construction of Anti-human c-Met VHH Phage Display Library Recombinant human c-Met-Fc fusion protein (SinoBiological, catalog number 10692-H02H) was emulsified with complete Freund's adjuvant at a volume ratio of 1:1 and used for the first subcutaneous multi-site immunization of Bactrian camels. Subsequently, every two weeks, recombinant human c-Met-Fc fusion protein was emulsified with incomplete Freund's adjuvant at a volume ratio of 1:1 for booster immunization. Serum titers of anti-human c-Met antibodies were detected after the 4th or 5th 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 reversed to cDNA. The variable region (VHH) sequence of the camel antibody was amplified using nested PCR.

[0282] The amplified VHH 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. The vector was then electroporated into Escherichia coli TG1 (Lucigen, catalog number No. 60502-1) to construct 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 library amplification. The amplification was carried out overnight at 28°C and 200 rpm with shaking. Centrifuge the bacterial culture and collect the supernatant. Add 1 / 4 volume of PEG6000 / NaCl solution (20% PEG6000 (w / v), 2.5 M NaCl) to the supernatant. Incubate on ice for 1-2 hours to precipitate the phage. Centrifuge to collect the phage precipitate. Resuspend in PBS and add 20% glycerol. Store at -80℃ as a VHH phage display library.

[0283] Example 2: Screening for anti-human c-Met VHH The VHH phage display library was screened by solid-phase panning. The panned single clones were cultured and the supernatant was prepared by isopropyl-β-D-thiogalactoside (IPTG) induction expression.

[0284] Selected clones were positively identified using an indirect ELISA method targeting human c-Met-His (SinoBiological, catalog number 10692-H08H). Positive clones that only bind to human c-Met-His and have relatively 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 through screening. Sequence analysis revealed the following amino acid sequences for VHH: 1B-3B11 (SEQ ID NO: 25), 1B-1B6 (SEQ ID NO: 26), 1B-1C7 (SEQ ID NO: 27), 1B-1A8 (SEQ ID NO: 28), 1B-1B2 (SEQ ID NO: 29), 3B-1C7 (SEQ ID NO: 30), 4&5B-2F01 (SEQ ID NO: 31, page 42 / 72 of the specification, CN 121570606 A), and 4&5C-12B04 (SEQ ID NO: 32).

[0285] Example 3: Preparation of anti-human c-Met VHH-Fc chimeric antibody The VHH sequence of the screened positive clones was linked to the human Fc region to construct VHH-Fc chimeric antibodies. Specifically, the VHH sequence obtained from sequencing in Example 2 was inserted into the pcDNA3.1(+) eukaryotic expression vector containing the human IgG1 Fc region, and these VHH-Fc chimeric antibodies were expressed using the Expifectamine™ CHO Transfection Kit transient expression system (Thermo Fisher Scientific Inc., catalog number A29129). At the same time, the sequences of VL1016-069 and VH1016-069 from patent application US20200079872A1 were inserted into the pcDNA3.1(+) eukaryotic expression vector containing the human IgG1 constant region (amino acid sequence is SEQ ID NO: 92), and the chimeric antibody 1016-069 was expressed as a control using the same method.

[0286] Sequence analysis revealed the amino acid sequences of 1B-3B11-Fc as shown in SEQ ID NO: 43, 1B-1B6-Fc as shown in SEQ ID NO: 44, 1B-1C7-Fc as shown in SEQ ID NO: 45, and 1B-1A8-Fc as shown in SEQ ID NO: 46.As shown in SEQ ID NO: 46, the amino acid sequence of 1B-1B2-Fc is shown in SEQ ID NO: 47, the amino acid sequence of 3B-1C7-Fc is shown in SEQ ID NO: 48, the amino acid sequence of chimeric antibody 4&5B-2F01-Fc is shown in SEQ ID NO: 49, and the amino acid sequence of chimeric antibody 4&5C-12B04-Fc is shown in SEQ ID NO: 50.

[0287] Example 4: Affinity of anti-human VHH-Fc chimeric antibody with human and cynomolgus monkey c-Met 4.1 Surface plasmon resonance technique was used to determine the affinity of the antibody with human and cynomolgus monkey c-Met. The affinity of the anti-human c-Met VHH-Fc chimeric antibody was detected using a biomolecular interaction analysis system (GE, Biacore T200 or Biacore 8K). Amino-conjugated Anti-hIgG (Fc) antibody (GE, catalog No. BR-1008-39) was introduced into the CM5 sensor chip. The anti-human c-Met VHH-Fc chimeric antibody was diluted to 1 μg / mL 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) and captured through 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) were diluted with running buffer to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM, respectively. Binding was performed at a flow rate of 50 μL / min for 200 s, followed by dissociation at 600–1400 s. Data analysis was conducted using software, and the binding rate constant ka (1 / Ms), dissociation rate constant kd (1 / s), and equilibrium dissociation constant KD (M) were calculated using a Langmuir 1:1 model. 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 showed high affinity for human c-Met protein and cross-reacted with cynomolgus monkey c-Met protein.

[0288] Table 1-1. Affinity of VHH-Fc chimeric antibody to human and cynomolgus monkey c-Met

[0289] Table 1-2. Affinity of VHH-Fc chimeric antibody to human and cynomolgus monkey c-Met

[0290] 4.2 Flow Cytometry Determination of Antibody-Cell Binding Instructions for Use 43 / 72 Page 46 CN 121570606 A Detection of Anti-human c-Met using Flow Cytometry The VHH-Fc chimeric antibody binds to target cells with different c-Met expression levels. Among them, NCI-H1993 cells (Beina Biotechnology, catalog number BNCC342186) are human lung adenocarcinoma cell lines with high c-Met expression, MKN45 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60488) are human gastric cancer cell lines with intermediate c-Met expression, KP4 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60541) are human pancreatic cancer cell lines with low c-Met expression, NCI-H1975 cells (Beina Biotechnology, catalog number BNCC100690) are human non-small cell lung adenocarcinoma cell lines with medium to low c-Met expression, and NCI-H292 cells (Beina Biotechnology, catalog number BNCC100671) are human epidermal lung cancer cell lines with medium to low c-Met expression. Two × 10⁵ target cells were incubated with serially diluted (initial concentration 100 nM, 5-fold serial dilution, 7 concentrations) anti-human c-Met VHH-Fc chimeric antibody. After incubation on ice for 1 hour, the cells were washed and then incubated with PE-labeled anti-human IgG Fc antibody (Jackson Immuno Research, catalog No. 109-116-170) on ice for 0.5 hours. After washing the cells, they 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. 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. 4&5B-2F01-Fc and 4&5C-12B04-Fc were superior to the control 1016-069.

[0291] Table 2-1. Binding of anti-human c-Met VHH-Fc chimeric antibody to target cells

[0292] Table 2-2. Binding of anti-human c-Met VHH-Fc chimeric antibody to target cells

[0293] Example 5: Epitope difference analysis among different clones of anti-human c-Met VHH Epitope competition was analyzed using a biomolecular interaction system (Fortebio, catalog number Octet RED96). An Anti-Penta-HIS (HIS1K) sensor (Fortebio, catalog number No. 18-5120) was used, with a running buffer.The c-Met-His protein (SinoBiological, catalog number 10692-H08H) was diluted to approximately 5 μg / mL. The sensor was then immersed in the diluted antigen sample, and the solidification height was controlled to approximately 1 nm by adjusting the binding time. It was then sequentially interacted with the first antibody A and the second 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 as follows: a value >60% indicated that the two antibodies did not compete at all; a value between 20% and 60% indicated that the two antibodies partially competed (possibly with epitope overlap); a value <20% indicated that the two antibodies were completely competitive. A self-reaction signal (underlined portion) <20% was considered valid.

