Antibody-drug conjugate, its preparation method and use

Abstract

Antibody-drug conjugates, as well as methods for their preparation and use, are provided, specifically antibody-drug conjugates for treating HER3-positive cancers. A fully human HER3 antibody is provided that has excellent binding activity against HER3-positive cells and can efficiently deliver drugs to HER3-positive cells. A drug-linker molecule conjugated to the antibody is provided, and the drug includes a DNA topoisomerase inhibitor. The resulting antibody-drug conjugate has a better drug-to-antibody ratio and has a good target killing effect against colon cancer, gastric cancer, breast cancer, and lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma). Methods for preparing the antibody-drug conjugate and the application of the antibody-drug conjugate in the treatment of HER3-positive cancers are provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of Chinese Patent Application No. 2023 / 10444522.X, filed on April 23, 2023, and Chinese Patent Application No. 2022 / 10467789.6, filed on April 29, 2022, and the entire disclosure of each of these is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application includes a computer - readable sequence listing submitted in XML file format together with this application, and the entire content thereof is incorporated herein by reference in its entirety. The sequence listing XML file submitted together with this application is entitled "14463 - 050 - 228_SEQ_LISTING.xml", created on April 20, 2023, and has a size of 59,247 bytes.

Background Art

[0003] (1) Technical Field This application relates to the field of targeted therapy, particularly antibody - drug conjugates and their preparation, as well as their methods and uses. (2) Description of Related Technologies

[0004] Cancer is one of the major causes of death in modern times. Cancer is a type of disease caused by the malignant transformation of healthy cells, which is caused by genetic changes such as chromosomal translocations, mutations in tumor suppressor genes and growth factor receptors, resulting in the malignant proliferation of cells. Incomplete apoptosis or programmed cell death further promotes the malignant transformation of cells that lead to cancer.

[0005] Human epidermal growth factor receptor 3 (also known as HER3 and ErbB3) is a receptor protein tyrosine kinase belonging to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases. The subfamily also includes HER1 (also known as EGFR), HER2, and HER4. Similar to the epidermal growth factor receptor, the transmembrane receptor HER3 consists of a ligand-binding extracellular domain (ECD), a dimerization domain within the ECD, a transmembrane domain, and a carboxy-terminal phosphorylation domain. In addition to these domains, HER1, HER2, and HER4 possess an intracellular protein tyrosine kinase domain (TKD), but HER3 lacks this domain and thus cannot undergo autophosphorylation.

[0006] The ligand-regulated protein (HRG) binds to the extracellular domain of HER3 and activates the receptor-mediated signaling pathway by promoting dimerization with other members of the human epidermal growth factor receptor (HER) family and phosphorylation of their intracellular domains. Dimerization of HER3 with other HER family members expands the signaling capacity of HER3 and serves as a means not only for signal diversification but also for signal amplification. For example, the HER2 / HER3 heterodimer induces one of the most important mitogenic signals among the HER family members. HER3 is overexpressed in many types of cancer, such as breast cancer, gastrointestinal cancer, and pancreatic cancer. Interestingly, a relationship has been shown between the expression of HER2 / HER3 and the progression from the non-invasive stage to the invasive stage. Therefore, agents that can interfere with HER3-mediated signal transduction are desired.

[0007] The HER3 target indications currently in clinical research cover hematological malignancies and solid tumors. The main treatment strategies focus on several directions such as monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs). Among them, regarding anti-HER3 antibody-drug conjugates, two are in international research and one is in the clinical research stage (Daiichi Sankyo U3-1402) for the treatment of non-small cell lung cancer (NSCLC), metastatic breast cancer (MBC), and colorectal cancer (CRC).

[0008] An ADC is composed of an antibody, a bioactive molecule, and a linker. The bioactive molecule is covalently bound to the antibody via the linker, and the antibody (e.g., a monoclonal antibody) can specifically recognize a specific target on the surface of tumor cells, so the ADC can be induced to the surface of cancer cells, whereby the ADC can enter the cells by the endocytosis effect, and then the bioactive molecule is released into the cancer cells to kill the cancer cells with minimal damage to normal tissue cells.

[0009] U3-1402 was developed by Daiichi Sankyo, uses the GGFG tetrapeptide as an enzyme-cleavable linker, and the cytotoxin is Dxd. According to reports, in the treatment of NSCLC with EGFR mutations, the Phase I with dose escalation (5.6 mg / kg, Q3W) showed an objective response rate (ORR) of 39%, a disease control rate (DCR) of 72%, and a median progression-free survival (mPFS) of 8.2 months, and showed a similar effect on patients with brain metastases. Regarding safety, adverse events mainly included hematotoxicity (thrombocytopenia, neutropenia, etc.) and interstitial pneumonia (5 - 7%).

Summary of the Invention

[0010] In one aspect, the present application provides an antibody-drug conjugate having a structure represented by the formula Ab-[M-L-E-D] x wherein Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3, also known as Erbb3), M is a binding site that binds to an antibody or an antigen-binding fragment thereof, L is a linker that connects the binding site M and E, E is a fragment that connects L and D, D is a fragment of a cytotoxic drug, x is selected from 1 to 10.

[0011] In some embodiments, the antibody or an antigen-binding fragment thereof is (1) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Chothia numbering system, a heavy chain variable region (VH) and / or a light chain variable region (VL): (1a) The following three CDRs: a CDR-H1 having the sequence set forth in SEQ ID NO: 1 or a variant thereof, a CDR-H2 having the sequence set forth in SEQ ID NO: 2 or a variant thereof, a CDR-H3 having the sequence set forth in SEQ ID NO: 3 or a variant thereof, a heavy chain variable region (VH) comprising, and / or the following three CDRs: a CDR-L1 having the sequence set forth in SEQ ID NO: 4 or a variant thereof, a CDR-L2 having the sequence set forth in SEQ ID NO: 5 or a variant thereof, a CDR-L3 having the sequence set forth in SEQ ID NO: 6 or a variant thereof, a light chain variable region (VL) comprising, or (1b) The following three CDRs: a CDR-H1 having the sequence set forth in SEQ ID NO: 19 or a variant thereof, a CDR-H2 having the sequence set forth in SEQ ID NO: 20 or a variant thereof, a CDR-H3 having the sequence set forth in SEQ ID NO: 21 or a variant thereof, a heavy chain variable region (VH) comprising, and / or the following three CDRs: a CDR-L1 having the sequence set forth in SEQ ID NO: 22 or a variant thereof, a CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, a CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a variant thereof, a light chain variable region (VL) comprising, or (1c) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, a CDR-H2 having the sequence set forth in SEQ ID NO: 37 or a variant thereof, and a CDR-H3 having the sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the sequence set forth in SEQ ID NO: 45 or a variant thereof, a CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, and a CDR-L3 having the sequence set forth in SEQ ID NO: 52 or a variant thereof, (1a), (1b), and the variant described in any item of (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each CDR of the VH and VL of (1a), (1b), and (1c), and the variant binds to HER3, or (2) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the Kabat numbering system: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 7 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 8 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 9 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 5 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 6 or a variant thereof, or (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 25 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 26 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a variant thereof, or (2c) Comprising a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 39 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 40 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 52 or a variant thereof, (2a), (2b), or a variant according to any item of (2c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) when compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequences of the respective CDRs of VH and VL of (2a), (2b), and (2c), and the variant binds to HER3, or (3) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are the heavy chain variable region (VH) and / or the light chain variable region (VL) defined by the IMGT numbering system: (3a) The following three CDRs: a CDR-H1 having the sequence set forth in SEQ ID NO: 10 or a variant thereof, a CDR-H2 having the sequence set forth in SEQ ID NO: 11 or a variant thereof, a CDR-H3 having the sequence set forth in SEQ ID NO: 12 or a variant thereof, and / or the following three CDRs: a CDR-L1 having the sequence set forth in SEQ ID NO: 13 or a variant thereof, a CDR-L2 having the sequence set forth in SEQ ID NO: 14 or a variant thereof, a CDR-L3 having the sequence set forth in SEQ ID NO: 6 or a variant thereof, or (3b) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the sequence set forth in SEQ ID NO: 27 or a variant thereof, a CDR-H2 having the sequence set forth in SEQ ID NO: 28 or a variant thereof, and a CDR-H3 having the sequence set forth in SEQ ID NO: 29 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the sequence set forth in SEQ ID NO: 30 or a variant thereof, a CDR-L2 having the sequence set forth in SEQ ID NO: 31 or a variant thereof, and a CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a variant thereof, or (3c) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the sequence set forth in SEQ ID NO: 41 or a variant thereof, a CDR-H2 having the sequence set forth in SEQ ID NO: 42 or a variant thereof, and a CDR-H3 having the sequence set forth in SEQ ID NO: 43 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the sequence set forth in SEQ ID NO: 44 or a variant thereof, a CDR-L2 having the sequence set forth in SEQ ID NO: 31 or a variant thereof, and a CDR-L3 having the sequence set forth in SEQ ID NO: 52 or a variant thereof, (3a), (3b), and (3c) The variant described in any of the items has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of the respective CDR of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

[0012] In an antibody-drug conjugate, the cytotoxic drug can be conjugated to an antibody or antigen-binding fragment via a linker (e.g., the "M-L-E" fragment of the present application).

[0013] In some embodiments, M is

Chemical formula

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Chemical formula

[0014] In some embodiments,

Chemical formula

Chemical formula

Chemical formula

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[0015] In some embodiments, the cytotoxic drug is selected from the group consisting of a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor. In some embodiments, the tubulin inhibitor is an auristatin compound or a maytansine compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD). In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, velotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof.

[0016] In some embodiments, the cytotoxic drug is selected from the compounds represented by Formulas I and II,

Chemical formula

[0017] In a further embodiment, the cytotoxic drug has the following structure:

Chemical formula

[0018] In some embodiments, the antibody-drug conjugate is ADC A-01 to ADC A-25, ADC B-01 to ADC B-05 shown below:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

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Chemical formula

[0019] In a further embodiment, HA is (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3, and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6, or (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 19, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 20, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21, and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24, or (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 36, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 37, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38, and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 (2a) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 7, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 8, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 9, and the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6, or (2b) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 25, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21, and the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24, or (2c) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 39, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 40, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38, and the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52, or (3a) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 10, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 11, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 12, and the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 13, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 14, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6, or (3b) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29, and the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 30, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24, which are included in a light chain variable region (VL), or (3c) An antibody comprising: the following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 41, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 42, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 43, and the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 44, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52, which are included in a light chain variable region (VL).

[0020] In a further embodiment, HA comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0021] In a further embodiment, HA comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0022] In a further embodiment, HA comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 47.

[0023] In a further embodiment, HA comprises: (1) a heavy chain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) set forth in SEQ ID NO: 50, and a light chain comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region (CL) set forth in SEQ ID NO: 51.

[0024] In a further embodiment, HA comprises a heavy chain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a heavy chain constant region (CH) set forth in SEQ ID NO: 50, and a light chain comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 33 and a light chain constant region (CL) set forth in SEQ ID NO: 51, or

[0025] In a further embodiment, HA comprises a heavy chain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a heavy chain constant region (CH) set forth in SEQ ID NO: 50, and a light chain comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 47 and a light chain constant region (CL) set forth in SEQ ID NO: 51.

[0026] In a further embodiment, HA comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0027] In a further embodiment, HA comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 34, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0028] In a further embodiment, HA comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 49.

[0029] In certain embodiments of the HA or antibody-drug conjugate disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine, or may lack either the C-terminal lysine or the C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the variable domain of the antibody or antigen-binding fragment may undergo cyclization to pyroglutamic acid.

[0030] Accordingly, the composition may comprise a population of antibody-drug conjugate species, each of which may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise cyclization of the N-terminal glutamine or glutamic acid or the N-terminal amino acid to pyroglutamic acid.

[0031] Thus, in certain embodiments, the present invention provides a composition comprising the ADCs disclosed herein, wherein the predominant ADC species in the composition is (i) an antibody lacking a lysine residue at the C-terminus of the heavy chain, (ii) an antibody having glutamine, glutamic acid, or pyroglutamic acid at the N-terminus of the heavy chain, or (iii) an antibody lacking a lysine residue at the C-terminus of the heavy chain and having glutamine, glutamic acid, or pyroglutamic acid at the N-terminus of the heavy chain.

[0032] Also provided herein are pharmaceutical compositions comprising the antibody-drug conjugates described herein and one or more pharmaceutically acceptable excipients.

[0033] Also provided herein is a method of treating cancer in a subject having high expression of HER3, comprising administering to the subject a therapeutically effective amount of the antibody-drug conjugate described herein or a pharmaceutical composition thereof. In certain embodiments, the cancer comprises solid tumors or hematological malignancies. In further embodiments, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0034] Also provided herein is the use of the antibody-drug conjugate described herein or a pharmaceutical composition thereof in the manufacture of a medicament for the treatment of cancer associated with high expression of HER3. In certain embodiments, the cancer comprises solid tumors or hematological malignancies. In further embodiments, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0035] Also provided herein is the use of the antibody-drug conjugate described herein or a pharmaceutical composition thereof in the treatment of cancer associated with high expression of HER3.

[0036] In certain embodiments, the cancer comprises solid tumors or hematological malignancies. In further embodiments, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0037] Also provided herein are the antibody-drug conjugates described herein, or the pharmaceutical compositions described herein, for the treatment of cancer associated with high expression of HER3. In certain embodiments, the cancer includes solid tumors or hematological malignancies. In further embodiments, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0038] In one embodiment, the cancer associated with high expression of HER3 includes solid tumors or hematological malignancies such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0039] The present invention further provides a pharmaceutically acceptable salt or solvate of any one of the foregoing antibody-drug conjugates.

[0040] The present invention further provides a composition comprising any one of the foregoing antibody-drug conjugates and a pharmaceutically acceptable carrier or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0062] Definition Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used in this specification are intended to refer to techniques commonly understood in the art, including variations of the techniques or substitutions of equivalent techniques that would be apparent to one of ordinary skill in the art. Further, all laboratory procedure steps of genomics, nucleic acid chemistry, and molecular biology used in this specification are routine steps widely used in the corresponding fields. The following terms are considered to be well understood by one of ordinary skill in the art, but the following definitions are provided for a better explanation of the present invention.

[0063] The term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair having a light chain (LC) and a heavy chain (HC)). Antibody light chains can be classified into κ (kappa) and λ (lambda) light chains. Heavy chains can be classified into μ, δ, γ, α or ε heavy chains, and thus the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in the binding of the antibody and the antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site respectively. The amino acid assignment to the regions or domains may follow various numbering systems known in the art.

[0064] The term "antibody" further includes embodiments in which the heavy chain constant domain may contain a C-terminal lysine or may lack either the C-terminal lysine or the C-terminal glycine-lysine dipeptide. The term further includes embodiments in which the N-terminal amino acid of the antibody variable domain has undergone cyclization to pyroglutamic acid. Thus, in the compositions containing the antibodies disclosed herein, the various species of antibodies therein may independently include a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or include cyclization of the N-terminal glutamine or glutamic acid or the N-terminal amino acid to pyroglutamic acid.

[0065] The term "complementary determining region" or "CDR" refers to the amino acid residues in the antibody variable region that are involved in antigen binding. Each of the variable regions of the heavy and light chains contains three CDRs named CDR1, CDR2, and CDR3. The exact boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, one of ordinary skill in the art will be able to readily identify the CDRs defined by each numbering system. Further, the correspondence between different numbering systems is well known to those of ordinary skill in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0066] In the present invention, the CDRs contained in the antibody of the present invention or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In certain embodiments, the CDRs contained in the antibody or its antigen-binding fragment are determined by the Chothia numbering system.

[0067] Disclosed at www.bioinf.org.uk:Prof.Andrew C.R.Martin’s Group, the CDRs in the antibody sequence containing amino acids that specifically interact with the amino acids in the epitope to which the antibody binds can be defined using the following general rules, which are reproduced below. There are rare examples where these generally constant features do not occur, however, the Cys residue is the most conserved feature. [Table 1]

[0068] V H The entire amino acid sequence of generally follows numbering according to Kabat, however, the three CDRs within the variable region can be defined according to any one of the aforementioned numbering schemes. In certain embodiments, the numbering of the amino acid positions in H can start from amino acid position 1 and continue continuously to the end of the sequence, or be continuous according to Kabat. Unless otherwise specified, the amino acid positions in H and L in this specification are defined according to continuous numbering.

[0069] The numbering of the amino acid positions in the heavy chain constant domain can start from amino acid position 1 and continue continuously to the end of the sequence, or be continuous according to Eu numbering. The IgG1 heavy chain constant domain amino acid sequence has 330 amino acids numbered continuously from 1 to 330. The corresponding sequence numbered according to Eu starts at position number 118 and ends at position number 447. Unless otherwise specified, the amino acid positions in the heavy and light chains in this specification are defined according to continuous numbering.

[0070] The term "framework region" or "FR" residue refers to the amino acid residues in the variable region of the antibody other than the CDR residues as defined above.

[0071] The term "antigen-binding fragment" of an antibody refers to a polypeptide of an antibody fragment, e.g., a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen to which the full-length antibody binds and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". Generally, reference is made to Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)), which is hereby incorporated by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced using recombinant DNA techniques or enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, F(ab')3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide bond stabilized Fv protein ("dsFv"), single domain antibodies (sdAb, nanobody), and such polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed by Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0072] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains, the term "dAb fragment" refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341:544 546 (1989)), and the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL, and CH1 domains. 2 The term "F(ab') fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge in the hinge region, and the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in an F(ab')2 fragment, which consists of an intact light chain and a heavy chain Fd fragment (consisting of VH and CH1 domains).

[0073] The term "Fv" refers to an antibody fragment composed of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. Generally, six CDRs are thought to be able to confer antigen-binding specificity to an antibody. However, even a variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind an antigen, although its affinity may be lower than that of a complete binding site.

[0074] The term "Fc" refers to an antibody fragment formed by the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain of an antibody, which are joined via disulfide bonds. The Fc fragment of an antibody has many different functions but is not involved in antigen binding.

[0075] The term "scFv" refers to a single polypeptide chain containing VL and VH domains, where VL and VH are connected by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH. Preferred linkers of the prior art consist of the repetitive GGGGS (SEQ ID NO: 53) amino acid sequence or variants thereof. For example, the amino acid sequence (GGGGS) 4A linker having (Accession No. 54) can be used, and its variants can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al (1999), J. Mol. Biol. 293: 41-56 and Roovers et al (2001), Cancer Immunol. In some cases, a disulfide bond may be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, the scFv may be NH 2 -VH-VH-COOH, NH 2 -VL-VL-COOH.

[0076] The term "single domain antibody (sdAb)" has the meaning generally understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen to which a full-length antibody binds (Holt, L. et al., Trends in Biotechnology, 21(11): 484-490, 2003). Single domain antibodies are also referred to as nanobodies.

[0077] Each of the above antibody fragments maintains the ability to specifically bind to the same antigen to which a full-length antibody binds and / or competes with a full-length antibody for specific binding to an antigen.

[0078] As used herein, unless otherwise clearly indicated, the term "antibody" when referred to includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0079] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods), and can be screened for specificity in the same manner as screening intact antibodies.

[0080] The terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably to refer to one antibody molecule from a group of highly homogeneous antibody molecules or antibody fragments, i.e., a population of antibody molecules that are identical except for naturally occurring mutations that may occur. An mAb is highly specific for a single epitope on an antigen. Polyclonal antibodies are usually relative to monoclonal antibodies that contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Furthermore, the modifier "monoclonal" merely indicates that the antibody is characterized as being obtained from a highly homogeneous antibody population and should not be construed to require that the antibody be prepared by any particular method.

[0081] The term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain is partially derived from an antibody (which may be from a particular species or may belong to a particular antibody class or subclass), and the other part of the light chain and / or heavy chain is derived from another antibody (which may be from the same or a different species or may belong to the same or a different antibody class or subclass), but still retains binding activity to the target antigen. For example, the term "chimeric antibody" can include an antibody whose heavy chain and light chain variable regions are derived from a first antibody (e.g., human) and whose heavy chain and light chain constant regions are derived from a second antibody (e.g., mouse). For example, an antibody produced by immunizing a fully human transgenic mouse can be referred to as a chimeric antibody, which consists of a fully human variable region and a mouse constant region.

[0082] The term "mouse antibody" refers to an antibody obtained by the following method: fusing B cells of an immunized mouse with myeloma cells, selecting mouse hybridoma cells that can proliferate infinitely and secrete antibodies, subsequently screening, preparing the antibodies, and purifying the antibodies, or an antibody secreted by plasma cells formed by the differentiation and proliferation of B cells after an antigen has invaded the mouse body.

[0083] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology to the sequence of a human antibody. Generally, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) is derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain antigen specificity, affinity, reactivity, immunocyte activity (including, but not limited to, the ability to increase reactivity and immunocyte activity, and the ability to enhance the immune response). The donor antibody can be an antibody derived from a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) that has the desired properties (e.g., antigen specificity, affinity, reactivity, the ability to increase immunocyte activity, and / or the ability to enhance the immune response).

[0084] The term "identity" is used to refer to the degree of identity between two polypeptides or between two nucleic acids. If the two sequences for comparison have the same monomer subunits of bases or amino acids at a particular site (e.g., if two DNA molecules each have adenine at a particular site, or if two polypeptides each have lysine at a particular site), the two molecules are identical at that site. The percent homology between two sequences is a function of the number of identical sites shared by the two sequences divided by the total number of sites being compared × 100. For example, if 6 out of 10 sites of two sequences match, these two sequences have 60% identity. For example, the DNA sequences: CTGACT and CAGGTT share 50% identity (3 out of 6 sites match). Generally, the comparison of two sequences is done in a way that yields the maximum identity. Such an alignment can be performed using a computer program such as the Align program (DNAStar, Inc.) based on the method of Needleman et al. (J. Mol. Biol. 48:443 - 453, 1970). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. Appl. Biosci., 4:11 - 17 (1988)), which is incorporated into the ALIGN program (version 2.0) and uses the PAM120 weight residue table, a 12-gap length penalty, and a 4-gap penalty. Further, the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444 - 453 (1970)) incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0085] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide containing an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions are those in which an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having chemical properties such as similar size, shape, charge, ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, valine, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0086] The 20 conventional amino acids included in this specification are expressed in a conventional manner. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0087] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well-known in the art (see, for example, Remington’s Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, etc. Agents for delaying absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid. Stabilizers have the meaning generally understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or their degradation products (such as lactalbumin hydrolysate).

[0088] As used herein, the term "solvate" means a physical association of an ADC disclosed herein with one or more solvent molecules. This physical association includes various degrees of ionic and covalent bonds (including hydrogen bonds). In certain instances, a solvate can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both the solution phase and isolable solvates. Non-limiting examples of solvates include ethanolates, methanolates, and the like. "Hydrate" is a solvate in which the solvent molecule is water.

[0089] One or more of the ADCs disclosed herein can optionally be converted to solvates. The preparation of solvates is generally known. Thus, for example, M. Caira et al., J. Pharmaceutical Sci, 93(3), 601 - 611(2004) describes the preparation of solvates of the antifungal drug fluconazole from ethyl acetate as well as from water. Similar preparations of solvates, hemisolvates, hydrates, etc. are described by E.C. van Tonder et al., AAPS PharmSciTechours, 5(1), article 12(2004); and A.L. Bingham et al., Chem.. Commun., 603 - 604(2001). A typical and non-limiting process involves dissolving a compound of the invention in a desired amount of a desired solvent (organic solvent or water or a mixture thereof) at a temperature higher than room temperature, cooling the solution at a rate sufficient to form crystals, and then isolating the crystals by standard methods. Analytical techniques such as IR spectroscopy indicate the presence of the solvent (or water) in the crystals as a solvate (or hydrate).

[0090] As used herein, the term "pharmaceutically acceptable salt" includes acid addition salts and basic salts.

[0091] Exemplary acid addition salts include acetates, ammonium, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates (also known as mesylates), naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates (also known as tosylates), and the like. Further, acids generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are, for example, those considered in P. Stahl et al., Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. 2 nd Revised Ed. (2011) Zurich: Wiley-VCH; S. Berge et al., Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) -33 201-217, Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York, and The Orange Book (Food & Drug Administration, Washington, D.C. on their website). These disclosures are incorporated herein by reference. In one embodiment, the acidic salt is an ammonium salt or a diammonium salt.

[0092] Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (e.g., organic amines) such as dicyclohexylamine, t-butylamine, choline, and salts with amino acids such as arginine and lysine. The basic nitrogen-containing group can be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, and butyl chloride, bromide, and iodide), dialkyl sulfates (e.g., dimethyl, diethyl, and dibutyl sulfate), long-chain halides (e.g., decyl, lauryl, and stearyl chloride, bromide, and iodide), and aralkyl halides (e.g., benzyl and phenethyl bromide).

[0093] All such acidic and basic salts are intended to be pharmaceutically acceptable salts within the scope of the present disclosure, and all acidic and basic salts are considered to be equivalent to the free form of the corresponding compounds for the purposes of the present disclosure.

[0094] The terms "including," "comprising," "having," "containing," or "involving" and other variations thereof as used herein are inclusive or open-ended and do not exclude other unenumerated elements or method steps.

[0095] The term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, arrest, or delay the occurrence of the disease (such as a tumor), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent an existing disease. The amount of the patient's disease and its complications. The determination of such an effective amount is completely within the ability of one of ordinary skill in the art. For example, the amount effective for therapeutic use depends on factors such as the severity of the disease being treated, the overall condition of the patient's own immune system, the overall condition of the patient such as age, weight, and gender, the mode of administration of the drug, and other treatments administered simultaneously. The therapeutically effective amount of an ADC can vary according to the following factors. The severity of the disease being treated, the overall condition of the patient's own immune system, the overall condition of the patient such as age, weight, and gender, the mode of administration of the drug, and other treatments administered simultaneously.

[0096] The term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the degree of the disease, stabilization of the disease state (i.e., not worsening), delay or deceleration of the progression of the disease, improvement or alleviation of the disease state, and reduction of symptoms (whether partial or complete), whether detectable or undetectable. Further, "treatment" can also refer to extending the survival period compared to the expected survival period without treatment. Treating or treatment can be therapeutic or prophylactic.

[0097] As used herein, the term "subject" refers to a mammal, such as a primate mammal (e.g., a human). In certain embodiments, the subject (e.g., a human) has a tumor or is at risk of having the above-mentioned disease.

[0098] The terms "cancer" and "tumor" are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that can invade adjacent tissues and metastasize to distant sites in the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.

[0099] The term "hematological malignancy" includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as splenic and lymph node neoplasms. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Hematological malignancies also include leukemias such as secondary leukemia or acute lymphoblastic leukemia. Hematological malignancies also include myelomas (e.g., multiple myeloma) and other blood and / or T-cell related cancers. The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched-chain hydrocarbon group such as "C 1~20 alkyl", "C 1~10 alkyl", "C 1~6 alkyl", "C 1~4 alkyl", "C 1~3 alkyl", etc., and specific examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc., but are not limited thereto.

[0100] The term "alkylene" refers to "C 1~20 alkylene", "C 1~10 alkylene", "C 3~10 alkylene", "C 5~8"alkylene", "C 1~6 "alkylene", "C 1~4 "alkylene", "C 1~3 A group obtained by removing two hydrogen atoms from a linear or branched hydrocarbon group such as "alkylene", for example, specific examples thereof include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene, or 1,6-hexylene.

[0101] The term "alkenylene" refers to a divalent group obtained by removing two hydrogen atoms from a linear or branched hydrocarbon group containing at least one carbon-carbon double bond. For example, "C 2~20 alkenylene", "C 3~10 alkenylene", "C 5~8 alkenylene", etc. are included. Examples thereof include, but are not limited to, ethenylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, etc.

[0102] The term "alkynylene" refers to a divalent group obtained by removing two hydrogen atoms from a linear or branched hydrocarbon group containing at least one carbon-carbon triple bond. For example, "C 2~20 alkynylene", "C 3~10 alkynylene", "C 5~8 alkynylene", etc. are included. Examples thereof include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentynylene, 1,4-pentynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, etc.

[0103] The term "heteroaliphatic ring" refers to a saturated or partially saturated cyclic structure containing at least one ring member selected from the group consisting of N, O, and S. Specific examples thereof include 5- to 6-membered heteroaliphatic rings, 5- to 6-membered nitrogen-containing heteroaliphatic rings, 5- to 6-membered oxygen-containing heteroaliphatic rings, etc., such as tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, etc., but are not limited thereto.

[0104] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from the group consisting of N, O, and S. Specific examples thereof include 5- to 6-membered heteroaromatic rings, 5- to 6-membered nitrogen-containing heteroaromatic rings, 5- to 6-membered oxygen-containing heteroaromatic rings, etc., such as furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, 1,2,4,5-tetrazine, etc., but are not limited thereto.

[0105] The term "aromatic ring system" refers to a monocyclic or polycyclic system containing at least one aromatic ring (e.g., benzene ring, etc.) or heteroaromatic ring (e.g., pyrimidine ring, etc.), and two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be connected by a single bond (e.g., bipyrimidinylphenyl, etc.), and the aromatic ring system may be divalent or polyvalent (e.g., trivalent or tetravalent), and may be, for example, a 5- to 20-membered aromatic ring system.

[0106] As used herein, the terms "about" or "approximately" when used in conjunction with a numerical variable generally mean that the value of the variable is within the experimental error range (e.g., within the 95% confidence interval of the mean value) or within a range of ±10% or more.

[0107] It should be noted that if there is a contradiction between the shown structure and the name of the structure, the shown structure is given more weight.

[0108] This application relates to an antibody-drug conjugate (ADC) for the treatment of HER3-positive cancer, having the structure represented by the general formula Ab-[M-L-E-D] x and exemplarily discloses an antibody-drug conjugate using the fully human antibody 22B6D2-hIgG1 as the targeting moiety. The ADC (or conjugate) of the present invention has an average drug-to-antibody ratio (DAR) that can be as high as 7.99 and exhibits a targeting killing effect against HER3-positive cancers such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma). The results herein show that the conjugate has a better drug-to-antibody ratio, the conjugate has excellent binding activity against HER3-positive cells, and has a very good target killing effect against HER3-positive cancers such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma). Therefore, this application provides an antibody-drug conjugate for the treatment of cancer with high expression of HER3, a pharmaceutical composition containing the antibody-drug conjugate, and its application in the treatment of HER3-high expressing cancer. Antibody-drug conjugate

[0109] In one aspect, this application provides an antibody-drug conjugate having the structure represented by the formula Ab-[M-L-E-D] x wherein Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3, also known as Erbb3), M is a binding site that binds to the antibody or an antigen-binding fragment thereof, L is a linker that connects the binding site M and E, E is a fragment that connects L and D, D is a fragment of a cytotoxic drug, x is selected from 1 to 10.

[0110] In some embodiments, the antibody or an antigen-binding fragment thereof (1) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Chothia numbering system: a heavy chain variable region (VH) and / or a light chain variable region (VL): (1a) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the sequence set forth in SEQ ID NO: 1 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 2 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 5 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 6 or a variant thereof, or (1b) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the sequence set forth in SEQ ID NO: 19 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 20 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a variant thereof, or (1c) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 37 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 52 or a variant thereof, including The variant according to any one of items (1a), (1b), and (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, preferably the substitutions are conservative substitutions, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each of the CDRs of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3, or (2) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the Kabat numbering system: (2a) The following three CDRs: a heavy chain variable region (VH) containing CDR-H1 having the sequence set forth in SEQ ID NO: 7 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 8 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 9 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) containing CDR-L1 having the sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 5 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 6 or a variant thereof, or (2b) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the sequence set forth in SEQ ID NO: 25 or a variant thereof, a CDR-H2 having the sequence set forth in SEQ ID NO: 26 or a variant thereof, and a CDR-H3 having the sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the sequence set forth in SEQ ID NO: 22 or a variant thereof, a CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, and a CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a variant thereof, or (2c) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the sequence set forth in SEQ ID NO: 39 or a variant thereof, a CDR-H2 having the sequence set forth in SEQ ID NO: 40 or a variant thereof, and a CDR-H3 having the sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the sequence set forth in SEQ ID NO: 45 or a variant thereof, a CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, and a CDR-L3 having the sequence set forth in SEQ ID NO: 52 or a variant thereof, (2a), (2b), and (2c) wherein the variant described in any of the items has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each CDR of the VH and VL of (2a), (2b), and (2c), and the variant binds to HER3, or (3) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the IMGT numbering system: (3a) The following three CDRs: a VH comprising CDR-H1 having the sequence set forth in SEQ ID NO: 10 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 11 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 12 or a variant thereof, and / or the following three CDRs: a VL comprising CDR-L1 having the sequence set forth in SEQ ID NO: 13 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 14 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 6 or a variant thereof, or (3b) The following three CDRs: a VH comprising CDR-H1 having the sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 28 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 29 or a variant thereof, and / or the following three CDRs: a VL comprising CDR-L1 having the sequence set forth in SEQ ID NO: 30 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 31 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a variant thereof, or (3c) The following three CDRs: a VH comprising CDR-H1 having the sequence set forth in SEQ ID NO: 41 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 42 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 43 or a variant thereof, and / or the following three CDRs: a VL comprising CDR-L1 having the sequence set forth in SEQ ID NO: 44 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 31 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 52 or a variant thereof, (3a), (3b), or (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, preferably the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequences of the respective CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof is (1) a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Chothia numbering system, the heavy chain variable region (VH) and / or the light chain variable region (VL): (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (1b) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (1c) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 36 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 37 or a variant thereof, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, (1a), (1b), and the variant described in any item of (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, or (2) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the Kabat numbering system: (2a) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (2b) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 25 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (2c) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 39 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 40 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, comprising (2a), (2b), or (2c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, or (3) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, where the CDRs are the heavy chain variable region (VH) and / or the light chain variable region (VL) defined by the IMGT numbering system: (3a) The following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof, in the heavy chain variable region (VH), and / or the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, in the light chain variable region (VL), or (3b) The following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29 or a variant thereof, in the heavy chain variable region (VH), and / or the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 30 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, in the light chain variable region (VL), or (3c) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 42 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 43 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 44 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, (3a), (3b), and the variant according to any item of (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution.

[0112] In some embodiments, the antibody or its antigen-binding fragment is (1a) the following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (1b) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (1c) Comprising the following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 37 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, (1a), (1b), and the variant according to any one of the items in (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity when compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) when compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, or (2a) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (2b) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 25 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (2c) Comprising the following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 39 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 40 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, (2a), (2b), or (2c) Any of the variants described in the item, when compared to the sequence from which it is derived, has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably the substitution is a conservative substitution, or (3a) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (3b) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 30 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (3c) The following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 42 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 43 or a variant thereof, and / or the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 44 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, and a variable light chain region (VL) containing the same, (3a), (3b), and the variant according to any one of the items of (3c), compared to the sequence from which it is derived, has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity, or the variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof is (1a) The following three CDRs: a variable heavy chain region (VH) containing CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3, and / or the following three CDRs: a variable light chain region (VL) containing CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6, or (1b) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 19, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24, or (1c) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 36, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 37, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52, Or (2a) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 7, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 8, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 9, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6, or (2b) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 25, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24, or (2c) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 39, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 40, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52, or (3a) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 10, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 11, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 12, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 13, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 14, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6, or (3b) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 30, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24, or (3c) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 41, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 42, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 43, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 44, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52, are included.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 19, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 20, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 36, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 37, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 7, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 8, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 9; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 25, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0119] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 39, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 40, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52.

[0120] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 10, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 11, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 12, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 13, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 14, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 30, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0122] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 41, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 42, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 43, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 44, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof (a) VH set forth in SEQ ID NO: 15 or a variant thereof, and / or VL set forth in SEQ ID NO: 16 or a variant thereof, (b) the VH or its variant described in SEQ ID NO: 32, and / or the VL or its variant described in SEQ ID NO: 33, or (c) the VH or its variant described in SEQ ID NO: 46, and / or the VL or its variant described in SEQ ID NO: 47, and the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each of the CDRs of the VH and VL in (a), (b), and (c), and the variant binds to HER3.

[0124] In some embodiments, the antibody or its antigen-binding fragment (a) a VH comprising the amino acid sequence described in SEQ ID NO: 15 or its variant, and a VL comprising the amino acid sequence described in SEQ ID NO: 16 or its variant, (b) a VH comprising the amino acid sequence described in SEQ ID NO: 32 or its variant, and a VL comprising the amino acid sequence described in SEQ ID NO: 33 or its variant, or (c) a VH comprising the amino acid sequence described in SEQ ID NO: 46 or its variant, and a VL comprising the amino acid sequence described in SEQ ID NO: 47 or its variant, and The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., one, two, three, four or five amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, and preferably the substitutions are conservative substitutions.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof (a) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16, (b) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33, or (c) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 47.

[0126] In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof comprises (b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof comprises (c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 47.

[0129] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 described for VH comprising the amino acid sequence of SEQ ID NO: 15, respectively, and a light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 described for VL comprising the amino acid sequence of SEQ ID NO: 16, respectively.

[0130] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 described for VH comprising the amino acid sequence of SEQ ID NO: 32, respectively, and a light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 described for VL comprising the amino acid sequence of SEQ ID NO: 33, respectively.

[0131] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 described for VH comprising the amino acid sequence of SEQ ID NO: 46, respectively, and a light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 described for VL comprising the amino acid sequence of SEQ ID NO: 47, respectively.

[0132] In some embodiments, the antibody or antigen-binding fragment thereof (a) A human immunoglobulin heavy chain constant region (CH) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions, e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived, a human immunoglobulin heavy chain constant region (CH) or a variant thereof, and (b) A human immunoglobulin light chain constant region (CL) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions, e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived, further comprising a human immunoglobulin light chain constant region (CL) or a variant thereof.

[0133] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, e.g., a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) set forth in SEQ ID NO: 50 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions, e.g., 1, 2, 3, 4 or 5 conservative amino acid substitutions) compared to SEQ ID NO: 50.

[0134] In some embodiments, the light chain constant region comprises a κ light chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) set forth in SEQ ID NO: 51 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions, e.g., 1, 2, 3, 4 or 5 conservative amino acid substitutions) compared to SEQ ID NO: 51.

[0135] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 50 and a light chain constant region (CL) set forth in SEQ ID NO: 51.

[0136] In some embodiments, the antibody or antigen-binding fragment thereof (1) a heavy chain comprising a VH set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) set forth in SEQ ID NO: 50, and a light chain comprising a VL set forth in SEQ ID NO: 16 and a light chain constant region (CL) set forth in SEQ ID NO: 51, (2) a heavy chain comprising a VH set forth in SEQ ID NO: 32 and a heavy chain constant region (CH) set forth in SEQ ID NO: 50, and a light chain comprising a VL set forth in SEQ ID NO: 33 and a light chain constant region (CL) set forth in SEQ ID NO: 51, or (3) a heavy chain comprising a VH set forth in SEQ ID NO: 46 and a heavy chain constant region (CH) set forth in SEQ ID NO: 50, and a light chain comprising a VL set forth in SEQ ID NO: 47 and a light chain constant region (CL) set forth in SEQ ID NO: 51.

[0137] In some embodiments, the antibody or antigen-binding fragment thereof comprises (1) a heavy chain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 50, and a light chain comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 51.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof comprises (2) a heavy chain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 50, and a light chain comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 33 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 51, or

[0139] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 50, and a light chain comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 47 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 51.

[0140] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0141] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 34, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0142] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 49.

[0143] In certain embodiments of the antibody, the heavy chain constant domain disclosed herein may comprise a C-terminal lysine, or may lack either the C-terminal lysine or the C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the variable domain of the antibody or antigen-binding fragment may undergo cyclization to pyroglutamic acid. Thus, in a composition comprising a particular antibody disclosed herein, the composition may comprise a population of antibody species, each of which may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or may comprise cyclization of the N-terminal glutamine or glutamic acid or the N-terminal amino acid to pyroglutamic acid.

[0144] In an antibody-drug conjugate, the cytotoxic drug may be conjugated to the antibody or antigen-binding fragment via a linker (e.g., the "M-L-E" fragment of the present application).

[0145] In some embodiments, M is

Chemical formula

[0146] In some embodiments, M is

Chemical formula

[0147] In some embodiments, M is

Chemical formula

Chemical formula

[0148] In some embodiments, M has the following structure:

Chemical formula

[0149] In some embodiments, M has the following structure:

Chemical formula

[0150] In some embodiments, L is the following: C 1~6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) s OCH 3 )、D-Val、Leu、Gly、Ala、Asn、Val-Cit、Val-Ala、Val-Lys、Val-Lys(Ac)、Phe-Lys、Phe-Lys(Ac)、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn、Ala-Ala-Ala、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Gly-Gly-Gly,

Chemical formula

[0151] In some embodiments, L is the following: C 1~6Alkylene, -NH-, Phe, Lys, Lys(COCH 2 CH 2 (OCH 2 CH 2 )sOCH 3 )、Gly, Gly-Gly-Phe-Gly,

Chem.

[0152] In some embodiments, L has the following structure:

Chem.

[0153] In some embodiments, L has the following structure:

Chem.

[0154] In some embodiments, L has the following structure:

Chem.

[0155] In some embodiments, E is a single bond, -NH-CH 2 -, -NH-CH 2 -O-CH 2 -CO-, or has the following structure:

Chem.

[0156] In some embodiments, E is a single bond, -NH-CH 2 -, -NH-CH 2 -O-CH2 -CO-、

Chem.

[0157] In some embodiments, E is -NH-CH 2 -O-CH 2 -CO-、

Chem.

[0158] In some embodiments, E is -NH-CH 2 -O-CH 2 -CO- or

Chem.

[0159] In some embodiments, M is selected from the following structures:

Chem.

Chem.

Chem.

[0160] In some embodiments,

Chem.

Chem.

[0161] In some embodiments, [Chemistry] has the following structure: [Chemistry] is selected from

[0162] In some embodiments, the cytotoxic drug is selected from the group consisting of tubulin inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors. In some embodiments, the tubulin inhibitor is an auristatin compound or a maytansine compound. In some embodiments, the DNA intercalator is pyrrolobenzodiazepine (PBD). In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, velotecan, or rubitecan) and a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof.

[0163] The cytotoxic drugs disclosed in the present application usually have hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amine (-NH 2 ), secondary amine group (-NR A H), or tertiary amine group (-NR B R C) containing various functional groups such as, and R in the present specification A , R B , and R C represent only non-hydrogen substituents on N, and the cytotoxic drug can be connected to the linker in the complex through these functional groups.