[0294] From the data in Table 3-1, it can be seen that the two 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, 1B-1C7-Fc and 1B-1B6-Fc can simultaneously bind to different epitopes of the c-Met antigen. From the data in Table 3-44 / 72 of the specification, page 47, CN 121570606 A 2, it can be concluded that the two antibodies 3B-1C7-Fc and 1B-1B2-Fc are in complete competition. Therefore, combined with 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.

[0295] From the data in Table 3-3, it can be concluded that 4&5B-2F01-Fc and 4&5C-12B04-Fc are in complete competition with 1016-069.

[0296] Table 3-1. Epitope Difference Analysis

[0297] Table 3-2. Epitope Difference Analysis

[0298] Table 3-3. Epitope Difference Analysis

[0299] Example 6: Construction, Expression, Purification and Affinity Detection of Humanized Anti-human c-Met VHH-Fc Antibodies The chimeric anti-human c-Met VHH-Fc antibodies 1B-1B6-Fc, 1B-1C7-Fc and 4&5C-12B04-Fc were respectively subjected to...Humanization. The humanized VHH sequence was inserted into the pcDNA3.1(+) eukaryotic expression vector containing the human IgG1 constant region, and these VHH-Fc humanized antibodies were expressed using the Expifectamine™ CHO Transfection Kit transient expression system (Thermo Fisher Scientific Inc., catalog No. A29129). Among them, 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 VHHs: 12B04-V1 and 12B04-V2, with amino acid sequences as shown in SEQ ID NO: 58, 59, and 50, respectively. As shown in 38 and 39.

[0300] The affinity of the obtained humanized antibody and chimeric antibody for human c-Met protein was detected using a biomolecular interaction analysis system (GE, Biacore 8K) with surface plasmon resonance technology. The amino-conjugated Anti-hIgG (Fc) antibody (GE, catalog No. BR-1008-39) was added to the CM5 sensor chip, and the anti-human c-Met VHH-Fc chimeric antibody was diluted to 2 μg / mL 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) and captured for 90 s through the experimental channel at a flow rate of 30 μL / min. Human c-Met-His protein (SinoBiological, catalog number 10692-H08H) 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. The binding signal curve was observed. (Instructions for use, pages 45 / 72, CN 121570606 A)

[0301] Humanized antibodies (1B-1B6-V1, 1B-1B6-V2, 1B-1B6-V3, 1B-1C7-V1, and 1B-1C7-V2), and intercalated...The affinity data of the chimeric antibodies (1B-1B6-Fc and 1B-1C7-Fc) to human c-Met protein are shown in Table 4.

[0302] Table 4. Binding affinity of anti-human c-Met VHH-Fc chimeric antibodies and humanized antibodies to human c-Met-His

[0303] It was found that 1B-1B6-Fc and its humanized antibody, 1B-1C7-Fc and its humanized antibody can specifically bind to c-Met-His protein.

[0304] Example 7: Construction, expression and purification of anti-EGFR / anti-c-Met multispecific antibodies The anti-EGFR / anti-c-Met multispecific antibody uses two humanized VHHs targeting c-Met in tandem. Modification of the Fc domain of the multispecific antibody: knock-into-hole (according to EU designations, one Fc polypeptide uses the Y349C, T366S, L368A, Y407V mutation; the other Fc polypeptide uses the S354C, T366W mutation), one of the Fc polypeptides in the Fc domain also contains a mutation that does not bind to protein A (according to EU designations, H435R and Y436F; or H435R).

[0305] 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, wherein the second antigen-binding part is an EGFR-binding antigen-binding domain in Fab form. Specifically, the anti-EGFR / anti-c-Met multispecific antibody has three polypeptide chains, one of which (named anti-c-Met-VHH-Fc) contains two tandem antigen-binding domains that bind c-Met (derived from chimeric antibodies and humanized antibodies of 1B-1B6-Fc, 1B-1C7-Fc, and 4&5C-12B04-Fc, as shown in Table 5). The amino acid sequences of this polypeptide chain of V12, V41, V35, V42, V67, V73, and V74 are shown in SEQ ID NO: 71, 73, 75, 77, 79, 81, and 83, respectively, and the nucleotide sequences are shown in SEQ ID NO: As shown in SEQ ID NO: 72, 74, 76, 78, 80, 82, and 84; the other two polypeptide chains form an antigen-binding domain containing Fab (the variable region sequence for EGFR binding is derived from patent document CN100497389C), one polypeptide chain is named anti-EGFR-HC-Fc (amino acid sequence as shown in SEQ ID NO: 85, nucleotide sequence as shown in SEQ ID NO: 86), and the other 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).(As shown in Figure 88). The nucleotide sequences encoding anti-c-Met-VHH-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. Using the CHOgro® high-yield expression system (catalog number: MIR 6270), the above expression vectors were co-transfected into CHO-S cells (named CHO FUT8- / - cells) with the FUT8 gene knocked out at a transfection ratio of anti-c-Met-VHH-Fc:anti-EGFR-HC-Fc:anti-EGFR-LC = 1.5:1:1.5, at a cell density of 4 × 10⁶ cells / mL. Cell culture supernatant was collected by centrifugation on day 10 post-transfection. Protein purification was performed using the ÄKTA pure protein purification system (GE Healthcare) via protein A affinity chromatography, CHT chromatography, and gel chromatography. Protein concentration was measured using a UV-Vis spectrophotometer (NanoDrop One C, Thermo Scientific). Electrophoresis (reducing SDS-PAGE), molecular weight analysis (TOF MS), and sequence analysis confirmed the obtained multispecific antibody with the expected structure and sequence.

[0306] Table 5. Composition of the antigen-binding domain of c-Met in the anti-EGFR / anti-c-Met multispecific antibody. Source: Specification, pages 46 / 72, 49, CN 121570606 A

[0307] Example 8: Affinity of anti-EGFR / anti-c-Met multispecific antibody to human c-Met. Following the method in Example 6, surface plasmon resonance 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 can specifically bind to human c-Met-His protein, and the affinity of humanized multispecific antibodies is comparable to that of chimeric multispecific antibodies.

[0308] Table 6. Binding affinity of anti-EGFR / anti-c-Met multispecific antibodies to human c-Met-His

[0309] Example 9: Binding of anti-EGFR / anti-c-Met multispecific antibodies to tumor cells expressing EGFR and c-Met was analyzed by flow cytometry. The binding affinity of anti-EGFR / anti-c-Met multispecific antibodies (including: Amivantamab, V42, V73) to A431 cells expressing EGFR and c-Met (high expression of EGFR, low expression of c-Met, source: Shanghai Institute of Cell Biology) and NCI-The H1975 cells (expressed in EGFR, low expression in c-Met, source: Beina Biotechnology) and NCI-H1993 cells (expressed in EGFR, high expression in c-Met, source: Beina Biotechnology) were combined, with hIgG1 (Baiying Biotechnology, catalog number B117901) as the negative control.