[0164] In some embodiments, the cytotoxic drug is linked to E in the antibody-drug conjugate through -OH, -SH, a primary amino group, a secondary amine group, or a tertiary amine group.

[0165] In some embodiments, the cytotoxic drug is selected from the following Formulas I and II, [Chemical formula] R 1 and R 2 are each independently selected from the group consisting of C 1~6 alkyl and halogen, R 3 is selected from the group consisting of H and -CO-CH 2 OH, R 4 and R 5 are each independently selected from the group consisting of H, halogen, and hydroxyl, or R 4 and R 5 together with the carbon atom to which they are attached form a 5- to 6-membered oxygen-containing heterocycle, R 6 is selected from the group consisting of hydrogen and -C 1~4 alkylene-NR a R b R 7 is selected from the group consisting of C 1~6 alkyl and -C 1~4 alkylene-NR a R b In each occurrence, R a and R b are each independently H, C 1~6 alkyl, -SO 2 -C 1~6Alkyl and -CO-C 1~6 is selected from the group consisting of alkyls.

[0166] In some embodiments, the cytotoxic drug is one of the following compounds:

Chemical formula

[0167] In some embodiments, the cytotoxic drug is one of the following compounds:

Chemical formula

[0168] The corresponding fragment of the cytotoxic drug after being connected to the linker is D in the formula Ab-[M-L-E-D] x In some embodiments, D is a monovalent structure obtained by removing one H from -OH, -NH 2 or a secondary amine group on the cytotoxic drug.

[0169] In some embodiments, D has the following structure:

Chemical formula

[0170] In some embodiments, the antibody-drug conjugate is ADC A-01 to ADC A-25, ADC B-01 to ADC B-05 shown below:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0171] In some embodiments, the antibody-drug conjugate is [Chemical formula] [Chemical formula] selected from wherein x is 3-8, or 3-4, or 7-8, and HA in each antibody-drug conjugate is (1) a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Chothia numbering system, the heavy chain variable region (VH) and / or the light chain variable region (VL): (1a) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (1b) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (1c) Comprising the following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 37 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, (1a), (1b), or (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably the substitutions are conservative substitutions, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each of the CDRs of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3, or (2) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Kabat numbering system for the heavy chain variable region (VH) and / or the light chain variable region (VL): (2a) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (2b) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 25 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26 or a variant thereof, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (2c) The following three CDRs: a heavy chain variable region (VH) comprising a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 39 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 40 or a variant thereof, and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, (1a), (1b), and the variant according to any one of the items of (1c), compared to the sequence from which it is derived, has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each CDR of VH and VL in (2a), (2b), and (2c), and the variant binds to HER3, or (3) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the IMGT numbering system: (3a) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof, and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (3b) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29 or a variant thereof, and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 30 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (3c) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 42 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 43 or a variant thereof, and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 44 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, selected from an antibody or an antigen-binding fragment thereof that includes (1a), (1b), or (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably the substitutions are conservative substitutions, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each of the CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

[0172] In a further embodiment, the HA in each antibody-drug conjugate is (a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof, and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof, (b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 or a variant thereof, and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 33 or a variant thereof, or (c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 or a variant thereof, and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 47 or a variant thereof, and is selected from an antibody or an antigen-binding fragment thereof comprising (1a), (1b), or the variant according to any item of (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100%, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequences of the respective CDRs of VH and VL of (a), (b), and (c), and the variant binds to HER3.

[0173] In a further embodiment, the HA in each antibody-drug conjugate is (a) VH comprising the amino acid sequence set forth in SEQ ID NO: 15, and VL comprising the amino acid sequence set forth in SEQ ID NO: 16, (b) VH comprising the amino acid sequence set forth in SEQ ID NO: 32, and VL comprising the amino acid sequence set forth in SEQ ID NO: 33, or (c) VH comprising the amino acid sequence set forth in SEQ ID NO: 46, and VL comprising the amino acid sequence set forth in SEQ ID NO: 47, and is selected from an antibody or an antigen-binding fragment thereof.

[0174] In some embodiments, the antibody-drug conjugate is

Chemical formula

Chemical formula

[0175] In some embodiments, the antibody-drug conjugate is [Chemical formula] wherein HA in each antibody-drug conjugate is (1) An antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 15, a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, a VL comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, (2) An antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 32, a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, a VL comprising the amino acid sequence set forth in SEQ ID NO: 33, and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, and (3) An antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 47, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 46, a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, a VL comprising the amino acid sequence set forth in SEQ ID NO: 47, and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, and is selected from an antibody or an antigen-binding fragment thereof comprising

[0176] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0177] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0178] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0179] In some embodiments, the antibody-drug conjugate is selected from the following

Chemical formula

[0180] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0181] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0182] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0183] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0184] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0185] In some embodiments, the antibody-drug conjugate is [Chemical formula] and HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 47, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, and an antibody or an antigen-binding fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 47 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51.

[0186] In some embodiments, the antibody-drug conjugate is [Chemical formula] and HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 18, and x is from 7 to 8.

[0187] In some embodiments, the antibody-drug conjugate is [Chemical formula] and HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 35, and x is from 7 to 8.

[0188] In some embodiments, the antibody-drug conjugate is [Chemical formula] and HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 48 and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 49, and x is from 7 to 8.

[0189] In some embodiments, the antibody-drug conjugate is

Chem.

[0190] In some embodiments, the antibody-drug conjugate is

Chem.

[0191] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0192] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0193] In some embodiments, the antibody-drug conjugate is [Chem.] and HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 18, and x is from 7 to 8.

[0194] In some embodiments, the antibody-drug conjugate is [Chem.] and HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 35, and x is from 7 to 8.

[0195] In some embodiments, the antibody-drug conjugate is [Chem.] and HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 48 and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 49, and x is from 7 to 8.

[0196] In some embodiments, the antibody-drug conjugate is [Chem.] and HA in each antibody-drug conjugate is (1) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, (2) An antibody or an antigen-binding fragment thereof comprising a VH containing the amino acid sequence set forth in SEQ ID NO: 32 and a VL containing the amino acid sequence set forth in SEQ ID NO: 33, for example, an antibody or an antigen-binding fragment thereof comprising a VH containing the amino acid sequence set forth in SEQ ID NO: 32, a CH containing the amino acid sequence set forth in SEQ ID NO: 50, a VL containing the amino acid sequence set forth in SEQ ID NO: 33, and a CL containing the amino acid sequence set forth in SEQ ID NO: 51, and (3) An antibody or an antigen-binding fragment thereof comprising a VH containing the amino acid sequence set forth in SEQ ID NO: 46 and a VL containing the amino acid sequence set forth in SEQ ID NO: 47, for example, an antibody or an antigen-binding fragment thereof comprising a VH containing the amino acid sequence set forth in SEQ ID NO: 46, a CH containing the amino acid sequence set forth in SEQ ID NO: 50, a VL containing the amino acid sequence set forth in SEQ ID NO: 47, and a CL containing the amino acid sequence set forth in SEQ ID NO: 51, and is selected from an antibody or an antigen-binding fragment thereof comprising

[0197] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0198] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0199] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0200] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0201] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0202] In some embodiments, the antibody-drug conjugate is

Chemical formula

[0203] In some embodiments, the antibody-drug conjugate (ADC) has an x of from 1 to 10, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10.

[0204] In some embodiments, the antibody-drug conjugate (ADC) has an x of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0205] In some embodiments, the antibodies disclosed herein are genetically engineered to contain one or more cysteine or non-standard amino acid substitutions at defined positions within the antibody. These cysteine or non-standard amino acid residues can then be conjugated to a drug-linker via the sulfhydryl group of the cysteine residue or the reactive group of the non-standard amino acid. Thus, the antibody-drug conjugates of the invention can contain one or more substitutions of amino acids in the heavy or light chains of the antibody by cysteine or non-standard amino acid residues, which are then conjugated to the drug-linkers disclosed herein. In certain embodiments, the amino acid positions that can be substituted are selected from positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain (numbering according to the Eu numbering scheme) and positions 165 and 168 of the light chain constant domain (numbering starting from amino acid 1 at the N-terminus). In certain embodiments, cysteine can be substituted for one or more of the amino acids at positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain (numbering according to the Eu numbering scheme) and positions 165 and 168 of the light chain constant domain (numbering starting from amino acid 1 at the N-terminus). In certain embodiments, the antibody-drug conjugate contains the S375C amino acid substitution that is conjugated to the drug-linker disclosed herein. In certain embodiments, the antibody contains the S375C amino acid substitution and the E152C amino acid substitution, each of which is conjugated to the drug-linker disclosed herein. In certain embodiments, the antibody contains the S375C amino acid substitution and the S168C amino acid substitution, each of which is conjugated to the drug-linker disclosed herein.In some embodiments, as described herein, the composition of the ADC has a DAR (drug-to-antibody ratio) value of 1 to 10, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10, preferably 3 to 9, such as 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 to 5.5, 3.5 to 6.0, 3.5 to 6.5, 3.5 to 7.0, 3.5 to 7.5, 3.5 to 8.0, 4.0 to 4.5, 4.0 to 5.0, 4.0 to 5.5, 4.0 to 6.0, 4.0 to 6.5, 4.0 to 7.0, 4.0 to 7.5, 4.0 to 8.0, 4.5 to 5.0, 4.5 to 5.5, 4.5 to 6.0, 4.5 to 6.5, 4.5 to 7.0, 4.5 to 7.5, 4.5 to 8.0, 5.0 to 5.5, 5.0 to 6.0, 5.0 to 6.5, 5.0 to 7.0, 5.0 to 7.5, 5.0 to 8.0, 5.5 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 5.5 to 7.5, 5.5 to 8.0, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.5, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.5, 7.0 to 7.5, 7.0 to 9.0 or 7.5 to 9.0.

[0206] Drug-linker One of ordinary skill in the art should understand that the antibody-drug conjugate (ADC) described in this application can be prepared modularly. For example, the free form of the "drug-linker" (which can be understood as M'-L-E-D, where M' is the structure of M before being covalently bound to the antibody or its antigen-binding fragment) can be obtained first, and then covalently bound to the antibody or its antigen-binding fragment to obtain the antibody-drug conjugate of this application. Correspondingly, one or more sulfhydryl (-SH), amino (-NH 2) Or a carboxyl (-COOH) group is connected to M' of the "drug-linker" in free form by a substitution reaction (e.g., by cleavage of a group such as -SO 2 Me or -Br) or an addition reaction.

[0207] In another aspect, the present invention provides a drug-linker having a structure represented by the formula M'-L-E-D, where M' is

Chemical formula

Chemical formula

[0208] In some embodiments, M' is

Chemical formula

[0209] In some embodiments, M' is

Chemical formula

Chemical formula

[0210] In some embodiments, M' is

Chemical formula

[0211] In some embodiments, M' is

Chemical formula

[0212] In some embodiments, M' is

Chemical formula

Chem.

Chem.

[0213] In some embodiments, the free form of the "drug-linker" is selected from A-01 to A-25, B-01 to B-05 shown below:

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0214] The present invention further provides an ADC comprising an antibody that binds to HER3 and is conjugated to a drug-linker selected from the group consisting of A-01, A-02, A-03, A-04, A-05, A-06, A-07, A-08, A-09, A-10, A-12, A13, A-14, A15, A-16, A-17, A-18, A-19, A-20, A-21, A-22, A-23, A-24, A-25, B-01, B-02, B-03, B-04, and B-05 via a cysteine residue.

[0215] In a further embodiment of the ADC, the antibody is (1) a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Chothia numbering system: a heavy chain variable region (VH) and / or a light chain variable region (VL): (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acids set forth in SEQ ID NO: 1 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 37 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, or (2) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the Kabat numbering system: (2a) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof, in a heavy chain variable region (VH), and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, in a light chain variable region (VL), or (2b) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 25 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, in a heavy chain variable region (VH), and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, in a light chain variable region (VL), or (2c) The following three CDRs: a CDR-H1 having the sequence set forth in SEQ ID NO: 39 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 40 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, in a heavy chain variable region (VH), and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, in a light chain variable region (VL), or (3) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the IMGT numbering system: (3a) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof, a heavy chain variable region (VH) comprising the same, and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, a light chain variable region (VL) comprising the same, or (3b) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29 or a variant thereof, a heavy chain variable region (VH) comprising the same, and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 30 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, a light chain variable region (VL) comprising the same, or (3c) An antibody or antigen-binding fragment selected from the group consisting of the following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 42 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 43 or a variant thereof, a heavy chain variable region (VH) comprising the same, and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 44 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, a light chain variable region (VL) comprising the same.

[0216] In a further embodiment of the ADC, the antibody or antigen-binding fragment is (a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof, and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof, (b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 or a variant thereof, and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 33 or a variant thereof, or (c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 or a variant thereof, and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 47 or a variant thereof, and is selected from the group consisting of.

[0217] In a further embodiment of the ADC, the antibody or antigen-binding fragment is (1) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, and an antibody or an antigen-binding fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, (2) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, and an antibody or an antigen-binding fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 33 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, and (3) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 47, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, and an antibody or an antigen-binding fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 47 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, and is selected from the group consisting of.

[0218] In a further embodiment of the ADC, the antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16, for example, a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, in an antibody or antigen-binding fragment.

[0219] In a further embodiment of the ADC, the antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33, for example, a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 33 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, in an antibody or antigen-binding fragment.

[0220] In a further embodiment of the ADC, the antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 47, for example, a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 47 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, in an antibody or antigen-binding fragment.

[0221] In a further embodiment, the antibody comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0222] In a further embodiment, the antibody comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 34, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0223] In a further embodiment, the antibody comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 49.

[0224] Antibody Preparation The antibodies described herein can be prepared by various methods known in the art, for example, by genetic engineering and recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. After inserting the obtained DNA molecules into an expression vector, the host cells are transfected. Then, the transfected host cells are cultured under specific conditions to express the antibodies of the present invention.

[0225] Binding In another aspect, the present application provides a method for binding a drug-linker described herein to an antibody described herein to produce an antibody-drug conjugate (ADC) described herein.

[0226] In certain embodiments, the antibodies described herein are bound to a drug-linker described herein via binding to lysine in the antibody.

[0227] In certain embodiments, the antibodies described herein are bound to a drug-linker described herein via binding to cysteine in the antibody. In certain embodiments, the cysteine is derived from a reduced intra-chain disulfide bond in the antibody. In certain embodiments, the cysteine is derived from a reduced inter-chain disulfide bond in the antibody.

[0228] In some embodiments, the antibody is conjugated to a drug-linker via binding to reduced inter-chain disulfide bonds in the antibody. For example, an IgG1 antibody comprises two heavy chains, each comprising four polypeptide chains, VH, CH1 and Fc (e.g., hinge, CH2 and CH3) domains, and two light chains, each comprising VL and CL domains connected by inter-chain cysteine disulfide (-S-S-) bonds (e.g., two heavy chain-light chain inter-chain disulfide bonds and two hinge heavy chain-heavy chain inter-chain disulfide bonds). In certain embodiments, when these disulfide bonds are cleaved under reducing conditions, eight reactive cysteine sulfhydryl moieties are generated. In certain embodiments, each of the eight reactive cysteine sulfhydryl moieties is a binding site for a linker-drug such that up to eight (x = 8) drug-linkers can be conjugated to the reduced antibody. In certain embodiments, any one of the four disulfide bonds is cleaved under reducing conditions, generating two reactive cysteine sulfhydryl moieties. In a further embodiment, each of the two reactive cysteine sulfhydryl moieties is a binding site for a linker-drug such that two (x = 2) linker-drugs can be conjugated to the reduced antibody. In certain embodiments, any two of the four disulfide bonds are cleaved under reducing conditions, generating four reactive cysteine sulfhydryl moieties. In a further embodiment, each of the four reactive cysteine sulfhydryl moieties is a binding site for a linker-drug such that four (x = 4) linker-drugs can be conjugated to the reduced antibody. In certain embodiments, any three of the four disulfide bonds are cleaved under reducing conditions, generating six reactive cysteine sulfhydryl moieties. In a further embodiment, each of the six reactive cysteine sulfhydryl moieties is a binding site for a linker-drug such that six (x = 6) linker-drugs can be conjugated to the reduced antibody.

[0229] In some embodiments, the inter-chain disulfide bond is between two cysteine residues that are cleaved under reducing conditions, resulting in two reactive cysteine sulfhydryl moieties. In further embodiments, the inter-chain disulfide bridge in an antibody is between a heavy chain and a light chain, for example, between C220 of the heavy chain by EU numbering and C214 of the kappa light chain, or between C220 of the heavy chain by EU numbering and C214 of the lambda light chain by Kabat numbering. Further, or alternatively, the inter-chain disulfide bridge in an antibody is between two heavy chains, for example, between C226 and / or C229 of the first heavy chain by EU numbering and C226 and / or C229 of the second heavy chain by EU numbering. In some embodiments, the cysteine residue is in the hinge region of the antibody. In some embodiments, the cysteine residue is at any one or more of positions 220, 226, or 229 (also referred to herein as C220, C226, or C229, respectively) in the heavy chain by EU numbering. In some embodiments, the cysteine residue is at position 214 (also referred to herein as C214, for example, position 214 in the kappa light chain by EU and Kabat numbering or position 214 in the lambda light chain by Kabat numbering) in the light chain by EU and / or Kabat numbering. In one embodiment, the cysteine residue is at each of positions 220, 226, and 229 of the heavy chain by EU numbering and position 214 of the light chain by EU or Kabat numbering. In one embodiment, the cysteine residue is at each of positions 220, 226, and 229 of the heavy chain by EU numbering and position 214 of the kappa light chain by EU and Kabat numbering. In one embodiment, the cysteine residue is at each of positions 220, 226, and 229 of the heavy chain by EU numbering and position 214 of the lambda light chain by Kabat numbering. In one embodiment, the cysteine residue is at any one or more of the following positions. (i) Any one, or any two, or any three, or all four of positions 220, 226, and 229 within the first heavy chain by EU numbering, (ii) Any one, or any two, or any three, or all four of positions 220, 226, and 229 within the second heavy chain by EU numbering, (iii) Position 214 of the first light chain by Kabat numbering, and / or (iv) Position 214 of the second light chain by Kabat numbering.

[0230] As used herein, C220, C226, and C229 refer to the amino acid residues (cysteine, Cys, C) of an immunoglobulin identified according to EU numbering. Thus, as will be understood by those skilled in the art, such numbering represents the amino acid residues of a polypeptide aligned with those identified in an immunoglobulin, such as that shown at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0231] As used herein, the cysteine residue at position 214 of the kappa light chain refers to the amino acid residue (cysteine, Cys, C) of an immunoglobulin identified according to Kabat numbering. Thus, as will be understood by those skilled in the art, such numbering represents the amino acid residues of a polypeptide aligned with those identified in an immunoglobulin, such as that shown at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html.

[0232] As used herein, the cysteine residue at position 214 of the lambda light chain refers to the amino acid residue (cysteine, Cys, C) of an immunoglobulin identified according to Kabat numbering. Thus, as will be understood by those skilled in the art, such numbering represents the amino acid residues of a polypeptide aligned with those identified in an immunoglobulin, such as that shown at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGLCnber.html.

[0233] In certain embodiments, the antibodies described herein contain four interchain disulfide bonds in the hinge region, which can be reduced, thereby cleaving the bonds and exposing reactive sulfhydryl moieties that can bind to maleimide moieties on drug-linkers such as those on the drug-linkers described herein.

[0234] In one embodiment, the present disclosure is a method of making an ADC described herein, for making an ADC, a) providing a solution comprising an antibody, b) contacting the solution of a) with a reducing agent, c) contacting the solution of b) with a solution comprising a drug-linker or a salt thereof described herein, and provides a method comprising the steps.

[0235] In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0236] Composition In another aspect, the present application provides a composition of an antibody-drug conjugate (ADC) described herein. Such a composition may comprise a plurality of ADCs described herein, each ADC comprising a drug-linker described herein, and x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In other words, each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drug-linkers. Thus, the composition can be characterized by a "drug-to-antibody" ratio (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art and include methods using reverse phase chromatography or HPLC-MS.

[0237] For example, in any embodiment, the ADC compositions described herein have a DAR of from about 1 to about 10, or any sub-range therebetween, such as from about 1 to 2, from about 1 to 3, from about 1 to 4, from about 1 to 5, from about 1 to 6, from about 1 to 7, from about 1 to 8, from about 1 to 9, from about 1 to 10, from about 2 to 3, from about 2 to 4, from about 2 to 5, from about 2 to 6, from about 2 to 7, from about 2 to 8, from about 2 to 9, from about 2 to 10, from about 3 to 4, from about 3 to 5, from about 3 to 6, from about 3 to 7, from about 3 to 8, from about 3 to 9, from about 3 to 10, from about 4 to 5, from about 4 to 6, from about 4 to 7, from about 4 to 8, from about 4 to 9, from about 4 to 10, from about 5 to 6, from about 5 to 7, from about 5 to 8, from about 5 to 9, from about 5 to 10, from about 6 to 7, from about 6 to 8, from about 6 to 9, from about 6 to 10, from about 7 to 8, from about 7 to 9, from about 7 to 10, from about 8 to 9, from about 8 to 10, or from about 9 to 10.