[0310] Logarithmic growth phase A431 cells, NCI-H1975 cells and NCI-H1993 cells were used to adjust the viable cell density to 5 × 106~1 × 107 cells / mL using RPMI medium (Hyclon, catalog number SH30809.01) containing 2% fetal bovine serum, and 50 μL / well was seeded in 96-well U-shaped cell culture plates (Costar, catalog number 3799). Different concentrations of anti-EGFR / anti-c-Met multispecific antibodies were prepared using the above medium, with the highest concentration being 500 nM, and 10 concentration gradients were prepared by 5-fold serial dilution. Different concentrations of antibodies were added to the above 96-well cell culture plates, 50 μL / well, mixed, and incubated at 4°C for 1 hour. After washing the cells with pre-chilled Running buffer (MACS, catalog number 130-091-221) and discarding the supernatant, 100 μL / well of pre-chilled fluorescently labeled goat anti-human IgG antibody (Jackson, catalog number 109-116-170) was added, mixed, and incubated at 4°C for 30 minutes. After washing, 40 μL / well of pre-chilled Running buffer was added to resuspend the cells, mixed, and flow cytometry was performed on an iQue3 flow cytometer, and data were analyzed using software.

[0311] Table 7 and Figures 3A-3C show the binding ability of anti-EGFR / anti-c-Met multispecific antibodies to A431 cells, NCI-H1975 cells, 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.

[0312] Table 7. EC50 values ​​and maximum binding amounts of anti-EGFR / anti-c-Met multispecific antibodies against EGFR and c-Met on the surface of tumor cells. Specification 47 / 72 pages 50 CN 121570606 A

[0313] " / " indicates that the curve cannot be fitted with numerical values.

[0314] Example 10: Inhibition of HGF-stimulated c-Met phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies. Western blotting was used to analyze the inhibition of HGF-stimulated c-Met phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies (including: Amivantamab, V42, V73). The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0315] HGF-α (amino acid sequence as shown in SEQ ID NO:The coding sequences of HGF-β (as shown in SEQ ID NO: 61) and HGF-β (amino acid sequence as shown in SEQ ID NO: 62) were inserted into the pcDNA3.1(+) eukaryotic expression vector, expressed using the Expifectamine™ 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.

[0316] Log-growing A549 cells (expressed in EGFR and c-Met, non-small cell lung cancer, source: Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were counted and the viable cell density was adjusted to 4 × 10⁵ cells / mL using RPMI medium (Hyclon, catalog number SH30809.01) containing 10% fetal bovine serum. 2 mL / well was added to a 6-well cell culture plate and cultured at 37°C in a 5% CO₂ incubator for 6 hours. The medium was then discarded, and 1 mL / well of RPMI medium was added for overnight starvation. The overnight starved 6-well cell culture plate was removed, the original medium was discarded, and anti-EGFR / anti-c-Met multispecific antibody was added to a final concentration of 100 nM, or HGF and anti-EGFR / anti-c-Met multispecific antibody were added simultaneously to a final concentration of 100 ng / mL. The plate was then cultured at 37°C in a 5% CO₂ incubator for 15 minutes. Cell culture plates were placed on ice, washed with pre-cooled PBS, and lysed for 30 minutes with lysis buffer containing protease inhibitors and phosphatase inhibitors. Proteins were collected and quantified using the BCA method. Western blotting was used to detect c-Met phosphorylation and downstream signaling pathways.

[0317] Figure 4 shows the inhibitory effect 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).

[0318] Example 11: Inhibition of EGF-stimulated EGFR phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies. Western blotting was used to analyze the inhibition of EGF-stimulated EGFR phosphorylation and downstream signaling pathways by anti-EGFR / anti-c-Met multispecific antibodies (including Amivantamab, V42, and V73). The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0319] Logarithmically growing A549 cells were taken, counted, and then the viable cell density was adjusted to 4 × 10⁵ cells / mL with complete culture medium.Add 1 mL / well of RPMI medium (Hyclon, catalog number SH30809.01) 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 to each well, starving the cells overnight. Remove the 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. The cell culture plates were placed on ice, washed with pre-cooled PBS, and lysed for 30 minutes with lysis buffer containing protease inhibitors and phosphatase inhibitors. Proteins were collected and quantified using the BCA method as described on pages 48 / 72 of the instruction manual (CN 121570606 A). Western blotting was used to detect EGFR phosphorylation and downstream signaling pathways.

[0320] Figure 5 shows the inhibitory effect 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).

[0321] Example 12: The activity of anti-EGFR / anti-c-Met multispecific antibodies competing with HGF ligands for c-Met binding was analyzed by ELISA. The activity of anti-EGFR / anti-c-Met multispecific antibodies (including: Amivantamab, V42, V73) competing with HGF ligands for c-Met binding was analyzed. The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0322] 2.0 mg of biotin (Thermo, catalog number 20217) was weighed and added to 590 µL of DMSO (sigma, catalog number D2650), and dissolved and mixed. Biotin solution was added at a ratio of 2.7 µL of biotin solution per 100 µL of HGF protein (prepared according to Example 10, concentration of 2 mg / mL) and mixed. The mixture was incubated 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, and 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...Incubate at room temperature for 2 hours (ng / mL). Wash the plate 3 times with PBST, add HRP-Avidin antibody (Invitrogen, catalog number 18-4100-51) and incubate at room temperature for 1 hour. Wash the plate 5 times with PBST, add TMB (Thermo, 00-4201-56) and incubate at room temperature in the dark for 10 minutes, add 1 M sulfuric acid stop solution, incubate at room temperature for 5 minutes, and read the absorbance value at 450 nm with a microplate reader using 630 nm as the reference wavelength.

[0323] Figures 6A-6C show the activity of anti-EGFR / anti-c-Met multispecific antibodies competing with HGF ligands for c-Met binding. The results show that the activity of V42 and V73 competing with HGF ligands for c-Met binding is basically equivalent to that of Amivantamab.

[0324] Example 13: Inhibitory activity of anti-EGFR / anti-c-Met multispecific antibodies against tumor cell proliferation was analyzed by microplate reader. The inhibitory activity of anti-EGFR / anti-c-Met multispecific antibodies (including: Amivantamab, V42, V73) against tumor cell proliferation was analyzed by microplate reader. The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0325] Logarithmically growing cells NCI-H292 (expressed in EGFR, low expression in c-Met, source: Beina Biotechnology) and KP4 (expressed in EGFR, low expression in c-Met, source: Kebai Biotechnology) were taken and the viable cell density was adjusted to 1.5 × 104~2 × 104 cells / mL with RPMI medium (Hyclon, catalog number SH30809.01) containing 2% fetal bovine serum. 100 μL / well was added to a 96-well plate (Costar, catalog number 3599). Anti-EGFR / anti-c-Met multispecific antibodies were prepared using RPMI medium. For NCI-H292 cells, the highest concentration was 2500 nM, with 10 concentration gradients achieved through 5-fold serial dilutions. For KP4 cells, the highest concentration was 555 nM, with 8 concentration gradients achieved through 3-fold serial dilutions. Different concentrations of antibody were added to the above-mentioned 96-well cell culture plates (100 μL per well) and incubated at 37 ℃ with 5% CO2. For NCI-H292 cells, 50 μL of HGF was added to the medium to a final concentration of 1 ng / mL, and the culture was carried out for 144 hours; for KP4 cells, the culture was carried out for 120 hours. CCK8 working solution was added to the cell culture plates at 20 μL per well and incubated at 37 ℃ with 5% CO2 for 2 hours. The absorbance was read at 450 nm using a microplate reader with 630 nm as the reference wavelength.

[0326] Figures 7A-7B and Table 8 show the inhibitory activity of anti-EGFR / anti-c-Met multispecific antibodies against tumor cell proliferation. The results show that in NCI-H292 cells and KP4 cells, V73 and V42 have better inhibitory activity against tumor cell proliferation than Amivantamab.

[0327] Table 8. IC50 values ​​of each antibody inhibiting the proliferation of NCI-H292 and KP4 cells. Instructions for use, pages 49 / 72, CN 121570606 A

[0328] Example 14: ADCC effect of anti-EGFR / anti-c-Met multispecific antibodies on tumor cells. The antibody-dependent cell-mediated cytotoxicity (ADCC) effect of anti-EGFR / anti-c-Met multispecific antibodies (including: Amivantamab, V42, V73) on tumor cells was studied using human PBMCs (peripheral blood mononuclear cells). The negative control was hIgG1 (Baiying Biotechnology, catalog number B117901).

[0329] Logarithmically growing NCI-H292 and KP4 cells were taken and the viable cell density was adjusted to 3 × 105 cells / mL with RPMI medium (Hyclon, catalog number SH30809.01) containing 2% fetal bovine serum as target cells. Anti-EGFR / anti-c-Met multispecific antibodies were prepared using RPMI medium to achieve a maximum concentration of 8 nM to 200 nM, with 8-10 concentration gradients achieved through 5-fold serial dilutions. After resuscitation, PBMCs were counted, and the viable cell density was adjusted to 1.5 × 10⁶ cells / mL using RPMI medium to serve as effector cells. The following groups were set up: a drug administration group (50 μL target cells + 100 μL effector cells + 50 μL antibody), a target cell group (50 μL target cells + 150 μL culture medium), an effector cell group (100 μL effector cells + 100 μL culture medium), a target cell + effector cell group (50 μL target cells + 100 μL effector cells + 50 μL culture medium), a blank control group (200 μL culture medium), a lysis buffer control group (200 μL culture medium + 20 μL lysis buffer), and a target cell maximum release group (50 μL target cells + 150 μL culture medium + 20 μL lysis buffer). All samples were added to 96-well plates (Costar, catalog number 3599) to achieve an effector-to-target ratio of 10:1. The cells were incubated at 37°C with 5% CO2 for 24 hours. Detection was performed using the CytoTox96® non-radioactive cytotoxicity assay kit (Promega, catalog number G1780). Finally, the absorbance at 490 nm was measured using a microplate reader.

[0330]

[0331] Figures 8A-8B and Table 9 show the ADCC effect of anti-EGFR / anti-c-Met multispecific antibodies on tumor cells.

[0332] Table 9. EC50 values ​​and maximum lysis rates of each antibody on NCI-H292 and KP4 cells

[0333] Example 15: Internalization activity of anti-EGFR / anti-c-Met multispecific antibodies in tumor cells was analyzed by flow cytometry. Anti-EGFR / anti-c-Met multispecific antibodies (including: Amivantamab, V42, ...) were analyzed.The internalization activity of V73 in A431 cells, NCI-H1975 cells and NCI-H441 cells (expressed in EGFR and c-Met, source: Beina Biotechnology) was measured, with hIgG1 (Baiying Biotechnology, catalog number B117901) as the negative control.

[0334] A431 cells, NCI-H1975 cells and NCI-H441 cells in logarithmic growth phase were taken, and the viable cell density was adjusted to 2 × 106 cells / mL with complete culture medium. 20 μL / well was seeded in 96-well V-type cell culture plates (Costar, catalog number 3894). The anti-EGFR / anti-c-Met multispecific antibody complex was prepared according to the instructions of Antibody Internalization Human Reagent (Sartorius, catalog number 90564) to achieve a final concentration of 100-167 nM, and incubated at 37°C for 15 minutes. Subsequently, it was diluted 5-fold, resulting in 10 concentration gradients. The complex solution was added to cell culture plates at 20 µL / well, mixed well, 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.

[0335] Figures 9A-9C and Table 10 show the internalization activity of the anti-EGFR / anti-c-Met multispecific antibody in A431 cells, NCI-H1975 cells, and NCI-H441 cells.

[0336] Table 10. Internalization activity of anti-EGFR / anti-c-Met multispecific antibody in A431 cells, NCI-H1975 cells and NCI-H441 cells. Specification 50 / 72 pages 53 CN 121570606 A

[0337] Example 16: Pharmacodynamic evaluation of anti-EGFR / anti-c-Met multispecific antibody in U-87 MG human glioma nude mouse subcutaneous xenograft model. SPF grade female BALB / c-nu nude mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were subcutaneously inoculated with U-87 MG cells (ATCC HTB-14™). After the tumor grew to 100-150 mm3, the mice were divided into model group (physiological saline), V42 group and V73 group, with 8 mice in each group.

[0338] 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 a week 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).

[0339] The calculation formulas for the detection indexes are as follows: Tumor volume (mm3) = 1 / 2 × (a × b2), where a represents the long diameter of the tumor and b represents the short diameter of the tumor.

[0340] Relative tumor proliferation rate T / C (%) = (T - T0) / (C - C0) × 100%, TGI (%) = 100% - T / C; where T and C are the tumor volumes of the treatment group and the model group at the end of the experiment respectively; T0 and C0 are the tumor volumes of the treatment group and the model group at the beginning of the experiment respectively.

[0341] If the tumor is smaller than the tumor volume at the beginning of the experiment, that is, when T < T0 or C < C0, it is defined as partial tumor regression (PR). When the tumor shows regression, TGI (%) = 100 - (T - T0) / T0 × 100%.

[0342] The results of the detection indexes are shown in Table 11. In the subcutaneous xenograft tumor model of U-87 MG human glioma nude mice, both V42 and V73 have significant tumor inhibitory effects.

[0343] Table 11. Effects of anti-EGFR / anti-c-Met multispecific antibody on subcutaneous xenograft tumors of U-87 MG human glioma nude mice

[0344] The P value is compared with the model group.

[0345] Example 17: Preparation of Linker-payload MC-GGFG-deuterated DXd (MC-GGFG-DDDXd) Description of the specification Page 51 / 72 54 CN 121570606 A

[0346] Step 1 Synthesis of Intermediate A Under nitrogen protection, addDissolve in mL of dichloromethane, 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 (excerpt from the instruction manual, pages 52 / 72, CN 121570606 A, hexane: dichloromethane = 1:1), and stir overnight. Filter, and dry the filter cake 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]+; CAS of intermediate B: 1599440-06-8.

[0348] Step 3 Synthesis of intermediate C Weigh 78 g of intermediate B and add it to a 3000 mL single-necked flask, add 800 mL of dichloromethane and 45 g of intermediate A, and place the 3000 mL single-necked flask in an ice-water bath to cool to 0°C. 16 g of lithium tert-butoxide was dissolved in 400 mL of dichloromethane to prepare a lithium tert-butoxide solution. The lithium tert-butoxide solution was added to a 3000 mL single-necked flask and reacted at 0 °C for 3 hours. The mixture was then cooled to room temperature, and 800 mL of water was added with stirring. The liquid was separated, and the organic phase was collected. The aqueous phase was extracted with 400 mL of dichloromethane, and the organic phases were combined. The organic phase was washed once with 800 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was then subjected to silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) to give 61 g of intermediate C. LC-MS (ESI) m / z: 437.34 [M+Na]+; the CAS of intermediate C is 2760715-83-9.

[0349] Step 4 Synthesis of compound D 31.2 g of intermediate C was added to 270 mL of deuterated methanol and 70 mL of heavy water and stirred in an ice bath. Then 5.5 g of NaOH was added and the mixture was moved to room temperature and stirred overnight. Extract the reaction mixture with 300 mL of ethyl acetate and 300 mL of water. Adjust the pH to 2-3 with 20 mL of glacial acetic acid. A solid precipitates out. Filter to obtain 20.3 g of compound D. ¹H-NMR (500 MHz, DMSO-d⁶) δ 8.70 (t, J = 6.6 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 7.5 Hz, 2H), 7.57 (t, J = 6.0 Hz, 1H), 7.42 (t, J = 7.5 Hz, 2H), 7.34 (t, J = 7.5 Hz, 2H), 4.61 (d, J = 6.6 Hz, 2H), 4.30 (d , J = 7.1 Hz, 2H), 4.23 (1H, m), 3.64 (d, J = 6.0 Hz, 2H); LC‑MS (ESI) m / z:409.08 [M+Na]+.