[0238] In certain embodiments, the ADC compositions described herein have a DAR of from about 3 to 9, such as from about 3.0 to 3.5, from about 3.0 to 4.0, from about 3.0 to 4.5, from about 3.0 to 5.0, from about 3.0 to 5.5, from about 3.0 to 6.0, from about 3.5 to 4.0, from about 3.5 to 4.5, from about 3.5 to 5.0, from about 3.5 to 5.5, from about 3.5 to 6.0, from about 3.5 to 6.5, from about 3.5 to 7.0, from about 3.5 to 7.5, from about 3.5 to 8.0, from about 4.0 to 4.5, from about 4.0 to 5.0, from about 4.0 to 5.5, from about 4.0 to 6.0, from about 4.0 to 6.5, from about 4.0 to 7.0, from about 4.0 to 7.5, from about 4.0 to 8.0, from about 4.5 to 5.0, from about 4.5 to 5.5, from about 4.5 to 6.0, from about 4.5 to 6.5, from about 4.5 to 7.0, from about 4.5 to 7.5, from about 4.5 to 8.0, from about 5.0 to 5.5, from about 5.0 to 6.0, from about 5.0 to 6.5, from about 5.0 to 7.0, from about 5.0 to 7.5, from about 5.0 to 8.0, from about 5.5 to 6.0, from about 5.5 to 6.5, from about 5.5 to 7.0, from about 5.5 to 7.5, from about 5.5 to 8.0, from about 6.0 to 6.5, from about 6.0 to 7.0, from about 6.0 to 7.5, from about 6.0 to 8.5, from about 6.5 to 7.0, from about 6.5 to 7.5, from about 6.5 to 8.5, from about 7.0 to 7.5, from about 7.0 to 9.0 or from about 7.5 to 9.0.

[0239] Pharmaceutical composition In another aspect, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate (ADC) described in any one of the foregoing embodiments, optionally a drug-linker described in any one of the foregoing embodiments, and one or more pharmaceutically acceptable adjuvants.

[0240] Examples of pharmaceutically acceptable adjuvants include, for example, pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable adjuvants further include salts and solvates.

[0241] The ADCs described herein are typically formulated with a pharmaceutically acceptable parenteral vehicle to form a unit injectable form for parenteral administration such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody-drug conjugate having the desired purity is mixed in the form of a lyophilizate or solution with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer (Remington’s Pharmaceutical Sciences (1980) 16 th edition, Osol, A. Ed.). The antibody-drug conjugates described herein, or pharmaceutical compositions comprising antibody-drug conjugates, can be administered via any route suitable for the individual being treated.

[0242] The ADCs and pharmaceutical compositions described herein can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention must be sterile and stable under the conditions of manufacture and storage. The preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared as follows: The required dose of the antibody of the present invention is incorporated into a suitable solvent and optionally other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, osmotic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, a sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) to facilitate storage and use. Such a sterile lyophilized powder can be dispersed in a suitable carrier (e.g., sterile water free of pyrogens) before use.

[0243] Furthermore, the ADCs described herein may be present in unit dosage form in a pharmaceutical composition to facilitate administration by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, local (e.g., powder, ointment, or drops), or nasal routes. However, in many therapeutic applications, the preferred route / mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or mode of administration will vary depending on the intended purpose. In some preferred embodiments, the ADCs and pharmaceutical compositions described herein are administered by intravenous infusion or injection.

[0244] In certain embodiments, the pharmaceutical composition may further comprise an additional active pharmaceutical agent. In certain embodiments, the additional pharmaceutically active agent is a drug having antitumor activity. In certain embodiments, the additional pharmaceutically active agent is selected from the group consisting of an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic agent, or any combination thereof. In certain embodiments, the ADCs and additional pharmaceutically active agents described herein are provided as separate components or as combined components. Accordingly, the antibodies or antigen-binding fragments thereof and additional pharmaceutically active agents of the invention can be administered simultaneously, separately, or sequentially.

[0245] Method of Use The antibody-drug conjugates, drug-linkers, or pharmaceutical compositions thereof described herein can be used to treat various diseases or conditions, such as solid tumors or hematological malignancies including colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma, such as cancers associated with high expression of HER3.

[0246] Accordingly, the present application provides the use of an antibody-drug conjugate (ADC), a drug-linker, or a pharmaceutical composition containing them, as described in any one of the foregoing embodiments, in the manufacture of a medicament for the treatment of cancers associated with high expression of HER3.

[0247] At the same time, the present application also provides a method for treating cancers associated with high expression of HER3, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate (ADC), a drug-linker, or a pharmaceutical composition containing them, as described in any one of the foregoing embodiments.

[0248] In certain embodiments, the antibody-drug conjugate (ADC), the drug-linker, or the pharmaceutical composition (e.g., in a subject) (1) inhibits the growth of cells (such as tumor cells), (2) Inhibition of tumor growth, (3) Induction and / or increase of antibody-dependent cellular cytotoxicity activity, (4) Inhibition of HER3-mediated signal transduction, (5) Prevention and / or treatment of HER3-mediated diseases / disorders, or (6) Sufficient in any combination of the above (1) to (5).

[0249] In certain embodiments, the HER3-mediated disease / disorder is a tumor, such as a HER3-expressing tumor. In certain embodiments, the tumor is selected from breast cancer, gastric cancer, lung cancer (such as non-small cell lung cancer), colorectal cancer, pancreatic cancer, squamous cell carcinoma of the head and neck, melanoma, ovarian cancer, prostate cancer, liver cancer, kidney cancer, bladder cancer, or any combination thereof.

Examples

[0250] The present invention will be further described below by way of examples, but the present invention is not limited to these examples. Those skilled in the art can make various modifications or improvements in accordance with the teachings of the present invention without departing from the principles and scope of the present invention.

[0251] Information on the sequences included in the present invention is described in Tables A and B below.

Table 2

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0252] The abbreviations used in this specification have the following meanings as shown in Table C below.

Table 4

[0253] The structures of the compounds described in the following examples were confirmed by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0254] Nuclear magnetic resonance ( 1 H-NMR) measurements were performed using a Bruker 400 MHz nuclear magnetic resonance spectrometer. The deuterated reagent was hexadeuterodimethyl sulfoxide (DMSO-d6), and the internal standard substance was tetramethylsilane (TMS).

[0255] The abbreviations of the nuclear magnetic resonance (NMR) spectra used in the examples are shown below. s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: hexadeuterodimethyl sulfoxide. The δ values were expressed in ppm. The mass spectrum (MS) was determined using an Agilent (ESI) mass spectrometer Agilent 6120B.

[0256] Example 1: N-((S)-10-Benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1-H-pyrrol-1-yl)hexanamide (A-01)

Chem.

[0257] Compound A-01-1 (0.40 g, 640.59 μmol, the synthesis of which can refer to Chinese Patent Application Publication No. 111936169A) and exatecan mesylate (0.37 g, 704.65 μmol) were dissolved in DMF (8 mL), HATU (0.32 g, 832.77 μmol) and DIPEA (0.25 g, 1.92 mmol) were added, and the reaction was carried out at 25 °C for 4 hours. DIPEA was removed under reduced pressure, and freeze-dried with water to remove most of the DMF to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography under the following conditions to obtain 273 mg of the title compound.

[0258] Chromatography column: Waters XBridge Prep C18 OBD 45mm×450mm×8.0μm

[0259] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% trifluoroacetic acid)

Table 5

[0260] The structural characteristic data was as follows: ESI-MS (m / z): 1034.4 [M+H] + .

[0261] Example 2: N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (1-8-A and 1-8-B)

Chemical formula

[0262] Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene (1-8-2)

[0263] At 25 °C, Compound 1-8-1 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL), concentrated sulfuric acid (25 mL) was added, and the mixture was heated to 50 °C. NBS (6.22 g, 34.97 mmol) was added portionwise at 50 °C, and the reaction was carried out at 50 °C for 2 h. The reaction was monitored by thin-layer chromatography (ethyl acetate: petroleum ether = 1:10). After the reaction solution was cooled to room temperature, it was added dropwise into ice water and extracted with toluene. The organic phases were combined, washed with sodium sulfite solution, water, and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by preparative high-performance liquid chromatography under the following conditions. The fractions were lyophilized to obtain 4.88 g of the title compound.

[0264] Chromatography column: C18 ODS 45 mm × 450 mm × 8.0 μm

[0265] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid) [Table 6]

[0266] Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline (1-8-3)

[0267] At 25 °C, Compound 1-8-2 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL), and platinum carbon (2.00 g, 19.48 mmol, content 5%) was added. Hydrogen substitution was carried out, and the reaction was carried out at 60 °C for 4 h under H 2 atmosphere. The reaction was monitored by HPLC-MS. The reaction solution was filtered and concentrated to obtain 3.68 g of the crude product of the title compound, which was directly used in the next reaction without further purification.

[0268] Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide (1-8-4)

[0269] At 20 °C, compound 1-8-3 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL), triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol) were added, and the mixture was reacted at 20 °C for 20 h. The reaction was monitored by HPLC-MS. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was slurried in a mixed solvent of ethyl acetate:petroleum ether = 1:5 to obtain 2.86 g of the title compound.

[0270] Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid (1-8-5)

[0271] At 20 °C, compound 1-8-4 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL), vinylacetic acid (708.31 mg, 8.23 mmol), DIPEA (1.95 g, 15.08 mmol) and tris(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added. After purging the reaction system with nitrogen, the mixture was heated to 70 °C and reacted for 5 h. The reaction was monitored by HPLC-MS. 1N sodium hydroxide solution was added to the reaction solution to adjust the pH to 8, ethyl acetate was added for extraction, the aqueous phase was adjusted to pH = 3 with 1N hydrochloric acid, and then extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.82 g of the title compound, which was used directly in the next reaction.

[0272] Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butanoic acid (1-8-6)

[0273] At 20 °C, compound 1-8-5 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL), and Pd / C (0.52 g, content 10%) was added. After the reaction system was purged with hydrogen, it was reacted at 40 °C for 2 hours under the protection of a hydrogen balloon, and the reaction was monitored by HPLC-MS. The reaction solution was filtered, and the filtrate was concentrated to obtain 2.43 g of the title compound, which was directly used in the next reaction without further purification.

[0274] Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-7)

[0275] Compound 1-8-6 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5 °C, and anhydrous trifluoroacetic acid (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise. The reaction was carried out at 5 °C for 4 hours, and the reaction was monitored by HPLC-MS. The reaction solution was added to water, adjusted to pH = 9 with 10 N sodium hydroxide, ethyl acetate was added for extraction, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column (ethyl acetate: petroleum ether = 0 - 20%) to obtain 1.53 g of the title compound.

[0276] Step 7: Synthesis of (Z)-N-(3-chloro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-8)

[0277] At 5 °C, potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL), and a THF solution (16 mL) of compound 1-8-7 (1.53 g, 6.08 mmol) was added dropwise. After 10 minutes, amyl nitrite (1.14 g, 9.73 mmol) was added dropwise, and the reaction was carried out at 5 °C for 1 hour, and the reaction was monitored by HPLC-MS. The reaction solution was adjusted to pH = 5 with 1 N hydrochloric acid, extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was slurried with methyl tert-butyl ether to obtain 1.20 g of the title compound.

[0278] Step 8: Synthesis of N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-9) At 20 °C, compound 1-8-8 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2N hydrochloric acid (8 mL), Pd / C (0.15 g, content 10%) was added, the system was purged with hydrogen, and then reacted at 5 °C for 2 hours under the protection of a hydrogen balloon. The reaction was monitored by HPLC-MS. The reaction solution was filtered and concentrated to obtain 0.52 g of the hydrochloride salt of the title compound, which was used directly in the next reaction without further purification.

[0279] Step 9: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (1-8-10)

[0280] At 20 °C, compound 1-8-9 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL), acetic anhydride (2 mL) was added, and the reaction was carried out at 20 °C for 2 hours. The reaction was monitored by HPLC-MS. The reaction solution was added to water and extracted with ethyl acetate. The organic phase was washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column (ethyl acetate: petroleum ether = 0 - 30%) to obtain 0.22 g of the title compound.

[0281] Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-8-11)

[0282] At 20 °C, compound 1-8-10 (450.97 mg, 1.46 mmol) was dissolved in methanol (16 mL), 2N hydrochloric acid (16 mL) was added, heated to 60 °C, reacted for 2 hours, and the reaction was monitored by HPLC-MS. The reaction solution was cooled, adjusted to pH = 8 by adding saturated sodium bicarbonate solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and 230.00 mg of the title compound was obtained, which was used directly in the next reaction without further purification.

[0283] Step 11: Synthesis of N-((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)propyl)acetamide (1-8-12)

[0284] Compound 1-8-11 (230.00 mg, 0.78 mmol) was dissolved in toluene (10 mL), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (230.00 mg, 0.87 mmol) and p-toluenesulfonic acid (26.73 mg, 0.16 mmol) were added, heated to 140 °C, reacted for 5 hours, and the reaction was monitored by HPLC-MS. The reaction solution was concentrated, and the crude product was purified by silica gel column (methanol:dichloromethane = 0~10%) to obtain 150.00 mg of the title compound.

[0285] Step 12: Synthesis of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[3’,4’:6,7]indolizino[1,2-b]quinolin-10,13-dione (1-2)

[0286] Compound 1-8-12 (40.00 mg, 0.081 mmol) was added to concentrated hydrochloric acid (1 mL), heated to 100 °C, and reacted for 5 hours. The reaction was monitored by HPLC-MS. The reaction solution was filtered, and the filtrate was purified by preparative high-performance liquid chromatography under the following conditions. The obtained solution was lyophilized to obtain the hydrochloride salt of the title compound 1-2. The hydrochloride salt of compound 1-2 was separated under the following purification conditions to obtain two isomers, which were named 1-2-A (trifluoroacetate, 5.00 mg, retention time, 9.85 minutes) and 1-2-B (trifluoroacetate, 7.00 mg, retention time, 10.62 minutes) according to their retention times.

[0287] Chromatography column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0288] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% trifluoroacetic acid)

Table 7

[0289] The structure characteristic data were as follows: 1-2-A: 1 1H-NMR (400 MHz, DMSO-d6) δ 8.42 (s, 3H), 8.27 (s, 1H), 7.36 (s, 1H), 6.59 (s, 1H), 5.78 - 5.63 (m, 1H), 5.50 - 5.36 (m, 3H), 5.10 - 5.06 (m, 1H), 3.20 - 3.04 (m, 2H), 2.56 (s, 3H), 2.26 - 2.13 (m, 2H), 1.93 - 1.79 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H). ESI-MS (m / z): 452.1 [M+H] + 。 1-2-B: 11H-NMR (400 MHz, DMSO-d6) δ 8.42 (s, 3H), 8.27 (s, 1H), 7.36 (s, 1H), 6.58 (s, 1H), 5.78 - 5.63 (m, 1H), 5.50 - 5.36 (m, 3H), 5.10 - 5.06 (m, 1H), 3.20 - 3.04 (m, 2H), 2.55 (s, 3H), 2.26 - 2.13 (m, 2H), 1.93 - 1.79 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H). ESI-MS (m / z): 452.0 [M+H] + 。

[0290] Project 13: Synthesis of 2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide and 2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-8-13-A and 1-8-13-B)

[0291] At 25 °C, the hydrochloride salt of compound 1-2 (40.00 mg, 81.91 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (30.91 mg, 98.29 μmol), HATU (62.25 mg, 163.81 μmol), and N,N-diisopropylethylamine (42.34 mg, 327.63 μmol) were added in sequence. The reaction was carried out at 25 °C for 0.5 h, and the reaction was monitored by HPLC-MS. After the reaction was completed, water was added to the reaction solution, and the mixture was extracted with dichloromethane / methanol (v / v = 10 / 1). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified and separated by preparative thin-layer chromatography (dichloromethane:methanol = 20:1) to obtain two isomers, which were named 1-8-13-A (15.00 mg, Rf value was 0.3) and 1-8-13-B (12.00 mg, Rf value was 0.35) based on the Rf values.

[0292] Step 14: Synthesis of N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (I-8-A and I-8-B)

[0293] At 25 °C, 1-8-13-A (15.00 mg) and 1-8-13-B (12.00 mg) were each dissolved in tetrahydrofuran (1 mL) in two reaction flasks, and tetrabutylammonium fluoride in a mixed solution of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) / glacial acetic acid (v / v = 13 / 1) (50 μL) was added dropwise. The reaction was carried out at 25 °C for 0.5 h, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography, and the prepared solution was freeze-dried to obtain the title compounds 1-8-A (6.94 mg) and 1-8-B (4.00 mg).

[0294] Chromatography column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0295] Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid)

Table 8

[0296] The structural characteristic data of 1-8-A were as follows: 1 1H-NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65 - 5.36 (m, 4H), 5.21 (q, J = 19.0 Hz, 2H), 3.95 (d, J = 5.7 Hz, 2H), 3.26 - 3.11 (m, 2H), 2.53 (s, 3H), 2.30 - 2.08 (m, 2H), 1.94 - 1.79 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 510.1 [M + H] + 。

[0297] The structural characteristic data of 1-8-B were as follows: 1H-NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 8.9 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.64 - 5.35 (m, 4H), 5.19 (q, J = 19.0 Hz, 2H), 3.97 (d, J = 5.2 Hz, 2H), 3.27 - 3.10 (m, 2H), 2.51 (s, 3H), 2.27 - 2.10 (m, 2H), 1.93 - 1.80 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 510.1 [M + H] + .

[0298] Example 3: Synthesis of N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:(6,7)indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide and N-((S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide (A-07-A and A-07-B)

Chemical Structure

[0299] Project 1: Synthesis of (S)-10-Benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentoxo-3-oxohetero-5,8,11,14,17-pentaazatricosane-22-carboxylic acid (A-07-3)

[0300] At 25 °C, compound A-07-2 (30.00 mg, 0.07 mmol) was dissolved in DMF (0.2 mL), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-inoate (A-07-1, 28.00 mg, 0.08 mmol) was added, and the mixture was reacted at 30 °C for 1 hour. The reaction was monitored by HPLC-MS. The reaction solution was directly purified by preparative high-performance liquid chromatography (conditions are as follows), and the fraction was lyophilized to obtain 20.00 mg of the title compound.

[0301] Chromatography column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0302] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 9

[0303] The structural characteristic data were as follows: ESI-MS (m / z): 691.0 [M+H 2 O] + 。

[0304] Project 2: Synthesis of N-((S)-10-Benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:(6,7)indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide and N-((S)-10-Benzyl-1-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide (A-07-A and A-07-B)

[0305] At 25 °C, the hydrochloride of 1-2 (30.00 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL), and A-07-3 (49.66 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol) and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added in sequence. The reaction was carried out at 25 °C for 0.5 h, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography under the following conditions, and the fractions were freeze-dried to obtain the title compound A-07. A-07 was separated under the following purification conditions to obtain two isomers, which were named A-07-A (11.04 mg, retention time was 7.5 min) and A-07-B (19.42 mg, retention time was 8.0 min) according to the retention time.

[0306] Chromatography column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0307] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 10

[0308] The structural characteristic data were as follows: A-07-A: ESI-MS (m / z): 1107.3 [M+H] + 。 A-07-B: ESI-MS (m / z): 1107.3 [M+H] + 。

[0309] Example 4: (S)-7-Ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

Chemical formula

[0310] Step 1: Synthesis of 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxol-5-yl)propan-1-one (2-2-2)

[0311] Compound 2-2-1 (1.90 g, 8.08 mmol) was slowly added to nitric acid (8 mL) at 0 °C, slowly heated to 25 °C, and reacted for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (acetonitrile / 0.5% aqueous formic acid) to obtain 1.50 g of the formate salt of the title compound.

[0312] The structural characteristic data were as follows: ESI-MS (m / z): 281.1 [M+H] + 。

[0313] Step 2: Synthesis of 1-(6-aminobenzene[d][1,3]dioxol-5-yl)-3-(isopropylamino)propan-1-one (2-2-3)

[0314] The formate salt of compound 2-2-2 (1.25 g, 4.46 mmol) was added to tetrahydrofuran (20 mL), 10% palladium on carbon (125.00 mg) was added, the mixture was purged with hydrogen three times, and reacted at 25 °C for 16 hours. The reaction solution was filtered through celite, and the filtrate was concentrated to dryness under reduced pressure to obtain 895.00 mg of the crude product of the title compound, which was used directly in the next reaction without purification.

[0315] The structure characteristic data were as follows: ESI-MS (m / z): 251.1 [M+H] + 。

[0316] Step 3: Synthesis of (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

[0317] Compound 2-2-3 (23.00 mg, 0.09 mmol) and (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H pyrano[3,4-f]indolizino-3,6,10(4H)-trione (21.00 mg, 0.09 mmol) were dissolved in toluene (4 mL), p-toluenesulfonic acid (1.40 mg, 0.009 mmol) was added, and the mixture was reacted at 140 °C for 12 hours, dried under reduced pressure, and the solvent was removed to obtain the crude product of the title compound. The crude product was purified by HPLC (mobile phase A: acetonitrile, mobile phase B: 0.05% aqueous formic acid solution), 3 drops of 3M hydrochloric acid were added to the prepared solution, and freeze-dried to obtain 8.70 mg of the hydrochloride salt of the title compound.