[0350] Step 5 Synthesis of Compound E Weigh 2.0 g ethatecan mesylate dihydrate and 1.63 g compound D and add them to a 100 mL round-bottom flask. Add 40 mL N,N-dimethylformamide and stir. Cool to 0 °C. Add 2.0 g 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and 1.82 g N,N-diisopropylethylamine in sequence, and react at 0 °C for 3 h. Pour the reaction solution into 120 mL ice water and stir for 1 h. Filter and dissolve the filter cake in dichloromethane. Perform chromatography using silica gel column chromatography (100 g 100-200 mesh silica gel, dichloromethane:methanol = 30:1, 2 L) to obtain 2.6 g compound E. 1H‑NMR (500 MHz, DMSO‑d6), δ 8.78 (t, J = 6.6 Hz, 1H), 8.47 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 11.0 Hz, 1H) , 7.68 (d, J = 7.5 Hz, 2H), 7.53 (t, J = 6.0 Hz, 1H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (m, 2H), 7.29 (s, 1H), 6.50 (brs, 1H) , 5.56 (m, 1H) , 5.39 (m, 2H) , 5.14 (m, 2H) , 4.64 (m, 2H) , 4.25 (d, J = 6.8 Hz, 2H) , 4.19 (m, 1H) , 3.62 (d, J = 6.0 Hz, 2H) , 3.15 (m, 2H), 2.35 (s, 3H), 2.17 (m, 2H), 1.83 (m, 2H), 0.85 (t, J = 7.3 Hz, 3H); LC‑MS (ESI) m / z: 804.84 [M+H]+.

[0351] Step 6: Preparation of Compound F. 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 a 100 mL round-bottom flask and allow it to naturally warm to room temperature. React for 3 h. Filter under nitrogen protection to obtain 1.45 g of compound F.Compound F. ¹H-NMR (500 MHz, DMSO-d6) δ 8.76 (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 = 7.1 Hz, 3H); LC-MS (ESI) m / z: 582.39 [M+H]+. Instructions for Use, Pages 53 / 72, CN 121570606 A

[0352] Step 7: Preparation of Compound H. 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 solution, stir for 0.5 h, let stand, separate the liquids, 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 (500 MHz, DMSO‑d6) δ 8.62 (t, J = 6.5 Hz, 1H) , 8.49 (d , J = 8.5 Hz, 1H) , 8.29 (t, J = 5.5 Hz, 1H) , 8.12 (d, J = 8.0 Hz, 1H) , 8.06 (t, J = 5.5 Hz, 1H) , 8.00 (t, J = 5.5 Hz, 1H) , 7.74 (d, J = 10.5 Hz, 1H) , 7.30 (s, 1H) , 7.27‑ 7.12 (m, 5H) , 6.98 (s, 2H) , 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 = 6.5 Hz, 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 .5 Hz , 14.0 Hz, 1H) , 2.77 (dd , J = 9.5 Hz, 13.5 Hz, 1H) , 2.36 (s, 3H) , 2.23‑2.14 (m, 2H) , 2.09 (t, J = 7 .5 Hz, 2H) , 1 .91‑1 .79 (m , 2H) , 1 .49‑1 .42 (m , 4H) , 1 .20‑ 1.14(m, 2H) , 0.87 (t, J = 7.5 Hz, 3H); HR-MS m / z: 1036.4194 [M+H]+.

[0353] Example 18: Preparation of ADC targeting EGFR and c-Met Example 18-1: Preparation of ADC V42-DDDXd-6(1) targeting EGFR and c-Met Reagents: Antibody is anti-EGFR / anti-c-Met multispecific antibody V42, Linker-payload is MC-GGFG-DDDXd synthesized in Example 17.

[0354] Experimental procedure: 1. Antibody reduction: Adjust the antibody concentration to about 8-10 g / L with 20 mM histidine buffer (containing L-histidine 1.43 mg / mL, L-histidine hydrochloride monohydrate 2.27 mg / mL, 5% sucrose, pH 6.0), and then adjust the pH to about 6.0 with 0.3 M disodium hydrogen phosphate aqueous solution. Adjust the temperature of the above solution to 30°C, add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution to the solution, so that the molar ratio of TCEP·HCl to antibody is 6.0:1. React in the dark for 2 hours under the conditions of 30°C and stirring at 50~60 rpm to obtain reaction solution 1.

[0355] 2. Coupling of antibody and linker-payload: Add DMSO to reaction solution 1 to make its final concentration 5% (v / v), and then add DMSO dissolved in Linker-payload with 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 under the conditions of 22°C and stirring at 50~60 rpm to obtain reaction solution 2.

[0356] 3. Conjugation termination and purification: After conjugation, reaction solution 2 was replaced with 20 mM histidine buffer (20 g / kg polysorbate 80 (II), 0.753 g / kg histidine, 3.1762 g / kg histidine hydrochloride, pH 5.8) using a 30 kDa ultrafiltration membrane to obtain antibody-drug conjugate V42-DDDXd-6 (1).

[0357] Referring to the method of Example 19, the DAR value of V42-DDDXd-6(1) was measured to be 6.0.

[0358] The structure of V42-DDDXd-6(1) is as follows: Specification 54 / 72 pages 57 CN 121570606 A.

[0359] Example 18-2: Preparation reagents of ADC V42-DDDXd-6(2) targeting EGFR and c-Met: The antibody is anti-EGFR / anti-c-Met multispecific antibody V42, and the linker-payload is MC-GGFG-DDDXd synthesized in Example 17.

[0360] Experimental Procedure: 1. Antibody Reduction: The antibody concentration was adjusted to approximately 18-20 g / L using 20 mM histidine buffer (containing 1.43 mg / mL L-histidine, 2.27 mg / mL L-histidine hydrochloride monohydrate, 5% sucrose, pH 6.0), and the pH was adjusted to approximately 6.5 using 0.3 M disodium hydrogen phosphate aqueous solution. The temperature of the above solution was adjusted to 30°C, and 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution was added to the solution so that the molar ratio of TCEP·HCl to antibody was 6.0:1. The reaction was carried out at 30°C and 50-60 rpm in the dark for 2 hours to obtain reaction solution 1.

[0361] 2. Coupling of antibody and linker-payload: DMSO was added to reaction solution 1 to make the final concentration 5% (v / v), and then DMSO-dissolved linker-payload with a concentration of 10 g / L was added to make the molar ratio of linker-payload to antibody 9.25:1. The reaction was carried out at 22°C and 50-60 rpm under light protection for 2 hours to obtain reaction solution 2.

[0362] 3. Coupling termination and purification: After the coupling was completed, reaction solution 2 was replaced with 20 mM histidine buffer (pH 5.8) using a 30 kDa ultrafiltration membrane to obtain antibody-drug conjugate V42-DDDXd-6(2).

[0363] Referring to the method of Example 19, the DAR value of V42-DDDXd-6(2) was measured to be 5.9.

[0364] The structure of V42-DDDXd-6(2) is as follows: .

[0365] Example 18-3: Preparation of ADC reference standard (Benchmark) AZD9592 targeting EGFR and c-Met: Instructions for use 55 / 72 pages 58 CN 121570606 A 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).

[0366] Experimental procedure: 1. Antibody reduction: The antibody was replaced with histidine buffer (L-histidine 1.43 mg / mL, L-histidine hydrochloride monohydrate 2.27 mg / mL) at pH 6.0, and the antibody concentration was adjusted to about 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 ratio of antibody to TCEP 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 and obtain reaction solution 1.

[0367] 2. Antibody-Connector-Load Conjugation: Add a 10 mg / mL adapter-loador solution dissolved in DMSO to reaction solution 1, making the molar ratio of antibody to adapter-loador 1:12. Incubate at 22°C in the dark for 1 hour, stirring gently to connect the antibody to the adapter-loador, obtaining reaction solution 2.

[0368] 3. Conjugation Termination and Purification: Ultrafilter reaction solution 2 with pH 6.0 histidine buffer to obtain antibody-drug conjugate AZD9592.

[0369] Referring to the method of Example 19, the DAR value of AZD9592 was measured to be 6.0.

[0370] The structure of AZD9592 is as follows: .

[0371] Example 19: Determination of DAR value of antibody-drug conjugate (ADC) The DAR value of ADC was determined by hydrophobic interaction chromatography (HIC). A neutral, high-salt mobile phase was used to enhance the hydrophobic properties of the protein molecules, thereby binding them to the hydrophobic bonds in the chromatographic column. Then, the elution was performed by gradually decreasing the salt concentration and gradually increasing the proportion of isopropanol, eluting substances with lower hydrophobicity first and those with higher hydrophobicity last.

[0372] The components of the ADC targeting EGFR and c-Met were separated using non-porous polystyrene / divinylbenzene (PS / DVB) packing material with bonded butyl groups. The column specifications were Sepax HIC-Butyl, 4.6 × 100 mm, 5 μm, and the column temperature was 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). 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 prepared using the initial proportioned mobile phase.The sample was diluted 1-fold to prepare the test solution. The injection volume was adjusted according to the sample concentration, and 50 μg of protein was injected for detection 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.

[0373] Data processing: The results were quantitatively analyzed using the area normalization method. The peak area percentages of the ADCs containing 0, 1, 2, 3, 4, 5, 6, 7, and 8 cytotoxic drugs were calculated, and the DAR values ​​were calculated. The calculation formula is: DAR value = (percentage of ADC peak area containing 0 cytotoxic drugs × 0 + percentage of ADC peak area containing 1 cytotoxic drug × 1 + percentage of ADC peak area containing 2 cytotoxic drugs × 2 + percentage of ADC peak area containing 3 cytotoxic drugs × 3 + percentage of ADC peak area containing 4 cytotoxic drugs × 4 + percentage of ADC peak area containing 5 cytotoxic drugs × 5 + percentage of ADC peak area containing 6 cytotoxic drugs × 6 + percentage of ADC peak area containing 7 cytotoxic drugs × 7 + percentage of ADC peak area containing 8 cytotoxic drugs × 8) / 100%.

[0374] Table 12. Gradient elution parameters

[0375] Example 20: Validation of antibody-drug conjugate aggregates. Gel chromatography was used to separate the components of ADC samples targeting EGFR and c-Met. Neutral pH buffer with 10% isopropanol was used as the mobile phase for elution, and the components were eluted in descending order of molecular weight. The chromatographic column was an ACQUITY UPLC Protein BEH SEC Column (200 Å, 1.7 μm, 4.6 × 300 mm), with a column temperature of 25℃. 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, then ultrapure water was added to 1000 mL, mixed well, and 900 mL was taken and 100 mL of isopropanol was added, mixed well, and filtered through a 0.22 μm filter membrane). 20 μg of the ADC sample was accurately injected into the HPLC system and detected at 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, with aggregates preceding the main peak, immunoglobulin monomers preceding the main peak, and low molecular weight impurities following the main peak. The detection results are shown in Table 13.

[0376] Table 13.Monomer, aggregate, and low molecular weight impurity content of ADCs targeting EGFR and c-Met

[0377] Example 21: Affinity of ADCs targeting EGFR and c-Met to human EGFR and human c-Met The affinity of ADCs for human EGFR-His protein or human c-Met-His protein was detected using a biomolecular interaction analysis system (Cytiva, Biacore 8K). Amino-conjugated Anti-hIgG (Fc) antibody (Cytiva, catalog No. BR-1008-39) was added to the CM5 sensor chip and diluted to 4 μg / mL 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). The antibody was captured through the experimental channel at a flow rate of 30 μL / min. Human EGFR-His or human c-Met-His was diluted with running 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. The Langmuir 1:1 model was used for fitting, and the binding rate constant ka (1 / Ms), dissociation rate constant kd (1 / s), and equilibrium dissociation constant KD (M) were calculated. The results are shown in Table 14.

[0378] Table 14. Affinity of antibody-drug conjugates

[0379] Example 22: Cell binding activity of ADCs targeting EGFR and c-Met was detected by flow cytometry. Human non-small cell lung cancer cells HCC827 (high EGFR expression, low c-Met expression, YS122C), NCI-H441, NCI-H292, and human colon adenocarcinoma cells HCA-7 (low EGFR expression, low c-Met expression, CBP60035) were diluted to 2 × 10⁶ cells / mL and added to 96-well plates at 50 μL / well. 50 μL / well of FACS buffer (Miltenyi Biotec, 130-091-221) was also added. A serially diluted ADC targeting EGFR and c-Met (V42-DDDXd-6(1)) with a final concentration of 115.4 nM was used as the starting concentration, and AZD9592 was used as the final concentration.(nM was the starting concentration, serially diluted 5-fold, for a total of 8 concentrations). After incubation at 4°C for 1 h, centrifuge at 1000 rpm for 5 min. Discard the supernatant, wash three times with pre-chilled FACS buffer, add 100 μL / well of goat anti-human IgG Fcγ-PE secondary antibody diluted 1:200 (v / v) (Jackson immunoresearch, catalog number 109-116-170), and incubate at 4°C 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 is 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 is shown in Table 15 below. The results showed that V42-DDDXd-6(1) could effectively bind to cells with different expression levels of EGFR and c-Met, and the binding activity was better than that of AZD9592.

[0380] Table 15. Binding of ADCs targeting EGFR and c-Met to cells

[0381] Example 23: Internalization activity of ADCs targeting EGFR and c-Met was detected by flow cytometry. The concentrations of HCC827, NCI-H292, HCA-7, and NCI-H441 cells were adjusted to 2 × 10⁶ cells / mL. 50 μL of each cell was seeded into 96-well plates, and 50 μL of FACS buffer (Miltenyi Biotec, catalog number 130-091-221) was added to each well. The ADCs targeting EGFR and c-Met were serially diluted (for HCC827 and HCA-7 cells, the starting concentrations were V42-DDDXd-6(1) at a final concentration of 115.4 nM and AZD9592 at a final concentration of 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 a final concentration of 23.08 nM and AZD9592 at a final concentration of 20 nM, with 5-fold serial dilutions for a total of 7 concentrations) and incubated at 4°C for 1 h. After incubation, remove the 96-well cell culture plate and centrifuge at 400 g for 5 min at 4°C, discarding the supernatant. 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 for 30 min.After washing the plate, 40 μL of cell culture medium was added to each well, mixed well, and incubated at 37°C for 2 h. The plate was then placed in a flow cytometer (Sartorius, iQUE3) to measure the fluorescence reading of the RL1 channel. The data were analyzed, and the results are shown in Figures 11A-11D. The calculated EC50 values ​​are 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.