[0318] The structure characteristic data were as follows: ESI-MS (m / z): 478.2 [M+H] + 。 1 1H-NMR (400 MHz, DMSO-d6): δ 9.38 (brs, 2H), 7.85 (s, 1H), 7.52 (s, 1H), 7.25 (s, 1H), 6.30 (d, J = 2.0 Hz, 2H), 5.43 (s, 2H), 5.30 (s, 2H), 3.57 - 3.45 (m, 2H), 3.40 - 3.25 (m, 1H), 3.22 - 3.06 (m, 2H), 1.95 - 1.77 (m, 2H), 1.27 (d, J = 6.4 Hz, 6H), 0.87 (t, J = 7.6 Hz, 3H).

[0319] Example 5: Synthesis of 4 - ((S)-2-(4 - aminobutyl)-35-(4 - ((6-(2 - (methylsulfonyl)pyrimidin - 5 - yl)hexa - 5 - inamide)methyl)-1H - 1,2,3 - triazol - 1 - yl)-4,8 - dioxo - 6,12,15,18,21,24,27,30,33 - nonaoxa - 3,9 - diazapentatriacontanamide)benzyl ((S)-4 - ethyl - 11-(2-(N - isopropylmethylsulfonamide)ethyl)-3,14 - dioxo - 3,4,12,14 - tetrahydro - 1H - pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 4 - yl)ethyl) carbonate (B - 01)

Chemical Structure

[0320] Step 1: Synthesis of (S)-4 - ethyl - 11-(2-(N - isopropylmethylsulfonamide)ethyl)-3,14 - dioxo - 3,4,12,14 - tetrahydro - 1H - pyrano[3’,4’:6,7 - indolizino[1,2 - b]quinolin - 4 - yl(4 - ((S)-2-(4 - (((4 - methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4 - ((6-(2 - (methylsulfonyl)pyrimidin - 5 - yl)hexa - 5 - inamide)methyl)-1H - 1,2,3 - triazol - 1 - yl)-4,8 - dioxo - 6,12,15,18,21,24,27,30,33 - nonaoxa - 3,9 - diazapentatriacontanamide)benzyl) carbonate (B - 01 - 2)

[0321] At room temperature, Compound B-01-1 (413.40 mg, 0.251 mmol, for its synthesis, refer to Chinese Patent No. 111295389(B)) was dissolved in dimethyl sulfoxide and water (2.0 mL:0.5 mL), cuprous bromide 72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (95.10 mg, 0.302 mmol) were added, and after stirring and reacting for 1 hour, filtration was carried out, and the filtrate was purified by preparative high performance liquid chromatography under the following conditions to obtain 30.00 mg of the title compound.

[0322] Chromatography column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0323] Mobile phase A: Acetonitrile, Mobile phase B: Water [Table 11]

[0324] The structural characteristic data were as follows: ESI-MS (m / z): 815.9 [(M - 273) / 2 + H] + .

[0325] Step 2: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl)ethyl) carbonate (B-01)

[0326] B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL), trifluoroacetic acid (0.2 mL) was added to the reaction solution, and the mixture was reacted at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure and purified by preparative high performance liquid chromatography under the following conditions to obtain 20.00 mg of the trifluoroacetate salt of the title compound.

[0327] Chromatography column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0328] Mobile phase A: acetonitrile, Mobile phase B: water (0.05% trifluoroacetic acid)

Table 12

[0329] The structure characteristic data were as follows: ESI-MS (m / z): 816.0 [M / 2 + H] + 11H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.10 (s, 2H), 8.38 (t, J = 5.56 Hz, 1H), 8.32 (d, J = 8.40 Hz, 1H), 8.22 - 8.20 (m, 2H), 8.09 (t, J = 5.68 Hz, 1H), 7.91 - 7.87 (m, 2H), 7.82 - 7.78 (m, 1H), 7.69 (brs, 3H), 7.61 (d, J = 8.56 Hz, 2H), 7.32 (d, J = 8.56 Hz, 2H), 7.06 (s, 1H), 5.56 (d, J = 16.96 Hz, 1H), 5.51 (d, J = 16.96 Hz, 1H), 5.47 (d, J = 19.28 Hz, 1H), 5.42 (d, J = 19.28 Hz, 1H), 5.14 (d, J = 12.20 Hz, 1H), 5.07 (d, J = 12.16 Hz, 1H), 4.48 (t, J = 5.24 Hz, 2H), 4.46 - 4.43 (m, 1H), 4.29 (d, J = 5.60 Hz, 2H), 4.08 - 3.95 (m, 5H), 3.79 (t, J = 5.28 Hz, 2H), 3.51 - 3.43 (m, 32H), 3.40 (s, 3H), 3.39 - 3.35 (m, 2H), 3.30 - 3.26 (m, 2H), 3.00 (s, 3H), 2.82 - 2.74 (m, 2H), 2.56 (t, J = 7.08 Hz, 2H), 2.29 (t, J = 7.36 Hz, 2H), 2.23 - 2.13 (m, 2H), 1.82 (p, J = 7.24 Hz, 2H), 1.78 - 1.63 (m, 2H), 1.61 - 1.49 (m, 2H), 1.42 - 1.27 (m, 2H), 1.15 (d, J = 6.80 Hz, 3H), 1.13 (d, J = 6.76 Hz, 3H), 0.90 (t, J = 7.32 Hz, 3H).

[0330] Example 6: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-Ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]-indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(2-(2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide)ethoxy)ethoxy)acetamide)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03) [Chemical formula]

[0331] Step 1: Synthesis of (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (B-03-3)

[0332] Compound (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-1, 12.32 g, 31.02 mmol) and 4-hydroxy-3-nitrobenzyl alcohol (Compound B-03-2, 5.00 g, 29.56 mmol) were dissolved in acetonitrile (200 mL). While stirring, silver oxide (27.40 g, 118.25 mmol) was added. After purging with nitrogen, the reaction was carried out at room temperature in the dark for 12 hours. The reaction mixture was monitored by HPLC-MS, filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. Then it was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:3) to obtain 12.80 g of the title compound.

[0333] The structure characteristic data were as follows: ESI-MS (m / z): 503 [M + 18] + .

[0334] Step 2: Synthesis of (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (B-03-4)

[0335] Compound B-03-3 (2.20 g, 4.53 mmol) was dissolved in ethyl acetate and tetrahydrofuran (50 mL each), and after adding PtO 2 (0.20 g), the reaction system was replaced with a hydrogen balloon three times and reacted under a hydrogen atmosphere for 2 hours. The reaction product was monitored by HPLC-MS, the reaction solution was directly filtered, the filter cake was rinsed with ethyl acetate, and the filtrate was evaporated to dryness under reduced pressure to obtain 2.02 g of the crude product of the title compound, which was used directly in the next reaction.

[0336] The structural characteristic data were as follows: ESI-MS (m / z): 456.1 [M+1] + 。

[0337] Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12-amido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (B-03-5)

[0338] Compound B-03-4 (456.00 mg, 1.00 mmol) and [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (385.91 mg, 1.00 mmol) were dissolved in dichloromethane (10 mL), and while stirring, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (495.22 mg, 2.00 mmol) was added and reacted with stirring for 2 hours. The reaction product was monitored by HPLC-MS, and after concentrating the reaction solution under reduced pressure, it was purified by silica gel column chromatography (methanol:dichloromethane = 1:20) to obtain 507.00 mg of the title compound.

[0339] The structural characteristic data were as follows: ESI-MS (m / z): 823.3 [M+1] + 。

[0340] Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12-amido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (B-03-6)

[0341] Compound B-03-5 (507.00 mg, 616.18 μmol) and diisopropylethylamine (238.91 mg, 1.85 mmol) were dissolved in dichloromethane (20 mL). Then, p-nitrophenyl chloroformate (372.60 mg, 1.85 mmol) was dissolved in dichloromethane (1 mL), and the resulting solution was slowly added dropwise to the reaction solution. After the addition was complete, the mixture was reacted at room temperature for 15 hours. The reaction was monitored by HPLC-MS, and after the reaction solution was concentrated under reduced pressure, it was purified by silica gel column chromatography (methanol:dichloromethane = 1:20) to obtain 496.00 mg of the title compound.

[0342] The structural characteristic data were as follows: ESI-MS (m / z): 988.5 [M+1] + 。

[0343] Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12-amido)-4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (B-03-7)

[0344] Compound B-03-6 (165.51 mg, 0.17 mmol), compound 2-2 (40.00 mg, 0.084 mmol) and 1-hydroxybenzotriazole (33.96 mg, 0.25 mmol) were dissolved in DMF (4 mL), and diisopropylethylamine (32.48 mg, 0.25 mmol) was added dropwise. The mixture was reacted with stirring for 12 hours, and the reaction was monitored by HPLC-MS. Water and ethyl acetate were added and stirred, and then left to stand for liquid separation. The organic phase was washed with saturated brine, dried, concentrated under reduced pressure to obtain 100.00 mg of the crude product of the title compound, which was used directly in the next reaction.

[0345] The structure characteristic data were as follows: ESI-MS (m / z): 1326.2 [M+1] + 。

[0346] Step 6: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03-8)

[0347] Compound B-03-7 (100.00 mg, 0.08 mmol) was dissolved in MeOH (5 mL). After adding 1 drop of dichloromethane, an aqueous solution (1 mL) of lithium hydroxide monohydrate (15.82 mg, 0.377 mmol) was added dropwise, and the reaction was carried out with stirring for 2 hours. The reaction mixture was monitored by HPLC-MS. An aqueous solution of 3N hydrochloric acid was added dropwise to the reaction solution to adjust the pH to 4, and the solution was concentrated under reduced pressure. Then, it was purified by preparative high-performance liquid chromatography under the following conditions, and the prepared solution was lyophilized to obtain 27.00 mg of the title compound.

[0348] Chromatography column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0349] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 13

[0350] The structure characteristic data were as follows: ESI-MS (m / z): 964.2 [M+1] + 。

[0351] Step 7: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]-indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(2-(2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide)ethoxy)ethoxy)acetamide)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03)

[0352] Compound B-03-8 (27.00 mg, 0.03 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (A-07-1, 11.26 mg, 0.03 mmol) were dissolved in DMF (1 mL), and diisopropylethylamine (3.62 mg, 0.03 mmol) was added dropwise with stirring. The reaction was carried out at room temperature for 4 hours. The reaction was monitored by chromatography combined with mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (conditions are as follows), and the preparative solution was lyophilized to obtain 11.70 mg of the title compound.

[0353] Chromatography column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0354] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid) [Table 14]

[0355] The structure characteristic data were as follows: ESI-MS (m / z): 1214.4 [M+1] + .

[0356] Example 7: Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (1-11-A and 1-11-B) [Chemical formula]

[0357] Step 1: Synthesis of 3-Bromo-4-chloro-5-fluoroaniline (1-5-02)

[0358] Compound 1-5-01 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL), and then N-chlorosuccinimide (1.69 g, 12.63 mmol) was slowly added. After the addition, the reaction was carried out at room temperature for 16 hours, and the reaction was monitored by HPLC-MS. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate: petroleum ether = 0 - 25%) to obtain 0.95 g of the title compound.

[0359] The structure characteristic data were as follows: 1 1H-NMR (400 MHz, DMSO-d6) δ 6.77 (dd, J = 2.5, 1.4 Hz, 1H), 6.51 (dd, J = 11.7, 2.5 Hz, 1H), 5.84 (s, 2H).

[0360] Step 2: Synthesis of N-(3-Bromo-4-chloro-5-fluorophenyl)acetamide (1-5-03)

[0361] Compound 1-5-02 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL), and acetic anhydride (648.13 mg, 6.35 mmol) was added under nitrogen protection. After the addition, the temperature was raised to 50 °C and the reaction was carried out for 15 hours, and the reaction was monitored by HPLC-MS. The reaction solution was quenched with methanol (5 mL) and then directly dried by evaporation under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate: petroleum ether = 0 - 40%) to obtain 1.01 g of the title compound.

[0362] The structure characteristic data were as follows: ESI-MS (m / z): 265.9 [M+H] + .

[0363] Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)but-3-enoic acid (1-5-04)

[0364] Compound 1-5-03 and 3-butenoic acid (387.65 mg, 4.50 mmol) were dissolved in a mixed solvent of 1,4-dioxane (24 mL) and water (8 mL). Then, N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tris(o-methylphenyl)phosphine (114.21 mg, 375.24 μmol), and palladium(II) acetate (42.12 mg, 187.62 μmol) were added. After the addition, the reaction system was purged with nitrogen three times, the temperature was raised to 100 °C under a nitrogen atmosphere, and the reaction was carried out for 16 hours. The reaction was monitored by HPLC-MS. After the reaction solution was cooled to room temperature, 1N aqueous sodium hydroxide solution (60 mL) and ethyl acetate (50 mL) were added, and the mixture was shaken and separated into layers. The lower aqueous layer was separated, the pH was adjusted to about 3 with 4 mol / L aqueous hydrochloric acid, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 1.00 g of the crude product of the title compound.

[0365] The structure characteristic data were as follows: ESI-MS (m / z): 272.0 [M+H] + 。

[0366] Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butanoic acid (1-5-05)

[0367] The crude product of compound 1-5-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL). Then, 10% palladium on carbon (0.10 g) was added. After the addition, the atmosphere of the reaction system was replaced with a hydrogen balloon three times, and the reaction was carried out for 4 hours under a hydrogen atmosphere. The reaction was monitored by HPLC-MS. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.00 g of the crude product of the title compound.

[0368] The structure characteristic data were as follows: ESI-MS (m / z): 274.0 [M+H] + 。

[0369] Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-06)

[0370] The crude product of compound 1-5-05 (1.00 g, 3.65 mmol) was dissolved in trifluoroacetic acid (5 mL), cooled to 5 °C, and then trifluoroacetic anhydride (3.84 g, 18.27 mmol, 2.54 mL) was slowly added. After the addition, the reaction was carried out at 5 °C for 2 hours, and the reaction was monitored by HPLC-MS. The reaction solution was slowly poured into water, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was dried by evaporation under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column chromatography to obtain 0.43 g of the title compound.

[0371] The structural characterization data were as follows: ESI-MS (m / z): 256.1 [M+H] + 。

[0372] Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-07)

[0373] Add tetrahydrofuran (16 mL) and tert-butanol (4 mL) to a reaction flask, cool to 5 °C in an ice bath, then add potassium tert-butoxide (415.18 mg, 3.70 mmol). Dissolve compound 1-5-06 (0.43 mg, 1.68 mmol) in tetrahydrofuran (1 mL) and slowly add it dropwise to the reaction solution. After 10 minutes, add isoamyl nitrite (315.24 mg, 2.69 mmol). After addition, react at 5 °C for 1 hour and monitor the reaction by HPLC-MS. Quench the reaction solution with saturated aqueous ammonium chloride, extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 455.00 mg of the crude product of the title compound.

[0374] The structural characteristic data were as follows: ESI-MS (m / z): 285.0 [M+H] + 。

[0375] Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydro-naphthalen-1-yl)acetamide (1-5-08)

[0376] Dissolve the crude product of compound 1-5-07 (0.40 g, 1.41 mmol) in methanol (10 mL), then add 3 mol / L aqueous hydrochloric acid (1 mL) and 10% palladium on carbon (40.00 mg). After addition, replace the reaction system with hydrogen three times and react at room temperature for 1 hour under a hydrogen atmosphere. Monitor the reaction by HPLC-MS. Filter the reaction solution and concentrate the filtrate to dryness under reduced pressure to obtain 0.43 g of the crude hydrochloride salt of the title compound.

[0377] The structural characteristic data were as follows: ESI-MS (m / z): 271.0 [M+H] + 。

[0378] Step 8: Synthesis of (9H-Fluoren-9-yl)methyl)(8-acetamido-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-09)

[0379] The crude hydrochloride salt of Compound 1-5-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL), and then sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL), and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol) were added. After the addition, the reaction was carried out at room temperature for 2 hours with stirring, and the reaction was monitored by HPLC-MS. The reaction solution was poured into water and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by a C18 reverse-phase column (acetonitrile: 0.05% formic acid in water = 20% - 100%) to obtain 301.00 mg of the title compound.

[0380] The structural characteristic data were as follows: ESI-MS (m / z): 493.2 [M+H] + 。

[0381] Step 9: Synthesis of (9H-Fluoren-9-yl)methyl)(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-10)

[0382] Compound 1-5-09 (300.00 mg, 608.61 μmol) was dissolved in dioxane (5 mL), and 1 mL of 12 mol / L concentrated hydrochloric acid was added. After the addition, the temperature was raised to 60 °C and the reaction was carried out for 2 hours, and the reaction was monitored by HPLC-MS. The reaction solution was poured into water and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by a flash silica gel column (ethyl acetate: petroleum ether = 0 - 50%) to obtain 198.00 mg of the title compound.

[0383] The structural characteristic data were as follows: ESI-MS (m / z): 451.1 [M+H] + 。

[0384] Step 10: Synthesis of (9H-fluoren-9-yl)methyl ((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)ethyl)carbamate (1-5-11)

[0385] (S)-4-Ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (138.72 mg, 526.96 μmol) and Compound 1-5-10 (198.00 mg, 439.13 μmol) were added to toluene (10 mL), then p-toluenesulfonic acid (75.53 mg, 439.13 μmol) was added. After the addition, the temperature was raised to 140 °C and the reaction was carried out for 4 hours. The reaction solution was directly dried by evaporation under reduced pressure at 140 °C to obtain a crude product, and the crude product was purified by a flash silica gel column (methanol:dichloromethane = 0~5%) to obtain 256.00 mg of the title compound.

[0386] The structural characteristic data were as follows: ESI-MS (m / z): 678.1 [M+H] + 。

[0387] Project 11: Synthesis of (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (1-5-A and 1-5-B)

[0388] Compound 1-5-11 (201.18 mg, 296.67 μmol) was dissolved in N,N-dimethylformamide (4 mL), and then diethylamine (108.49 mg, 1.48 mmol) was added. After the addition, the reaction was carried out at room temperature for 0.5 h, and the reaction was monitored by HPLC-MS. The reaction solution was evaporated under reduced pressure to remove ethylenediamine, the pH was adjusted to 2 - 3 using 1 mol / L aqueous hydrochloric acid solution, and the reaction solution was directly purified by preparative high performance liquid chromatography to obtain the title compounds 1-5-A (44.00 mg) and 1-5-B (43.00 mg).

[0389] Chromatography column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0390] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 15

[0391] 1-5-A (6-minute LCMS peak appears early, retention time: 1.276 min)

[0392] The structure characteristic data was as follows: 1H-NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (d, J = 19.3 Hz, 1H), 5.44 (s, 2H), 5.38 (d, J = 19.3 Hz, 1H), 4.43 - 4.38 (m, 1H), 3.28 - 3.10 (m, 2H), 2.22 - 2.12 (m, 1H), 2.12 - 2.02 (m, 1H), 1.93 - 1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0393] ESI-MS (m / z): 456.1 [M+H] + 。

[0394] The structural characteristic data of 1-5-B (the 6-minute LCMS peak appeared late, retention time: 1.300 minutes) were as follows: 1 H-NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 10.3 Hz, 1H), 7.32 (s, 1H), 5.61 (d, J = 19.4 Hz, 1H), 5.44 (s, 2H), 5.32 (d, J = 19.4 Hz, 1H), 4.44 - 4.36 (m, 1H), 3.33 - 3.25 (m, 1H), 3.22 - 3.11 (m, 1H), 2.23 - 2.13 (m, 1H), 2.11 - 2.03 (m, 1H), 1.96 - 1.82 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 456.1 [M+H] + 。

[0395] 6-minute LCMS conditions:

[0396] Chromatography column: Waters SunFire C18 OBD 4.6 mm × 50 mm × 5.0 μm

[0397] Mobile phase A: 0.05% acetonitrile, Mobile phase B: water (0.05% formic acid)

Table 16

[0398] Project 12: Synthesis of N-((S)-10-benzyl-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:(6,7)indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide and N-((S)-10-benzyl-1-(((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide (1-5-12-A and 1-5-12-B)

[0399] Compound 1-5-A (36.00 mg, 79.70 μmol) with a single configuration and Compound A-07-3 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL). Then, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After the addition, the reaction was carried out at room temperature for 1 hour, and the reaction was monitored by HPLC-MS. The reaction solution was directly purified by high-performance liquid chromatography to obtain the title compound 1-5-12-A in a single configuration (51.00 mg).

[0400] Chromatography column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0401] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 17

[0402] The structural characteristic data were as follows: ESI-MS (m / z): 1111.0 [M+H] + 。

[0403] Compound 1-5-B (36.00 mg, 79.70 μmol) and compound A-07-3 (64.43 mg, 95.64 μmol) in a single configuration were dissolved in N,N-dimethylformamide (2 mL). Then, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After the addition, the reaction was carried out at room temperature for 1 hour, and the reaction was monitored by HPLC-MS. The reaction solution was directly purified by high-performance liquid chromatography to obtain the title compound 1-5-12-B in a single configuration (52.00 mg).

[0404] Chromatography column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0405] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 18

[0406] The structural characteristic data were as follows: ESI-MS (m / z): 1111.0 [M+H] + 。

[0407] Project 13: Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (1-11-A and 1-11-B)

[0408] Compound 1-5-12-A (40.00 mg, 35.99 μmol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL). Then, an ethyl acetate solution of 4 mol / L hydrochloric acid (1 mL) was added. After the addition, the reaction was carried out at room temperature for 0.5 h, and the reaction was monitored by HPLC-MS. The reaction solution was directly concentrated to dryness under reduced pressure to obtain a crude product, and the crude product was purified by high-performance liquid chromatography to obtain the title compound 1-11-A in a single stereoconfiguration (4.75 mg).

[0409] Chromatography column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0410] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 19

[0411] The structural characteristic data were as follows: 11H-NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 5.67 - 5.60 (m, 1H), 5.49 (t, J = 5.8 Hz, 1H), 5.43 (s, 2H), 5.21 (s, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.32 - 3.22 (m, 2H), 2.28 - 2.15 (m, 2H), 1.93 - 1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 514.0 [M+H] + .

[0412] Compound 1-5-12-B (40.00 mg, 35.99 μmol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL). Then, 4 mol / L hydrochloric acid ethyl acetate solution (1 mL) was added. After the addition, the mixture was reacted at room temperature for 0.5 h, and the reaction was monitored by HPLC-MS. The reaction solution was directly concentrated to dryness under reduced pressure to obtain a crude product, and the crude product was purified by high performance liquid chromatography to obtain the title compound 1-11-B in a single stereoconfiguration (8.24 mg).

[0413] Chromatography column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0414] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid) [Table 20]

[0415] The structure characteristic data were as follows: 11H-NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 9.0 Hz, 1H), 8.05 (d, J = 10.3 Hz, 1H), 7.34 (s, 1H), 6.55 (s, 1H), 5.68 - 5.58 (m, 1H), 5.53 (t, J = 5.8 Hz, 1H), 5.43 (d, J = 2.9 Hz, 2H), 5.20 (d, J = 7.3 Hz, 2H), 3.97 (d, J = 5.7 Hz, 2H), 3.31 - 3.21 (m, 2H), 2.26 - 2.15 (m, 2H), 1.92 - 1.82 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 514.0 [M+H] + 。

[0416] Example 8: Synthesis of N-((10S,19S)-10-benzyl-1-(((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:(6,7)indolizino[1,2-b]quinolin-1-yl)amino)-30-(2-(methylsulfonyl)pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontan-29-yl)-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amide N-((10S,19S)-10-benzyl-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7-indolizino[1,2-b]quinolin-1-yl)amino)-30-(2-(methylsulfonyl)pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontan-29-yl)-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amide (A-17-A)

Chemical formula

[0417] Engineering 1:N 6 Synthesis of -(((9H-Fluoren-9-yl)methoxy)carbonyl)-N2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-yl)-D-lysine(2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeoxaoctadecane-38-amide)-6-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)hexanoic acid (A-17-03)

[0418] The hydrochloride salt of compound A-17-02 (389.68 mg, 962.45 μmol) was dissolved in dichloromethane (8 mL), DIPEA (518.28 mg, 4.01 mmol, 713.88 μL) and compound A-17-01 (550.00 mg, 802.04 μmol) were added, and the mixture was reacted at 25 °C for 1.5 hours. The pH of the reaction solution was adjusted to neutral with dilute hydrochloric acid, and the solvent was dried under reduced pressure. The concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-03 (450.00 mg).

[0419] Chromatography column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0420] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 21

[0421] Project 2: Synthesis of (40S,49S)-40-(4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)butyl)-49-benzyl-38,41,44,47,50,53-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,56-tridecaoxa-39,42,45,48,51,54-hexaazaoctapentacontan-58-oic acid (A-17-04)

[0422] Compound A-17-03 (50.00 mg, 118.09 μmol) was dissolved in DMF (2.5 mL), and HATU (49.39 mg, 129.89 μmol), compound A-07-2 (133.07 mg, 141.70 μmol), and DIPEA (45.78 mg, 354.26 μmol, 63.06 μL) were added, followed by reaction at 25 °C for 1 hour. The solvent was dried under reduced pressure, and the concentrate was purified by preparative high-performance liquid chromatography to obtain the title compound A-17-04 (40.00 mg).

[0423] Chromatography column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0424] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 22

[0425] Process 3: Synthesis of (9H-fluoren-9-yl)methyl ((40S)-40-(((10S)-10-benzyl-1-(((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azatetracontane-44-yl)carbamate (A-17-05)

[0426] Compound A-17-04 (29.86 mg, 59.50 μmol) was dissolved in DMF (3 mL), and HATU (27.15 mg, 71.40 μmol), compound 1-5-A (80.00 mg, 59.50 μmol) and DIPEA (38.45 mg, 297.51 μmol, 52.96 μL) were added, and the reaction was carried out at 25 °C for 1 hour. The solvent was dried under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-05 (50.00 mg).

[0427] Chromatography column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0428] Mobile phase A: acetonitrile, mobile phase B: water (0.05% formic acid)

Table 23

[0429] Project 4: N-((10S,19S)-23-Amino-10-benzyl-1-(((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosane-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane-38-amide N-((10S,19S)-23-Amino-10-benzyl-1-(((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4]:6,7-indreazino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxo-5,8,11,14,17-pentaaza230 Alk-19-yl)-2,5,8,11,14,17,20,23, 26,29,32,35-dodecyloxaoctatriacontane-38-amide (A-17-06) Synthesis

[0430] Compound A-17-05 (20.00 mg, 11.22 μmol) was dissolved in DMF (2.5 mL) and diethylamine (0.5 mL), and reacted at 25 °C for 2 hours. The solvent was dried under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the formate salt (10.00 mg) of the title compound A-17-06.

[0431] Chromatography column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0432] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 24

[0433] Project 5: N-((10S,19S)-10-Benzyl-1-(((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:(6,7)indolizino[1,2-b]quinolin-1-yl)amino)-30-(2-(methylsulfonyl)pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amide-N-((10S,19S)-10-Benzyl-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7-indolizino[1,2-b]quinolin-1-yl)amino)-30-(2-(methylsulfonyl)pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amide (A-17-A) Synthesis

[0434] The formates of Compound A-17-06 (7.00 mg, 4.49 μmol) and Compound A-07-1 (3.28 mg, 8.97 μmol) were dissolved in DMF (1 mL), DIPEA (1.74 mg, 13.46 μmol, 2.40 μL) was added, and the reaction was carried out at 25 °C for 2 hours. The solvent was dried under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-A (5.60 mg).

[0435] Chromatography column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0436] Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 25

[0437] Example 9: Synthesis of N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxyl-5,8,11,14-tetraazapentadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amide (A-05)

Chemical formula

[0438] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-inoate (A-07-1, 0.66 g, 1.80 mmol) and A-07-2 (0.75 g, 1.77 mmol) were added to DMF (19 mL), and after heating at 35 °C for 16 hours, (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (1-4 (i.e., 1-2-A), 1.00 g, 1.77 mmol) was added, and the mixture was cooled to 5 - 15 °C in ice water. DMTMM (0.98 g, 3.53 mmol) was added, DIPEA (1.14 g, 8.84 mmol) was added dropwise, and the reaction was carried out at 25 °C for 16 hours. The reaction solution was poured into DCM (600 mL), IPA (60 mL), and water (100 mL), the mixture was stirred for 10 minutes, then the DCM phase was separated, washed with brine (100 mL), concentrated to obtain a crude product, and purified by preparative high-performance liquid chromatography. After purification, the product was lyophilized to obtain 0.98 g of compound A-05 (i.e., A-07-A in Example 3).

[0439] The method for separating and purifying A-05 is as follows. Column: Waters SunFire Prep C18 OBD (5μm * 19mm * 150mm) Mobile phase A: Acetonitrile, Mobile phase B: Water (0.05% formic acid)

Table 26

[0440] The structural characteristic data of A-05 were as follows: MS m / z (ESI): 1107.3 [M+H] + 。 11H NMR (400 MHz, DMSO) δ 9.10 (s, 2H), 8.66 - 8.63 (m, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.34 - 8.31 (m, 1H), 8.21 - 8.19 (m, 1H), 8.17 - 8.09 (m, 2H), 8.08 - 8.04 (m, 1H), 7.30 (s, 1H), 7.26 - 7.15 (m, 5H), 6.55 (s, 1H), 5.56 - 5.55 (m, 1H), 5.48 - 5.35 (m, 2H), 5.25 - 5.10 (m, 2H), 4.64 (d, J = 6.4 Hz, 2H), 4.45 - 4.44 (m, 1H), 4.06 - 3.98 (m, 2H), 3.77 - 3.52 (m, 6H), 3.41 (s, 3H), 3.25 - 3.12 (m, 2H), 3.03 - 3.00 (m, 1H), 2.83 - 2.72 (m, 1H), 2.58 - 2.56 (m, 2H), 2.48 (s, 3H), 2.33 - 2.30 (m, 2H), 2.21 - 2.13 (m, 2H), 1.91 - 1.76 (m, 4H), 0.87 (t, J = 7.2 Hz, 3H).

[0441] To detect the compounds A - 07 - A and A - 07 - B prepared in Example 3, and the compound A - 07 - A prepared in Example 3 and the compound A - 05 prepared in Example 9, the following HPLC conditions were used.

[0442] Equipment: Agilent 1260 High Performance Liquid Chromatography VWD Detector

[0443] Chromatography column: Waters Xbridge C18 4.6 * 100 mm * 3.5 μm

[0444] Mobile phase A: 0.01 M ammonium phosphate aqueous solution / acetonitrile = 90 / 10, Mobile phase B: acetonitrile [Table 27]

[0445] As shown in Figure 8A, the retention times of Compound A-07-A and Compound A-07-B were 6.6 minutes and 6.8 minutes, respectively, indicating that the above HPLC conditions can well separate the isomers.

[0446] As shown in Figure 8B, for Compound A-07-A prepared in Example 3 and Compound A-05 prepared in Example 9, a single peak with a retention time of 6.6 minutes was present, indicating that Compound A-07-A and Compound A-05 are the same compound.

[0447] Example 10: Synthesis of (1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-9-yl 2-(4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)phenyl)acetate (B-04) [Chemical formula]

[0448] Step 1: Preparation of 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)ethyl)amino)-2-oxoethyl acetate (B-04-1).

[0449] (1S,9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione (2g, 3.65 mmol) was dissolved in DMF (50 mL), DIPEA (1.18 g, 9.12 mmol, 1.59 mL) was added dropwise, and acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) was added dropwise with stirring in an ice bath. The stirring reaction was continued for 1 hour. The reaction solution was added to 0.1 M dilute hydrochloric acid aqueous solution to precipitate a solid, which was then filtered. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column (methanol / dichloromethane = 0% - 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol).

[0450] The structural characteristic data were as follows: SI-MS (m / z): 552.2 [M+1]+.

[0451] Step 2: Preparation of (1S,9S)-1-(2-acetoxyacetamido)-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-9-yl 2-(4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)phenyl)acetate (B-04-2).

[0452] Ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3’,4’:6,7]indeno[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (500 mg, 0.905 mmol) and DMAP (885.33 mg, 7.25 mmol) were dissolved in dry dichloromethane (5 mL), cooled to 0 °C under nitrogen protection, and triphosgene (268.81 mg, 0.905 mmol) was added dropwise. The dichloromethane solution (5 mL) was continuously stirred and reacted for 0.5 h. A dichloromethane solution (5 mL) of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatritrityl chloride)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44 g, 1.36 mmol) was slowly added dropwise, allowed to return to room temperature naturally, and reacted for 4 h. The reaction solution was quenched with water, extracted three times with dichloromethane (100 mL × 3), the organic phases were combined, washed with saturated brine, dried, concentrated, and the title compound (498 mg, 0.304 mmol) was purified by silica gel column (MeOH / DCM = 0% - 5%).

[0453] The structure characteristic data were as follows: ESI-MS m / z: 1352.8 [M+1]+.

[0454] Step 3: Synthesis of (1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-9-yl 2-(4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxo-3,9-diazapentatriacontanamide)phenyl)acetate (B-04-3)

[0455] (1S,9S)-1-(2-Acetoxyacetamido)-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-9-yl 2-(4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)phenyl)acetate (B-04-2) (200 mg, 0.122 mmol) was dissolved in THF (3 mL) and MeOH (3 mL), and an aqueous solution (1 mL) of sodium carbonate (25.88 mg, 0.224 mmol) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. Dilute hydrochloric acid was added to the reaction solution to neutralize the reactants, and the solution was concentrated under reduced pressure and directly proceeded to the next step.

[0456] The structural characteristic data were as follows: ESI-MS m / z: 1596.7 [M+1]+.

[0457] Step 4: Synthesis of (1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-9-yl 2-(4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)phenyl)acetate (B-04-4)

[0458] (1S,9S)-5-Chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-9-yl 2-(4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)phenyl)acetate (B-04-3) (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.5 mL) was added, and the reaction was continued for 1 hour. After adding saturated aqueous sodium hydrogen carbonate solution to the reaction mixture for neutralization, liquid separation was performed, and the organic phase was concentrated to obtain a crude product. After purification by a reverse-phase chromatography column (acetonitrile / 1% aqueous formic acid solution = 0% - 50%) and lyophilization, the title compound (95 mg, 69.35 μmol) was obtained.

[0459] The structure characteristic data were as follows: ESI-MS m / z: 1323.6 [M+1]+.

[0460] Step 5: Synthesis of (1S,9S)-5-Chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-9-yl 2-(4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)phenyl)acetate (B-04)

[0461] (1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-9-yl 2-(4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamide)phenyl)acetate (B-04-4) (90 mg, 0.066 mmol) and N-ethenyl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL), cuprous bromide (9.42 mg, 0.066 mmol) was added, and stirring was continued for 2 hours. The reaction solution was directly filtered, the crude product was concentrated, purified by preparative high performance liquid chromatography, and lyophilized to obtain the title compound (42.2 mg, 24.69 μmol).

[0462] The structural characterization data were as follows: ESI-MS m / z: 1628.7 [M+1]+.

[0463] The preparative high performance liquid chromatography method is as follows.

[0464] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0465] Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid)

Table 28

[0466] Example 11: Preparation of Antibody

[0467] Human-mouse chimeric antibodies, 22B6D2 (heavy chain variable region, SEQ ID NO: 15, light chain variable region, SEQ ID NO: 16), 47A3E3 (heavy chain variable region, SEQ ID NO: 32, light chain variable region, SEQ ID NO: 33) and 100H7D3 (heavy chain variable region, SEQ ID NO: 46, light chain variable region, SEQ ID NO: 47) were obtained by immunizing H2L2 mice (provided by Harbour BioMed, and the antibodies produced by the mice were chimeric antibodies consisting of complete human variable regions and mouse constant regions) by hybridoma screening, combining the above heavy chain variable region sequences with the human IgG1 heavy chain constant region (SEQ ID NO: 50) respectively, and combining the above light chain variable region sequences with the human IgG1 light chain constant region (SEQ ID NO: 51) respectively, thereby forming three complete fully human antibodies (see Table 1). After codon optimization, they were constructed into the pTT5 plasmid, and the pTT5 plasmids corresponding to each antibody heavy chain and light chain were co-transfected into CHO-S cells, and the expressed antibodies in the supernatant were purified using Protein A to obtain the corresponding antibodies.

[0468] The HER3 control antibody was derived from U1-59 of Patent Application No.: CN200680049887. After codon optimization, the nucleotide sequences of the antibody heavy chain and light chain were synthesized, cloned into the pTT5 vector, and then expressed and purified according to the above method.

[0469]

Table 29

[0470] Example 12: Binding of Compounds Containing Cell Bioactive Molecules and Linkers to Antibodies

[0471] The antibodies 22B6, 47A3 and 100H7 involved in the antibody-drug conjugates prepared in the following examples were the antibody 22B6D2-hIgG1, 47A3E3-hIgG1 and 100H7D3-hIgG1 described in Section 2, respectively.

[0472] The binding for sample preparation was described as follows. Take 0.46 mL of antibodies 22B6, 47A3, 100H7, U1-59 and hIgG1 (each concentration adjusted to 11.0 mg / mL), dilute with 0.1 M sodium dihydrogen edetate solution (pH 7.7), then adjust the pH to 7.7 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution, mix well, and let stand at room temperature for 90 minutes. To the solution obtained as above, add a "drug-linker" compound 4.0 to 10 times the mass dissolved in dimethyl sulfoxide, mix well, let stand at room temperature for 2 hours, then use a NAP-5 gel column (Cytiva) to replace the buffer with 10 mM pH 6.0 histidine buffer, add sucrose and Tween 20, mix well to obtain an antibody-drug conjugate (i.e., an ADC compound). See Table 2.

[0473] Determine the drug-to-antibody ratio (DAR value) of the conjugated sample as follows: Use LC-MS to determine the molecular weight of the ADC sample and calculate the drug-to-antibody ratio (DAR value). Subject the conjugated ADC sample to molecular weight analysis by LC-MS.

[0474] Chromatography determination conditions: Liquid chromatography column: Thermo mAbPac RP 3.0 * 100 mm, Mobile phase A: 0.1% FA / H 2 O, mobile phase B: 0.1% FA / ACN, Flow rate: 0.25 mL / min, sample chamber temperature: 8 °C, column temperature: 60 °C, injection volume: 2 μL,

Table 30

[0475] Mass spectrometry conditions: Mass spectrometer model: AB Sciex Triple TOF 5600+ GS1 35, GS2 35, CUR 30, TEM 350, ISVF 5500, DP 250, CE 10, acquisition time 0.5 s m / z 600 - 4000, with a total of 40 time bins.

[0476]

Table 31

Chemical formula

[0477]

Table 32

[0478]

Table 33

[0479] The calculated drug - antibody ratio of ADC 22B6 - A - 07 - A - 1 was DAR = 7.99. See Table 4. 2. The molecular weight of ADC 22B6 - A - 07 - A - 2 was determined by LC - MS, and the drug - antibody ratio (DAR value) was calculated. The molecular weight analysis of the bound ADC 22B6 - A - 07 - A - 2 by LC - MS is shown in Table 5.

Chemical formula

[0480]

Table 34

[0481]

Table 35

[0482] The calculated drug-antibody ratio of ADC 22B6-A-07-A-2 was DAR = 3.56. See Table 6. 3. The molecular weight of ADC 47A3-A-07-A was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the bound ADC 47A3-A-07-A by LC-MS is shown in Table 7.

Chemical formula

[0483]

Table 36

[0484]

Table 37

[0485] The calculated drug-antibody ratio of ADC 47A3-A-07-A was DAR = 6.27. See Table 8. 4. The molecular weight of ADC 100H7-A-07-A was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the bound ADC 100H7-A-07-A by LC-MS is shown in Table 9.

[0486]

Table 38

[0487]

Table 39

[0488] The calculated drug-antibody ratio of ADC 100H7-A-07-A was DAR = 8.01. See Table 10. 5. The molecular weight of ADC U1-59-A-07-A was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the coupled ADC U1-59-A-07-A by LC-MS is shown in Table 11.

Chemical formula

[0489]

Table 40

[0490]

Table 41

[0491] The calculated drug-antibody ratio of ADC U1-59-A-07-A was DAR = 8.02. See Table 12. 6. The molecular weight of ADC hIgG1-A-07-A was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the conjugated ADC hIgG1-A-07-A by LC-MS is shown in Table 13.

Chemical formula

[0492]

Table 42

[0493]

Table 43

[0494] The calculated drug-antibody ratio of ADC hIgG1-A-07-A(8) was DAR = 8.03. See Table 14. 7. The molecular weight of ADC 22B6-A-01 was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the conjugated ADC 22B6-A-01 by LC-MS is shown in Table 15.

Chemical formula

[0495]

Table 44

[0496]

Table 45

[0497] The calculated drug-antibody ratio of ADC 22B6-A-01 was DAR = 7.93. See Table 16. 8. The molecular weight of ADC U1-59-A-01 was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the coupled ADC U1-59-A-01 by LC-MS is shown in Table 17.

Chemical formula

[0498]

Table 46

[0499]

Table 47

[0500] The calculated drug-antibody ratio of ADC U1-59-A-01 was DAR = 7.89. See Table 18. 9. The molecular weight of ADC 22B6-B-03-1 was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the conjugated ADC 22B6-B-03-1 by LC-MS is shown in Table 19. [Chemical formula]

[0501] [Table 48]

[0502] [Table 49]

[0503] The calculated drug-antibody ratio of ADC 22B6-B-03-1 was DAR = 8.0. See Table 20. 10. The molecular weight of ADC 22B6-B-03-2 was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the conjugated ADC 22B6-B-03-2 by LC-MS is shown in Table 21. [Chemical formula]

[0504] [Table 50]

[0505] [Table 51]

[0506] The calculated drug-antibody ratio of ADC 22B6-B-03-2 was DAR = 3.14. See Table 22. 11. The molecular weight of ADC hIgG1-B-03-1 was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the conjugated ADC hIgG1-B-03-1 by LC-MS is shown in Table 23.

Chemical formula

[0507]

Table 52

[0508]

Table 53

[0509] The calculated drug-antibody ratio of ADC hIgG1-B-03-1 was DAR = 7.90. See Table 24. 12. The molecular weight of ADC hIgG1-B-03-2 was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the conjugated ADC hIgG1-B-03-2 by LC-MS is shown in Table 25.

Chemical formula

[0510]

Table 54

[0511]

Table 55

[0512] The calculated drug-antibody ratio of ADC hIgG1-B-03-2 was DAR = 4.20. See Table 26. 13. The molecular weight of ADC 22B6-B-01 was determined by LC-MS, and the drug-to-antibody ratio (DAR value) was calculated. The molecular weight analysis of the conjugated ADC 22B6-B-01 by LC-MS is shown in Table 27.