[0382] Table 16. Internalization activity of ADCs targeting EGFR and c-Met

[0383] Example 24: Killing of tumor cells by ADCs targeting EGFR and c-Met To detect the killing effect of ADCs targeting EGFR and c-Met on EGFR and c-Met positive tumor cells, the killing activity was detected using natural human non-small cell lung cancer cells HCC827, NCI-H292, human gastric cancer cells MKN45 (low EGFR expression, high c-Met expression, Nanjing Kebai Biotechnology, catalog number: CBP60488), human colon adenocarcinoma cells HCA-7, and human pancreatic cancer cells BxPC3 (high EGFR expression, low c-Me expression, Pronosai, catalog number: CL-0042).

[0384] Take cells in the logarithmic growth phase, adjust the cell density of HCC827, MKN45, NCI-H292 and BxPC3 cells to 2 × 104 cells / mL, and the cell density of HCA-7 cells to 4 × 104 cells / mL, respectively, add 100 μL / well to a 96-well plate, and culture at 37 ℃ 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 a concentration of 25 nM and serially diluted 4-fold to a total of 10 concentrations. For MKN45, NCI-H292, and BxPC3 cells, V42-DDDXd-6(1) and AZD9592 were started at a concentration of 100 nM and serially diluted 4-fold to a total of 10 or 11 concentrations. For HCA-7 cells, V42-DDDXd-6(1) and AZD9592 were started at a concentration of 25 nM and serially diluted 4-fold to a total of 9 concentrations. After removing adherent cells from the culture, the experimental group was given 50 μL / well of a diluted ADC targeting EGFR and c-Met, while the control group was given 50 μL / well of the corresponding complete culture medium. Cells were cultured for 96 h, 120 h, or 144 h, and then analyzed using the CellCounting-Lite 2.0 Luminescent Cell Viability Assay Kit (Vazyme, catalog number: [missing information]).The detection of DD1101-03 was performed as follows: 75 μL of CellCounting-Lite detection solution (Vazyme, catalog number: DD1101-03) was added to each well of a 96-well plate, shaken to mix, and incubated at room temperature in the dark for 10 min. Then, 170 μL of each well was transferred to an opaque white plate, air bubbles were removed, and the chemiluminescence value was read using a microplate reader (PE, Envision 2105). The cell killing rate was calculated.

[0385]

[0386] The data were analyzed, and the results are shown in Figures 12A-12E. The calculated EC50 is shown in Table 17 below. The results showed that V42-DDDXd-6(1) had a better killing effect on tumor cells than AZD9592.

[0387] Table 17. Killing effect of EGFR and c-Met-targeting ADCs on tumor cells

[0388] Example 25: Bystander effect of EGFR and c-Met-targeting ADCs on tumor cells To detect the bystander effect of EGFR and c-Met-targeting ADCs, HCC827 cells were used as positive cells and Jurkat cells as negative cells, and the detection was performed based on FACS. HCC827 and Jurkat cells in logarithmic growth phase were taken, and the viable cell density of HCC827 cells was adjusted to 2 × 104 cells / mL. 1 mL of each cell was added to a 6-well plate and cultured overnight at 37°C in a 5% CO2 cell culture incubator. The ADC was pre-diluted to 1.6 nM (final concentration) and 2 mL of each cell plate was added to the cell plate. The viable cell density of Jurkat cells was adjusted to 1 × 104 cells / mL and 1 mL of each cell plate was added to the corresponding cell plate. After 5 days of incubation, cells were removed from 6-well plates, digested with trypsin, counted, and collected from different sample wells. The cell density was adjusted to 1 × 10⁶ cells / mL with FACS buffer, and 100 μL / well was added to a 96-well V plate. Fixed Viability Stain 780 (BD, catalog number: 565388) was added to the 96-well plate for cell viability identification. After incubation at room temperature for 15 min, the cells were washed twice, and then EGFR direct labeling antibody (abcam, catalog number: 130738) was added and incubated at 4℃ for 40 min. After incubation, the cells were washed twice, resuspended in FACS buffer, and the signal was detected by flow cytometry (Invitrogen, model: Attune® NxT).

[0389] The data were analyzed, and the results are shown in Figure 13. The results show that V42-DDDXd-6(1) has a bystander effect.

[0390] Example 26: Pharmacodynamic evaluation of EGFR and c-Met-targeting ADCs in NCI-H292 human lung cancer cell xenograft tumor model in nude miceSPF-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 × 10⁶ cells / mouse. When the average tumor volume reached about 100 mm³, the animals were divided into 6 groups of 5 mice each. The specific grouping and administration regimens are shown in Table 18: Table 18. Grouping and Administration Regimens

[0391] The day of grouping is day 0. On day 1 of grouping, the mice were administered the drug once via the tail vein. The tumor volume was measured twice a week, and the weight of the mice was also measured and recorded. The general performance of the mice was observed and recorded daily. After the experiment, the tumors were removed, weighed, and photographed.

[0392] The detection indicators and calculation formulas are as follows: Tumor volume, TV (mm³) = 1 / 2 × (a × b²); where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0393] Relative tumor volume, RTV = TVt / TV0; where TV0 is the tumor volume on day 0 and TVt is the tumor volume at each measurement.

[0394] Relative tumor proliferation rate, T / C (%) = TRTV / CRTV × 100%; where TRTV is the RTV of the treatment group; CRTV is the RTV of the solvent control group.

[0395] Tumor growth inhibition rate, TGI (%) = (1 - TW / TW0) × 100%; where TW is the tumor weight of the treatment group, and TW0 is the tumor weight of the solvent control group.

[0396] Body weight change rate, WCR (%) = (Wtt - Wt0) / Wt0 × 100%; where Wt0 is the mouse body weight on day 0, and Wtt is the mouse body weight at each measurement.

[0397] The results of each detection 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 both low and high doses of human lung cancer xenograft tumors in mice NCI-H292. At the same dose of cytotoxic drugs, V42-DDDXd-6(1) was more effective than AZD9592.

[0398] Table 19. Effects of ADCs targeting EGFR and c-Met on each index in the NCI-H292 human lung cancer cell subcutaneous xenograft tumor model in nude mice. Instructions for use 60 / 72 pages 63 CN 121570606 A

[0399] Note: Compared with the solvent control group (group 1), *P<0.05; N / A indicates not applicable.

[0400] Example 27: Pharmacodynamic evaluation of EGFR and c-Met-targeting ADCs in a nude mouse xenograft model of HCA-7 human colon cancer cells. HCA-7 human colon cancer cells were subcutaneously inoculated in the right axilla of SPF-grade female BALB / c nude mice (source: Experimental Animal Center of Hangzhou Medical College), 2 × 10⁶ cells / mouse. When the average tumor volume reached approximately 150 mm³, the animals were divided into 6 groups of 5 mice each. The specific grouping and administration regimens are shown in Table 20: Table 20.Mouse grouping and dosage

[0401] Day 0 was the day of grouping. The mice were administered the drug via tail vein on day 1 of grouping, twice a week for a total of 4 times. Tumor volume was measured twice a week, and the weight of the mice was recorded. The general performance of the mice was observed and recorded daily. After the experiment, the tumors were removed, weighed, and photographed.

[0402] The detection indicators and calculation formulas are as follows: Tumor volume, TV (mm3) = 1 / 2 × (a × b2); where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0403] Relative tumor volume, RTV = TVt / TV0; where TV0 is the tumor volume on day 0 and TVt is the tumor volume at each measurement.

[0404] Relative tumor proliferation rate, T / C (%) = TRTV / CRTV × 100%; where TRTV is the RTV of the treatment group and CRTV is the RTV of the solvent control group.