Chemical formula

[0513]

Table 56

[0514]

Table 57

[0515] The calculated drug-to-antibody ratio of ADC 22B6-B-01 was DAR = 8.07. See Table 28. 14. The molecular weight of ADC hIgG1-B-01 was determined by LC-MS, and the drug-to-antibody ratio (DAR value) was calculated. The molecular weight analysis of the conjugated ADC hIgG1-B-01 by LC-MS is shown in Table 29.

Chemical formula

[0516]

Table 58

[0517]

Table 59

[0518] The calculated drug-to-antibody ratio of ADC hIgG1-B-01 was DAR = 8.06. See Table 30. 15. The molecular weight of ADC hIgG1-A-01 was determined by LC-MS, and the drug-antibody ratio (DAR value) was calculated. The molecular weight analysis of the conjugated ADC hIgG1-A-01 by LC-MS is shown in the following table.

[0519]

Table 60

[0520] Example 13a: Binding of ADC 22B6-B-04

[0521] 0.854 ml of 22B6 antibody (23.43 mg / ml) was diluted with 42.68 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjusted to pH 7.57 using 1 M Na2HPO4 solution, then 20 mM TCEP (tris(2-carboxyethyl)phosphine, 73.68 μL, pH 7.60) was added and mixed well, and left to stand at room temperature for 1.5 hours. Then, 12-fold mass of B-04 dissolved in dimethyl sulfoxide (164 μl, 10 mM) was slowly added, mixed well, left to stand at room temperature for 2 hours, and then the buffer was replaced with 20 mM pH 6.0 histidine buffer using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-01 with a DAR value of 8.62 measured by mass spectrometry.

[0522] Example 13b: Binding of ADC 22B6-B-04 and ADC hIgG1-B-04

[0523] Alternatively, ADC 22B6-B-04 was prepared as follows: 2.667 ml of 22B6 antibody (18.75 mg / ml) was diluted with 133.35 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjusted to pH 7.63 using 1 M Na2HPO4 solution, then 10 mM TCEP (tris(2-carboxyethyl)phosphine, 184.19 μL, pH 7.60) was added and mixed well, and left standing at room temperature for 1.5 hours. Then, 10-fold mass of B-04 dissolved in dimethyl sulfoxide (341.73 μl, 10 mM) was slowly added, mixed well, left standing at room temperature for 2 hours, and then the buffer was replaced with 20 mM pH 6.0 histidine buffer using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-02 with a DAR value of 6.87 measured by mass spectrometry.

[0524] Example 13c: Binding of ADC hIgG1-B-04

[0525] The ADC hIgG1-B-04 sample was prepared using the same preparation method as in Example 13b, replacing it with hIgG1. The DAR value measured by mass spectrometry was 7.02.

[0526] Example 14: Detection of the activity of antibody-drug conjugates

[0527] 1. Detection of the cell affinity of anti-human HER3 antibodies and their drug conjugates

[0528] The affinity of anti-human HER3 ADC for MDA-MB-453, NCI-H358, KPL-4, and A431 cells was detected by flow cytometry (Beckman, model Cytoflex).

[0529] The attached MDA-MB-453, NCI-H358, KPL-4, and A431 cells were digested with Tryple (manufacturer: Gibco) solution, counted, an appropriate amount of cells was collected, washed twice with 1×PBS, resuspended in 1% BSA in PBS solution, and then transferred to a 96-well flat-bottom plate at 50 μl per well. The candidate antibody and its drug conjugate were diluted with 1% BSA in PBS solution starting from 5 μg / mL by serial dilution at 3-fold or 2.5-fold. Then, 50 μl of the diluted antibody or antibody-drug conjugate was added to the flat-bottom plate containing the cells, incubated at 4°C for 40 minutes, the cells were washed twice with PBS, 50 μl of the diluted secondary antibody was added to each well, mixed, incubated at 4°C for 30 minutes, the cells were washed twice with PBS, resuspended in 400 μl of PBS, and detected by flow cytometry. Data processing: After exporting the average fluorescence signal value, it was input into GraphPad Prism 6 software to calculate the EC50. The results are shown in Table 32 and Figures 1A, 1B, 1C, and 1D. The above results indicate that the affinity of the ADCs obtained by binding the antibodies of the present invention (e.g., 22B6, 47A3, and 100H7) to cells is significantly higher than that of the ADCs obtained by binding the control antibody U1-5, and the affinity of the naked antibodies of the present invention (e.g., 22B6) to cells is significantly higher than that of the naked control antibody U1-59. Furthermore, when the naked antibody was formed into an ADC by binding, the affinity to cells was hardly affected, and the affinity of 22B6 formed into an ADC by binding was still the highest.

[0530]

Table 61

[0531] 2. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugate

[0532] MDA-MB-453, HCC1569, and HEK293T-hHER3 cells were digested with TrypLE (manufacturer: Gibco) solution, counted, an appropriate amount of cells was collected, diluted with growth medium, and seeded in a 96-well plate at 5000 cells or 3000 cells (100 μl) / well, and cultured overnight at 37 °C, 5% CO 2 Then, the next day, the ADC was diluted from 150 μg / ml in growth medium by 2.5-fold serial dilution. Then, 100 μl of the diluted ADC was collected and added to the 96-well plate containing the cells. The plate was placed in an incubator and incubated at 37 °C and 5% CO 2 for 6 days. After that, 20 μl of CCK8 was added to each well, incubated at 37 °C for 2 - 5 hours, the OD450nm absorbance value was read with a microplate reader, and the original data was input into Graph Prism 6 to calculate the IC 50 As shown in Table 33 and Figures 2A, 2B, and 2C, the ADCs formed by the antibodies of the present invention (for example, 22B6-A-01, 22B6-A-07-A-1) have a target-specific killing effect, and the IC 50 of 22B6-A-07-A-1 against tumor cells is 6 - 8 times different from the IC 50 of hIgG1-A-01 or hIgG1-A-07-A. hIgG1-A-01 and hIgG1-A-07-A do not show an obvious killing effect against overexpressing cells (HEK293T-hHER3), and the IC 50 of ADCs such as 22B6-A-01 and 22B6-A-07-A-1 is more significantly different from the IC 50 of hIgG-A-01 or hIgG-A-07-A.

[0533]

Table 62

[0534] 3. Bystander effect of anti-human HER3 antibody-drug conjugate

[0535] HEK293T and HEK293T-hHER3 cells were digested with 0.25% Trypsin-EDTA (manufacturer: Gibco), counted, an appropriate amount of cells was collected, washed twice with 1×PBS, and after adjusting the cell density to 1×10 6 cells / ml, Cell Trace Violet was added at a final concentration of 5 μM for labeling, mixed well, incubated in an incubator for 20 minutes, then the cells were washed with growth medium and counted, and seeded into 24-well plates (500 μl / well) according to the following ratios: HEK293T labeled:HEK293T = 4000:12000 cells / well, HEK293T labeled:HEK293T-hHER3 = 4000:12000 cells / well, HEK293T-hHER3 labeled:HEK293T-hHER3 = 4000:12000 cells / well, and cultured overnight at 37 °C, 5% CO 2 2

[0536] The next day, the ADC was diluted with growth medium to concentrations of 20 nM, 4 nM, and 0.8 nM, then 500 μl of the diluted ADC was collected and added to the corresponding 24-well plates containing cells, and the plates were placed in an incubator at 37 °C, 5% CO 2 for 4 days of culture,

[0537] HEK293T and HEK293T-hHER3 cells were digested with 0.25% trypsin-EDTA, added to growth medium containing PI at a final concentration of 3 μM, mixed well, incubated at room temperature for 15 minutes, and then detected by flow cytometry (Thermo, model: Attune NxT). The results are shown in Figures 3A, 3B, and 3C. 22B6-A-07-A-1 and U1-59-A-01 basically did not kill HEK293T negative cells, but had a significant specific killing effect on HEK293T-HER3. 22B6-A-07-A-1 and U1-59-A-01 had a significant killing effect on negative cells under the condition of coexistence of negative cells HEK293T and positive cells HEK293T-HER3, indicating that 22B6-A-07-A-1 and U1-59-A-01 have bystander killing activity.

[0538] 4. Detection of Plasma Stability of Anti-Human HER3 Antibody-Drug Conjugate

[0539] 3.59 μl of 20.9 mg / ml 22B6-A-07-A-1 ADC sample was accurately weighed, transferred to an EP tube, and 496 μl of human, cynomolgus monkey, and mouse blank plasma were added respectively, and mixed well to obtain human, cynomolgus monkey, and mouse plasma samples containing 22B6-A-07-A-1 ADC. The samples were placed in a thermostatic incubator and incubated at 37 °C. After 1, 3, 6, 24, 48, 96, and 168 hours, 50 μl of each sample was taken, 200 μl of 0.5% formic acid-acetonitrile was added to precipitate proteins, centrifuged at 13,000 rpm for 10 minutes at 4 °C, and then the supernatant was taken and stored at -80 °C. After all samples were collected, the concentration of the cytotoxic drug was detected by LC-MS / MS. The results are shown in Figure 4, indicating that the release of the cytotoxic drug did not exceed 5%, demonstrating that the anti-human HER3 antibody-drug conjugate (e.g., 22B6-A-07-A-1) of the present invention is stable in the plasma of different species.

[0540] 5. Detection of In Vivo Drug Efficacy of Anti-Human HER3 Antibody-Drug Conjugate in H358 Model

[0541] NCI-H358 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37 °C, 5% CO 2 and. Logarithmically growing NCI-H358 cells were collected, resuspended in PBS at an appropriate concentration, and subcutaneously inoculated into female BALB / c-nu mice to establish a non-small cell lung cancer model. When the average tumor volume was about 130 - 150 mm 3When it was, the animals were randomly divided into 11 groups according to tumor size, including a vehicle control group (i.e., negative control group), hIgG1-A-01 1 mg / kg group, U1-59-A-01 1 mg / kg group, ADC 22B6-A-01 1 mg / kg group, hIgG1-B-01 1 mg / kg group, ADC 22B6-B-01 1 mg / kg group, hIgG1-A-07-A 1 mg / kg group, ADC 22B6-A-07-A-1 1 mg / kg and 3 mg / kg groups, hIgG1-B-03-1 1 mg / kg group, ADC 22B6-B-03-1 1 mg / kg group. All animals were administered by injection via the tail vein a total of 3 times on days 0, 1, 4, and 10. After administration, the tumor diameter was measured with calipers twice a week, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 wherein, a and b represent the major axis and minor axis of the tumor, respectively, and the death of the animals was observed and recorded daily. The specific results are shown in Table 34, Figure 5A, and Figure 5B.

[0542] The tumor growth inhibition rate TGI (%) was calculated using the following formula and used to evaluate the tumor inhibitory efficacy. TGI (%) = [1 - (VT end - VT start ) / (VC end - VC start )] × * 100% wherein, VT end represents the average value of the tumor volume at the end of the experiment in the treatment group. VT start represents the average value of the tumor volume at the start of administration in the treatment group. VC end represents the average value of the tumor volume at the end of the experiment in the negative control group. VC start represents the average value of the tumor volume at the start of administration in the negative control group.

[0543] From the experimental results, it was found that the ADC of the present invention has a significant tumor growth inhibitory effect on the NCI-H358 non-small cell lung cancer xenograft model. Compared with the vehicle control group, after 3 administrations, the data on the 32nd day showed that the tumor growth inhibition rates (TGI) of the ADC 22B6-A-01 1 mg / kg group and the 22B6-B-01 1 mg / kg group were 79.80% and 64.68%, respectively, the TGI of the 22B6-A-07-A-1 1 mg / kg group and the 3 mg / kg group were 81.22% and 111.53%, respectively, and the TGI of the 22B6-B-03-1 1 mg / kg group was 121.45%. The TGI of the positive control U1-59-A-01 1 mg / kg group was 44.51%. On the 32nd day, there was no death of animals and no significant weight loss of animals in each of the treatment groups, and no obvious drug toxicity was observed. During the treatment period, the mice showed good tolerance to the ADC of the present invention.

[0544]

Table 63

Number

Number

[0545] 6. Detection of in vivo drug efficacy of anti-human HER3 antibody-drug conjugate in N87 model

[0546] NCI-N87 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum under 5% CO 2, cultured at 37°C. NCI-N87 cells in the logarithmic growth phase were collected, resuspended in PBS to an appropriate concentration, and subcutaneously inoculated into female BALB / c-nu mice to establish a human gastric cancer model. When the average tumor volume was approximately 120 mm 3 , the animals were randomly divided into groups including a vehicle control group, an hIgG1-A-07-A 3 mg / kg group, an ADC 22B6-A-07-A-1 3 mg / kg group, an ADC 47A3-A-07-A 3 mg / kg group, and an ADC 100H7-A-07-A 3 mg / kg group according to tumor size. All animals were administered by injection via the tail vein a total of 3 times on days 0, 4, and 7. After administration, the tumor diameter was measured twice a week with calipers, the tumor volume was calculated according to the following formula: V = 0.5a × b2, where a and b represent the major and minor axes of the tumor, respectively, and the death of the animals was observed and recorded daily. The specific results are shown in Table 35, Figure 6A, and Figure 6B.

[0547] From the experimental results, compared with the vehicle control group, after 3 administrations, the data on day 32 showed that the ADC 22B6-A-07-A-1 3 mg / kg group, the 47A3-A-07-A 3 mg / kg group, and the 100H7-A-07-A 3 mg / kg group had growth inhibition rates (TGI) of 66.59%, 47.24%, and 36.04%, respectively. The ADC 22B6-A-07-A-1 3 mg / kg group and the 47A3-A-07-A 3 mg / kg group showed a significant inhibitory effect on tumor growth in the NCI-N87 gastric cancer xenograft model. On day 32, there was no death of animals and no significant weight loss of animals in each of the treatment groups, and no obvious drug toxicity was observed. During the treatment period, the mice showed good tolerance to the ADC of the present invention.

[0548]

Table 64

[0549] 7. Detection of in vivo drug efficacy of anti-human HER3 antibody-drug conjugate in MDA-MB-453 model

[0550] The human breast cancer cell line MDA-MB-453 was cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37 °C and 5% CO 2 2. The logarithmic growth phase MDA-MB-453 cells were collected, resuspended to an appropriate concentration by adding PBS and Matrigel at a final concentration of 50%, and subcutaneously inoculated into female NCG immunodeficient mice to establish a human breast cancer xenograft tumor model. When the average volume of the tumor was about 150 mm 3 3, the animals were randomly divided into groups according to tumor size, including a vehicle control group, an hIgG1-A-07-A 3 mg / kg group, an ADC 22B6-A-07-A-1 1 mg / kg group, a 3 mg / kg group, and a 10 mg / kg group, an hIgG1-A-01 3 mg / kg group, a U1-59-A-01 3 mg / kg group and a 10 mg / kg group, an hIgG1-B-03-2 3 mg / kg group, and an ADC 22B6-B-03-2 3 mg / kg group. After grouping, the animals were administered a single dose by injection via the tail vein on day 0. After administration, the tumor volume and body weight of the mice were observed and measured regularly, and the specific results are shown in Table 36 and Figures 7A and 7B.

[0551] The ADC of the present invention had a significant inhibitory effect on the tumor growth of NCG mice subcutaneously transplanted with human breast cancer MDA-MB-453. The data collected on the 32nd day after a single administration showed that, compared with the vehicle control group, the TGI values of the 22B6-A-07-A-1, 1 mg / kg group, 3 mg / kg group, and 10 mg / kg group were 62.07%, 118.30%, and 200.00% respectively, the tumors in the 10 mg / kg group completely regressed from the 19th day, and no growth was observed until the 32nd day. The TGI value of the 22B6-B-03-2 3 mg / kg group was 148.58%, and the TGI values of the positive control U1-59-A-01 3 mg / kg group and 10 mg / kg group were 76.11% and 180.65% respectively. During the treatment period, the mice showed good tolerance to the ADC of the present invention.

[0552]

Table 65

[0553] 8. Detection of the cell affinity of anti-human HER3 antibodies and their drug conjugates

[0554] The affinity of the anti-human HER3 ADC for MDA-MB-453 cells was detected by flow cytometry (Beckman, model Cytoflex).

[0555] The attached MDA-MB-453 cells were digested with Tryple (manufacturer: Gibco) solution, counted, an appropriate amount of cells was collected, washed twice with 1×PBS, resuspended in 1% BSA solution, and then transferred to a 96-well flat-bottom plate at 50 μl per well. The candidate antibody and its drug conjugate were diluted with 1% BSA by serial three-fold dilution starting from 15 μg / mL, and then 50 μl of the diluted antibody or antibody-drug conjugate was added to the flat-bottom plate containing the cells, incubated at 4°C for 40 minutes, the cells were washed twice with PBS, then 50 μl of the diluted secondary antibody was added to each well, mixed, incubated at 4°C for 30 minutes, the cells were washed twice with PBS, resuspended in 200 μl of PBS, and detected by flow cytometry. Data processing: After exporting the average fluorescence signal value, it was input into GraphPad Prism 6 software to calculate the EC50. The results are shown in Table 37 and Figure 9. The above results indicated that the affinity of the ADC obtained by binding the antibody of the present invention (e.g., 22B6) to cells was significantly higher than that of the ADC obtained by binding the control antibody U1-59.

[0556]

Table 66

[0557] 9. Detection of the endocytosis effect of anti-human HER3 antibody conjugates

[0558] The endocytosis effect of the anti-human HER3 ADC in MDA-MB-453 cells was detected by flow cytometry (Beckman, model Cytoflex).

[0559] Subsequently, the cells were digested with trypsin-EDTA (0.25%, Shanghai Basal Media Technologies) solution, counted, and after adjusting the cell concentration to 1×105 / ml with complete medium, 100 μl of the cell suspension was added to a 96-well plate (the number of cells was 1×104 / well), and the 96-well plate was incubated at 37 °C for 24 hours in a constant-temperature CO2 incubator. Next, the 96-well plate was taken out, the medium was discarded, 50 μl of fresh complete culture medium was added to each well, the bispecific antibody and the control antibody were diluted by serial dilution with complete culture medium, and the final concentrations were 0.55, 1.64, 4.94, 14.81, 44.44, 133.33, 400, 1200 ng / ml, a total of 8 concentrations. The pHrodo solution (Thermo) was diluted to 12 μg / ml with complete medium (the final concentration of pHrodo was 3 μg / ml), the serially diluted antibody candidates and the diluted pHrodo solution were mixed at a ratio of 1:1 (30 μl:30 μl), incubated at room temperature in the dark for 30 minutes, 50 μl of the mixture of the candidate antibody and the pHrodo solution was added to the 96-well plate, cultured at 37 °C and 5% CO2 for 24 hours, the 96-well plate was taken out, the medium was discarded, washed once with sterile PBS, then the cells were digested with 100 μl / well of trypsin-EDTA (0.25%), neutralized with 100 μl of complete culture medium, and then the cells were separated by pipetting up and down and detected by flow cytometry. Data processing: After exporting the average fluorescence signal value, it was input into GraphPad Prism 6 software to calculate the EC50. The results are shown in Table 38 and Figures 10 and 11, which indicated that the endocytosis effect of 22B6 and its conjugated ADC was better than or not worse than that of the control antibody U1-59 and its conjugated ADC.

[0560]

Table 67

[0561] 10. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugate

[0562] The MDA-MB-453 cells were digested with TrypLE (manufacturer: Gibco) solution, counted, an appropriate amount of cells was collected, diluted with growth medium, seeded into a 96-well plate at 5000 cells (100 μl) / well, and incubated at 37 °C, 5% CO 2 overnight. The next day, the ADC was diluted by 2.5-fold serial dilution starting from 150 μg / ml in growth medium, then 100 μl of the diluted ADC was collected and added to the 96-well plate containing the cells. The plate was placed in an incubator and incubated at 37 °C and 5% CO2 for 6 days. Then, 20 μl of CCK8 was added to each well and incubated at 37 °C for 2 - 5 hours. The OD450nm absorbance value was read with a microplate reader, and the original data was input into Graph Prism 6 to calculate the IC50. As shown in Table 39 and Figure 12, both 22B6-A-07-A-1 and 22B6-B-04-2 have a significant killing effect on MDA-MB-453 cells.

[0563]

Table 68

[0564] Although the present invention is described herein with reference to the illustrated embodiments, it should be understood that the present invention is not limited thereto. Those skilled in the art and those having access to the teachings herein will recognize additional modifications and embodiments within its scope. Accordingly, the present invention is limited only by the appended claims.

Claims

**Claim 1** Formula Ab−[M−L−ED] ｘ An antibody-drug conjugate having a structure represented by the formula, wherein Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3, M is a binding site that binds to the antibody or an antigen-binding fragment thereof, L is a linker that connects the binding site M and E, E is a fragment that connects L and D, D is a fragment of a cytotoxic drug, x is an antibody-drug conjugate selected from 1 to 10. **Claim 2** The antibody or an antigen-binding fragment thereof is (1) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Chothia numbering system, a heavy chain variable region (VH) and / or a light chain variable region (VL): (1a) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, a heavy chain variable region (VH), and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, a light chain variable region (VL), or (1b) The following three CDRs: a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof, a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof, a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, a heavy chain variable region (VH), and / or the following three CDRs: a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, a light chain variable region (VL), or (1c) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 37 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, the variant according to any one of items (1a), (1b), and (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared to the amino acid sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions when compared to the amino acid sequence from which it is derived, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each CDR of the VH and VL of (1a), (1b), and (1c), and the variant binds to HER3, or (2) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the Kabat numbering system: (2a) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (2b) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 25 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (2c) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 39 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 40 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, (2a), (2b), and the variant described in any item of (2c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the amino acid sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions compared to the amino acid sequence from which it is derived, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each CDR of the VH and VL of (2a), (2b), and (2c), and the variant binds to HER3, or (3) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the IMGT numbering system: (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 30 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (3c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 42 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 43 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 44 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, and The antibody-drug conjugate according to claim 1, wherein the variant according to any one of items (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the amino acid sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions compared to the amino acid sequence from which it is derived, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each of the CDRs of the VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

3. The antibody or antigen-binding fragment thereof is (a) VH comprising the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof, and / or VL comprising the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof, (b) VH comprising the amino acid sequence set forth in SEQ ID NO: 32 or a variant thereof, and / or VL comprising the amino acid sequence set forth in SEQ ID NO: 33 or a variant thereof, or (c) VH comprising the amino acid sequence set forth in SEQ ID NO: 46 or a variant thereof, and / or VL comprising the amino acid sequence set forth in SEQ ID NO: 47 or a variant thereof, and the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the amino acid sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions compared to the amino acid sequence from which it is derived, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each of the CDRs of the VH and VL of (a), (b), and (c), and the variant binds to HER3, the antibody-drug conjugate according to claim 1 or 2.

4. The antibody or antigen-binding fragment thereof is (a) A human immunoglobulin heavy chain constant region (CH) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived, a human immunoglobulin heavy chain constant region (CH) or a variant thereof, and (b) A human immunoglobulin light chain constant region (CL) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived, a human immunoglobulin light chain constant region (CH) or a variant thereof, The antibody-drug conjugate according to any one of claims 1 to 3, further comprising.

5. The antibody or antigen-binding fragment thereof is (1) A heavy chain comprising a VH containing the amino acid sequence set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) containing the amino acid sequence set forth in SEQ ID NO: 50, and a VL containing the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region (CL) containing the amino acid sequence set forth in SEQ ID NO: 51, (2) A heavy chain comprising a VH containing the amino acid sequence set forth in SEQ ID NO: 32 and a heavy chain constant region (CH) containing the amino acid sequence set forth in SEQ ID NO: 50, and a VL containing the amino acid sequence set forth in SEQ ID NO: 33 and a light chain constant region (CL) containing the amino acid sequence set forth in SEQ ID NO: 51, or (3) A heavy chain comprising a VH containing the amino acid sequence set forth in SEQ ID NO: 46 and a heavy chain constant region (CH) containing the amino acid sequence set forth in SEQ ID NO: 50, and a VL containing the amino acid sequence set forth in SEQ ID NO: 47 and a light chain constant region (CL) containing the amino acid sequence set forth in SEQ ID NO: 51, The antibody-drug conjugate according to any one of claims 1 to 4, comprising a light chain.

6. M is 【Chemical 1】 and In the formula, ring A is a 5- to 6-membered heteroaliphatic ring or a 5- to 20-membered aromatic ring system, and the heteroaliphatic ring and the aromatic ring system are optionally substituted with one or more groups selected from the group consisting of oxy(=O), halogen, cyano, amino, carboxyl, sulfhydryl group, and C １～６ alkyl, and M １ is a single bond and C １～２０ alkylene, C ２～２０ alkenylene, and C ２～２０ alkynylene, and the antibody-drug conjugate according to any one of claims 1 to 5.

7. M is 【Chemical Formula 2】 wherein ring A is a 5-membered heteroaliphatic ring, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings and a benzene ring with a single bond, and the heteroaliphatic ring is optionally substituted with one or more groups selected from the group consisting of oxy(=O), halogen, and C １～４ alkyl, and M １ is a single bond, C ３～１０ alkylene, C ３～１０ alkenylene, and C ３～１０ alkynylene, and the antibody-drug conjugate according to any one of claims 1 to 6.

8. M is [Chemical Formula 3] and ring A is [Chemical Formula 4] selected from the group consisting of M １ is a single bond, C ５～８ alkylene, C ５～８ alkenylene, and C ５～８ alkynylene, and the antibody-drug conjugate according to any one of claims 1 to 7, which is selected from the group consisting of.

9. M is 【Chemical Formula 5】 The antibody-drug conjugate according to any one of claims 1 to 7, which is.

10. M is ​ The antibody-drug conjugate according to any one of claims 1 to 7, which is.

11. L is the following: C １～６ alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH ２ CH ２ (OCH ２ CH ２ ) ｓ OCH ３ )), D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, 【Chemical Formula 7】 a structure composed of one or more selected from, wherein R' is hydrogen, C １～６ alkyl or -(CH ２ CH ２ O) ｒ -containing alkyl, r is an integer selected from 1 to 10, and s is an integer selected from 1 to 20. The antibody-drug conjugate according to any one of claims 1 to 10.

12. L is the following: C １～６ alkylene, -NH-, Phe, Lys, Lys(COCH ２ CH ２ (OCH ２ CH ２ )sOCH ３ )), Gly, Gly-Gly-Phe-Gly, 【Chemical 8】 It is a structure composed of one or more selected from, and s is an integer selected from 1 to 20. The antibody-drug conjugate according to any one of claims 1 to 11.

13. L has the following structure: 【Chemical Formula 9】 Selected from The antibody-drug conjugate according to any one of claims 1 to 11.

14. L has the following structure: 【Chemical Formula 10】 Selected from The antibody-drug conjugate according to any one of claims 1 to 11.

15. L has the following structure: 【Chemical 11】 An antibody-drug conjugate according to any one of claims 1 to 11, selected from

16. E is a single bond, -NH-CH ２ -, -NH-CH ２ -O-CH ２ -CO-, or has the following structure: [Chemical Formula 12] An antibody-drug conjugate according to any one of claims 1 to 15, selected from

17. E is a single bond, -NH-CH ２ -, -NH-CH ２ -O-CH ２ -CO- 【Chemical 13】 An antibody-drug conjugate according to any one of claims 1 to 16, which is

18. E is, -NH-CH ２ -O-CH ２ -CO- 【Chemical Formula 14】 An antibody-drug conjugate according to any one of claims 1 to 16, which is

19. E is -NH-CH ２ -O-CH ２ -CO- or 【Chemical Formula 15】 An antibody-drug conjugate according to any one of claims 1 to 16, which is

20. 【Fig. 16】 has the following structure: 【Chemical 17】 【Chemical 18】 【Chemical Formula 19】 An antibody-drug conjugate according to any one of claims 1 to 19, selected from

21. The antibody-drug conjugate according to any one of claims 1 to 20, wherein the cytotoxic drug is selected from the group consisting of a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor, or a pharmaceutically acceptable salt, ester, or analog thereof.

22. The antibody-drug conjugate according to any one of claims 1 to 21, wherein the tubulin inhibitor is an auristatin compound or a maytansine compound, or a pharmaceutically acceptable salt, ester, or analog thereof.

23. The antibody-drug conjugate according to any one of claims 1 to 21, wherein the DNA intercalator is pyrrolobenzodiazepine (PBD), or a pharmaceutically acceptable salt, ester, or analog thereof.

24. The antibody-drug conjugate according to any one of claims 1 to 21, wherein the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor, or a pharmaceutically acceptable salt, ester, or analog thereof.

25. The antibody-drug conjugate according to claim 24, wherein the topoisomerase I inhibitor is camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, velotecan, or rubitecan, or a pharmaceutically acceptable salt, ester, or analog thereof.

26. The antibody-drug conjugate according to claim 24, wherein the topoisomerase II inhibitor is doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide, or a pharmaceutically acceptable salt, ester, or analog thereof.

27. The antibody-drug conjugate according to any one of claims 1 to 21, wherein the RNA polymerase inhibitor is α-amanitin, or a pharmaceutically acceptable salt, ester or analog thereof.

28. The cytotoxic drug is selected from the compounds represented by Formulas I and II, 【Chemical 20】 wherein R １ and R ２ are each independently selected from the group consisting of C １～６ alkyl and halogen, R ３ is selected from the group consisting of H and -CO-CH ２ OH, and R ４ and R ５ are each independently selected from the group consisting of H, halogen and hydroxyl, or R ４ and R ５ together with the carbon atom to which they are attached form a 5- to 6-membered oxygen-containing heterocycle, R ６ is selected from the group consisting of hydrogen and -C １～４ alkylene-NR ａ R ｂ and is selected from the group consisting of R ７ is selected from the group consisting of C １～６ alkyl and -C １～４ alkylene-NR ａ R ｂ and is selected from the group consisting of In each occurrence, R ａ and R ｂ are each independently H, C １～６ alkyl, -SO ２ -C １～６ alkyl and -CO-C １～６ alkyl, and is selected from the group consisting of, the antibody-drug conjugate according to any one of claims 1 to 21.

29. The cytotoxic drug is the following compound: 【Chemical 21】 selected from the group consisting of, The antibody-drug conjugate according to any one of claims 1 to 21, wherein the corresponding fragment of the cytotoxic drug is after being connected to the linker D of the formula.

30. D is a monovalent structure obtained by losing one H from -OH, -NH ２ or a secondary amine group on the cytotoxic drug, the antibody-drug conjugate according to any one of claims 1 to 29.

31. D is 【Chemical 22】 selected from, the antibody-drug conjugate according to any one of claims 1 to 29.

32. The antibody-drug conjugate is selected from the group consisting of the following ADC A-01 to ADC A-25, ADC B-01 to ADC B-05: 【Chemical 23】 【Chemical 24】 【Chemical 25】 【Chemical 26】 【Chemical 27】 【Chemical 28】 【Chemical 29】 【Chemical 30】 【Chemical 31】 selected from the group consisting of, HA in each antibody-drug conjugate represents an antibody containing VH having the amino acid sequence set forth in SEQ ID NO: 15 and VL having the amino acid sequence set forth in SEQ ID NO: 16 or an antigen-binding fragment thereof, 【Chemical 32】 represents a specific mode in which the sulfhydryl group in the antibody or its antigen-binding fragment binds to the linker, the antibody-drug conjugate according to any one of claims 1 to 31.

33. The antibody or its antigen-binding fragment contains VH having the amino acid sequence set forth in SEQ ID NO: 15 and CH having the amino acid sequence set forth in SEQ ID NO: 50, and VL having the amino acid sequence set forth in SEQ ID NO: 16 and CL having the amino acid sequence set forth in SEQ ID NO: 51, the antibody-drug conjugate according to any one of claims 1 to 32.

34. The antibody-drug conjugate contains the following formula: 【Chemical Formula 33】 【Chemical 34】 including, HA in each antibody-drug conjugate is (1) an antibody containing VH having the amino acid sequence set forth in SEQ ID NO: 15 and VL having the amino acid sequence set forth in SEQ ID NO: 16 or an antigen-binding fragment thereof, for example, an antibody containing VH having the amino acid sequence set forth in SEQ ID NO: 15 and CH having the amino acid sequence set forth in SEQ ID NO: 50, and an antibody containing VL having the amino acid sequence set forth in SEQ ID NO: 16 and CL having the amino acid sequence set forth in SEQ ID NO: 51 or an antigen-binding fragment thereof, (2) an antibody containing VH having the amino acid sequence set forth in SEQ ID NO: 32 and VL having the amino acid sequence set forth in SEQ ID NO: 33 or an antigen-binding fragment thereof, for example, an antibody containing VH having the amino acid sequence set forth in SEQ ID NO: 32 and CH having the amino acid sequence set forth in SEQ ID NO: 50, and an antibody containing VL having the amino acid sequence set forth in SEQ ID NO: 33 and CL having the amino acid sequence set forth in SEQ ID NO: 51 or an antigen-binding fragment thereof, and (3) An antibody or an antigen-binding fragment thereof comprising VH described in SEQ ID NO: 46 and VL described in SEQ ID NO: 47, for example, an antibody or an antigen-binding fragment thereof comprising VH described in SEQ ID NO: 46 and CH described in SEQ ID NO: 50, and VL described in SEQ ID NO: 47 and CL described in SEQ ID NO: 51, selected from The antibody-drug conjugate according to any one of claims 1 to 33, wherein x is 3 to 8. **Claim 35** The antibody-drug conjugate comprises the following formula: 【Chemical 35】 HA is an antibody or an antigen-binding fragment thereof comprising HC described in SEQ ID NO: 17 and LC described in SEQ ID NO: 18, The antibody-drug conjugate according to any one of claims 1 to 33, wherein x is 7 to 8. **Claim 36** The antibody-drug conjugate according to any one of claims 1 to 35, wherein (i) the C-terminus of the heavy chain lacks a lysine residue, (ii) the N-terminus of the heavy chain is glutamine, glutamic acid, or pyroglutamic acid, or (iii) the C-terminus of the heavy chain lacks a lysine residue and the N-terminus of the heavy chain is glutamine, glutamic acid, or pyroglutamic acid. **Claim 37** A drug-linker compound having a structure represented by the formula M’-L-E-D, wherein M’ is 【Chemical 36】 and Lg is a leaving group for a nucleophilic substitution reaction, or is hydroxyl (-OH), a sulfhydryl group (-SH) or amino (-NH ２ ), ring A is a 5- to 6-membered heteroaliphatic ring or a 5- to 20-membered aromatic ring system, and the heteroaliphatic ring and the aromatic ring system are optionally substituted with one or more groups selected from the group consisting of oxy (=O), halogen, cyano, amino, carboxyl, sulfhydryl group and C １～６ alkyl, M １ is a single bond, C １～２０ alkylene, C ２～２０ alkenylene and C ２～２０ alkynylene, and is selected from the group consisting of: L is a linker connecting the binding sites M and E, E is a fragment connecting L and D, D is a fragment of a cytotoxic drug. **Claim 38** Lg is halogen, methylsulfonyl, fluorophenol, or 【Chemical 37】 The drug-linker compound according to claim 37. **Claim 39** The drug-linker compound according to claim 37, wherein Lg forms an unsaturated double bond together with adjacent atoms on ring A. **Claim 40** M’ is 【Chemical 38】 wherein Lg is methylsulfonyl or Lg together with an adjacent atom on ring A forms a carbon-carbon double bond, ring A is a 5-membered heteroaliphatic ring, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings and a benzene ring with single bonds, and the heteroaliphatic ring is optionally substituted with one or more groups selected from the group consisting of oxy(=O), halogen, and C １～４ alkyl, and M １ is a single bond, C ３～１０ alkylene, C ３～１０ alkenylene, or C ３～１０ alkynylene, and the drug-linker compound according to claim 37. **Claim 41** M’ is 【Chemical 39】 where 【Chemical 40】 selected from the group consisting of M １ is a single bond, C ５～８ alkylene, C ５～８ alkenylene, and C ５～８ alkynylene, the drug-linker compound according to claim 37, which is selected from the group consisting of. **Claim 42** M’ is 【Chemical Formula 41】 The drug-linker compound according to claim 37, selected from the group consisting of **Claim 43** M’ is 【Chemical 42】 The drug-linker compound according to claim 37. **Claim 44** A-01 to A-25, B-01 to B-05 shown below: 【Chemical 43】 【Chemical 44】 【Chemical 45】 【Chemical 46】 【Chemical 47】 【Chemical 48】 【Chemical 49】 The drug-linker compound according to any one of claims 37 to 43, selected from the group consisting of **Claim 45** An antibody-drug conjugate comprising an antibody that binds to HER3, which is bound to the drug linker according to any one of claims 37 to 44 via one or more cysteine residues of the antibody. **Claim 46** An antibody-drug conjugate comprising an antibody that binds to HER3 conjugated to a drug-linker selected from the group consisting of A-01, A-02, A-03, A-04, A-05, A-06, A-07, A-08, A-09, A-10, A-12, A13, A-14, A15, A-16, A-17, A-18, A-19, A-20, A-21, A-22, A-23, A-24, A-25, B-01, B-02, B-03, B-04, and B-05 via one or more cysteine residues of the antibody.

47. The antibody that binds to HER3 is (1) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Chothia numbering system: a heavy chain variable region (VH) and / or a light chain variable region (VL): (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (1c) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 37 or a variant thereof, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, selected from the group consisting of (1a), (1b), and (1c) wherein the variant according to any of the items has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each CDR of the VH and VL of (1a), (1b), and (1c), and the variant binds to HER3 or (2) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are a heavy chain variable region (VH) and / or a light chain variable region (VL) defined by the Kabat numbering system: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3, and / or the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6, or (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 25 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 21, and / or the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24, or (2c) Selected from the group consisting of: a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 39 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 40 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 38, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52, The variant according to any one of items (1a), (1b), and (1c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each of the CDRs of the VH and VL of (2a), (2b), and (2c), and the variant binds to HER, or (3) A heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are the heavy chain variable region (VH) and / or the light chain variable region (VL) defined by the IMGT numbering system: (3a) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, or (3b) The following three CDRs: a heavy chain variable region (VH) comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29 or a variant thereof, and / or the following three CDRs: a light chain variable region (VL) comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 30 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 24 or a variant thereof, or (3c) Selected from the group consisting of: a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 42 or a variant thereof, CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 43 or a variant thereof, and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 44 or a variant thereof, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 52 or a variant thereof, (1a), (1b), and (1c) wherein the variant according to any one of the items has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of each CDR of the VH and VL of (3a), (3b), and (3c), and the variant binds to HER3. The antibody-drug conjugate according to claim 45 or 46. [

48. ] The antibody that binds to HER3 is a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof, and / or a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof, b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 or a variant thereof, and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 33 or a variant thereof, or c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 or a variant thereof, and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 47 or a variant thereof, selected from the group consisting of: (1a), (1b), and (1c) wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably the substitutions are conservative substitutions, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity to the amino acid sequence of the CDRs of the VH and VL of (a), (b), and (c), and the variant binds to HER3, the antibody-drug conjugate according to claim 45 or 46.

49. The antibody that binds to HER3 is 1) (i) a heavy chain (HC) comprising a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 15 and a heavy chain constant domain (CH) comprising the amino acid sequence set forth in SEQ ID NO: 50, and (ii) a light chain (LC) comprising a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant domain (CL) comprising the amino acid sequence set forth in SEQ ID NO: 51, an antibody or an antigen-binding fragment thereof, and 2) (i) a HC comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 32 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50, and (ii) a LC comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 33 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, an antibody or an antigen-binding fragment thereof, and An antibody-drug conjugate selected from the group consisting of: (3)(i) an HC comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and a CH comprising the amino acid sequence set forth in SEQ ID NO: 50; and (ii) an LC comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 47 and a CL comprising the amino acid sequence set forth in SEQ ID NO: 51, or an antigen-binding fragment thereof.

50. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 36 or 45 to 49, and one or more pharmaceutically acceptable excipients.

51. A pharmaceutical composition comprising the drug-linker compound according to any one of claims 37 to 44, and one or more pharmaceutically acceptable excipients.

52. A method of treating cancer in a subject having high expression of HER3, the method comprising administering to the subject a therapeutically effective amount of the antibody-drug conjugate according to any one of claims 1 to 36 or 45 to 49, or the pharmaceutical composition according to claim 50.

53. The method according to claim 52, wherein the cancer comprises a solid tumor or a hematological malignancy.

54. The method according to claim 52, wherein the cancer is colon cancer, gastric cancer, breast cancer, lung cancer or lymphoma.

55. The method according to claim 52, wherein the lung cancer is non-small cell lung cancer.

56. The method according to claim 54, wherein the lung cancer is lung adenocarcinoma.

57. Use of the antibody-drug conjugate according to any one of claims 1 to 36 or 45 to 49, or the pharmaceutical composition according to claim 50, in the manufacture of a medicament for the treatment of cancer associated with high expression of HER3.

58. The use according to claim 57, wherein the cancer comprises a solid tumor or a hematological malignancy.

59. The use according to claim 57, wherein the cancer is colon cancer, gastric cancer, breast cancer, lung cancer or lymphoma.

60. The use according to claim 59, wherein the lung cancer is non-small cell lung cancer.

61. The use according to claim 59, wherein the lung cancer is lung adenocarcinoma.

62. Use of the antibody-drug conjugate according to any one of claims 1 to 36 or 45 to 49, or the pharmaceutical composition according to claim 50, in the treatment of cancer associated with high expression of HER3.

63. The use according to claim 62, wherein the cancer comprises a solid tumor or a hematological malignancy.

64. The use according to claim 62, wherein the cancer is colon cancer, gastric cancer, breast cancer, lung cancer or lymphoma.

65. The use according to claim 64, wherein the lung cancer is non-small cell lung cancer.

66. The use according to claim 64, wherein the lung cancer is lung adenocarcinoma.

67. The antibody-drug conjugate according to any one of claims 1 to 36 or 45 to 49, or the pharmaceutical composition according to claim 50, for the treatment of cancer associated with high expression of HER3.

68. The antibody-drug conjugate according to claim 67, wherein the cancer includes solid tumors or hematological malignancies.

69. The antibody-drug conjugate according to claim 67, wherein the cancer is colon cancer, gastric cancer, breast cancer, lung cancer or lymphoma.

70. The antibody-drug conjugate according to claim 69, wherein the lung cancer is non-small cell lung cancer.

71. The antibody-drug conjugate according to claim 69, wherein the lung cancer is lung adenocarcinoma.

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