[0405] Tumor growth inhibition rate, TGI (%) = (1 - TW / TW0) × 100%; where TW is the tumor weight of the treatment group and TW0 is the tumor weight of the solvent control group.

[0406] Weight change rate, WCR (%) = (Wtt - Wt0) / Wt0 × 100%; where Wt0 is the mouse weight on day 0 and Wtt is the mouse weight at each measurement.

[0407] The results of each detection index on day 21 are shown in Table 21 and Figure 15 below. There was no obvious toxicity in each group; among them, the high dose of V42-DDDXd-6(2) had inhibitory activity on mouse HCA-7 human colon cancer xenograft tumors.

[0408] Table 21. Effects of ADC-EGFR cMet on various indicators in a nude mouse subcutaneous xenograft model of HCA-7 human colon cancer cells. (Pages 61 / 72, CN 121570606 A)

[0409] Note: *P<0.05 compared with the solvent control group; N / A indicates not applicable.

[0410] The sequence information of this disclosure is summarized in Table S4 below.

[0411] Specification page 62 / 72, page 65, CN 121570606 A

[0412] Specification page 63 / 72, page 66, CN 121570606 A

[0413] Specification page 64 / 72, page 67, CN 121570606 A

[0414] Specification page 65 / 72, page 68, CN 121570606 A

[0415] Specification page 66 / 72, page 69, CN 121570606 A

[0416] Specification page 67 / 72, page 70, CN 121570606 A

[0417] Specification page 68 / 72, page 71, CN 121570606 A

[0418] Specification page 69 / 72, page 72, CN 121570606 A

[0419] Description

[0420] Description 70 / 72 pages 73 CN 121570606 A

[0421]

[0422] For purposes of description and disclosure, all patents, patent applications and other identified publications are expressly incorporated herein by reference. These publications are provided only 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 correctness of the dates of these documents or their contents. Moreover, in any country, any reference to these publications herein does not constitute an endorsement that such publications are part of the common general knowledge in the art.

[0423] Although this disclosure has been described in detail above with general description and specific embodiments, some modifications or improvements may be made to it based on this disclosure, which will be apparent to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this disclosure are within the scope of protection claimed by this disclosure. Instruction manual pages 72 / 72; 75 CN 121570606 A; Figure 1A; Figure 1B; Figure 1C; Instruction manual drawing 1 / 14 pages; 76 CN 121570606 A; Figure 1D; Figure 1E; Figure 1F; Instruction manual drawing 2 / 14 pages; 77 CN 121570606 A; Figure 1G; Figure 2; Figure 3A; Instruction manual drawing 3 / 14 pages; 78 CN 121570606 A; Figure 3B; Figure 3C; Instruction manual drawing 4 / 14 pages; 79 CN 121570606 A; Figure 4; Figure 5; Figure 6A; Instruction manual drawing 5 / 14 pages; 80 CN 121570606 A; Figure 6B; Figure 6C; Figure 7A; Instruction manual drawing 6 / 14 pages; 81 CN 121570606 A; Figure 7B; Figure 8A; Figure 8B; Instruction manual drawing 7 / 14 pages; 82 CN 121570606 A; Figure 9A; Figure 9B; Figure 9C Instruction manual figures 8 / 14, page 83, CN 121570606 A, Figure 10A, Figure 10B, Figure 10C; Instruction manual figures 9 / 14, page 84, CN 121570606 A, Figure 10D, Figure 11A, Figure 11B; Instruction manual figures 10 / 14, page 85, CN 121570606 A, Figure 11C, Figure 11D, Figure 12A; Instruction manual figures 11 / 14, page 86, CN 121570606 A, Figure 12B, Figure 12C; Instruction manual figures 12 / 14, page 87, CN121570606 A Figures 12D, 12E, 13, pages 13 / 14 of the drawings of the specification; 88 CN 121570606 A Figures 14, 15, page 14 / 14 of the drawings of the specification; 89 CN 121570606 A Abstract The present application belongs to the field of biomedicine, and provides an antibody-drug conjugate targeting EGFR and c-Met. 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.

Claims

1. An antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of the antibody drug conjugate, the stereoisomer thereof, the pharmaceutically acceptable salt thereof, comprising a multispecific antibody, wherein, The multispecific antibody comprises (i) a first antigen binding moiety that binds to a first antigen; (ii) a second antigen binding moiety that binds to a second antigen; and (iii) a third antigen binding moiety that binds to the first antigen; wherein the first antigen is c-Met and the second antigen is EGFR, the first and third antigen binding moieties are single variable domains and each independently comprises any one of: (1) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, wherein X1 is selected from S or T, preferably T; (2) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; (3) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; (4) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12; (5) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; (6) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18; (7) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21; or (8) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

24. the first and third antigen binding moieties each independently comprises any one of:

2. The antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of the antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof according to claim 1, wherein, (1) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, wherein X1 is selected from S or T, preferably T; ​ (2) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; or (8) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

24.

3. The antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of the antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof according to claim 2, wherein, the first antigen binding moiety comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; and the third antigen binding moiety comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, wherein X1 is selected from S or T; preferably, the X1 is T.

4. The antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of the antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof according to claim 2, wherein, the first antigen binding moiety comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; and the third antigen binding moiety comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

24.

5. An antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of the antibody drug conjugate, the stereoisomer thereof, the pharmaceutically acceptable salt thereof, comprising a multispecific antibody, wherein, the multispecific antibody comprises (i) a first antigen binding moiety that binds a first antigen; (ii) a second antigen binding moiety that binds a second antigen; and (iii) a third antigen binding moiety that binds the first antigen; wherein the first antigen is c-Met and the second antigen is EGFR, the first and third antigen binding moieties are single variable domains and each independently comprises CDR1, CDR2, and CDR3 of a single variable domain having an amino acid sequence of SEQ ID NO: 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, or 39.

6. The antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of the antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof according to claim 5, wherein, the first and third antigen binding moieties each independently comprise CDR1, CDR2, and CDR3 of a single variable domain having an amino acid sequence of SEQ ID NO: 35, 36, or 38.

7. The antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of the antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof according to claim 6, wherein, the first antigen binding moiety comprises CDR1, CDR2, and CDR3 of a single variable domain having an amino acid sequence of SEQ ID NO: 36, and the third antigen binding moiety comprises CDR1, CDR2, and CDR3 of a single variable domain having an amino acid sequence of SEQ ID NO:

35.

8. The antibody drug conjugate, stereoisomer thereof, pharmaceutically acceptable salt thereof, or solvate of the antibody drug conjugate, stereoisomer thereof, pharmaceutically acceptable salt thereof according to claim 6, wherein, the first antigen binding moiety comprises a CDR1, a CDR2, and a CDR3 of a single variable domain of the amino acid sequence of SEQ ID NO: 36, and the third antigen binding moiety comprises a CDR1, a CDR2, and a CDR3 of a single variable domain of the amino acid sequence of SEQ ID NO:

38.

9. The antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate of the antibody drug conjugate, a stereoisomer thereof, a pharmaceutically acceptable salt thereof according to any one of claims 1-8, wherein, the first and third antigen binding moieties each independently comprise an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 40, 41, 35, 36, 26, 27, 25, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, or 42.

10. The antibody drug conjugate, stereoisomer thereof, pharmaceutically acceptable salt thereof, or solvate of said antibody drug conjugate, stereoisomer thereof, pharmaceutically acceptable salt thereof according to any one of claims 1-9, wherein, the first antigen binding moiety comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 41.

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