Antibody drug conjugate and preparation method and application thereof

Abstract

The invention relates to an antibody drug conjugate and a preparation method and application thereof. The present invention relates to antibody drug conjugates for use in the treatment of HER3 positive cancers. The invention provides a fully human-derived HER3 antibody which has excellent binding activity to HER3 positive cells and can be used for efficiently delivering a medicine to the HER3 positive cells. The present application also provides a drug-linker molecule for coupling to the antibody, the drug comprising a DNA topoisomerase inhibitor. The prepared antibody drug conjugate has a better drug-antibody coupling ratio, and has a very good targeted killing effect on colon cancer, gastric cancer, breast cancer and lung cancer (such as non-small cell lung cancer, particularly lung adenocarcinoma). Therefore, the invention further provides a preparation method of the antibody drug conjugate and application of the antibody drug conjugate in treatment of HER3 positive cancers.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese application No. 202310444522.X, filed on April 23, 2023, and Chinese application No. 202210467789.6, filed on April 29, 2022, the disclosures of each of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence table in computer readable form, which has been submitted with this application in XML file format, the entire contents of which are incorporated herein by reference. The sequence table XML file submitted with this application is named "14463-050-228_SEQ_LISTING.xml", created on April 20, 2023, and is 59,247 bytes in size.

[0005] Background of the Invention (1) Technical field

[0006] The present application relates to the field of targeted therapy, and specifically to antibody-drug conjugates and preparation methods and uses thereof. (2) Background technology

[0007] Cancer is one of the leading causes of death in modern times. It is a class of diseases caused by the malignant transformation of healthy cells, caused by genetic changes such as chromosomal translocations, mutations in tumor suppressor genes, growth factor receptors, leading to malignant proliferation of cells. Defective apoptosis or programmed cell death further promotes the malignant transformation of cells leading to cancer.

[0008] Human epidermal growth factor receptor 3 (also known as HER3 and ErbB3) is a receptor tyrosine kinase that belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes HER1 (also known as EGFR), HER2, and HER4. Similar to the EGFR, the transmembrane receptor HER3 consists of an extracellular domain (ECD) that binds to the ligand, a dimerization domain within the ECD, a transmembrane domain, and a carboxyl-terminal phosphorylation domain. In addition to these domains, HER1, HER2, and HER4 also carry an intracellular protein tyrosine kinase domain (TKD), while HER3 lacks this domain and cannot autophosphorylate.

[0009] Ligand regulatory protein (HRG) binds to the extracellular domain of HER3 and activates receptor-mediated signal transduction pathways by promoting dimerization with other human epidermal growth factor receptor (HER) family members and transphosphorylation of their intracellular domains. The dimer formation of HER3 with other HER family members expands the signal transduction potential of HER3, and not only serves as a means of signal diversification, but also as a means of signal amplification. For example, HER2 / HER3 heterodimers induce one of the most important mitogenic signals in HER family members. HER3 is overexpressed in various types of cancers, such as breast cancer, gastrointestinal cancer, and pancreatic cancer. Interestingly, the relationship between the expression of HER2 / HER3 and the progression from the non-invasive stage to the invasive stage has been shown. Therefore, it is desirable to interfere with the medicaments of HER3-mediated signal transduction.

[0010] Currently, the HER3 target indications under clinical research cover hematological tumors and solid tumors. The main treatment strategies focus on several directions such as monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs). Among them, there are currently two anti-HER3 antibody-drug conjugates under international research, and one has entered the clinical research stage (Daiichi Sankyo U3-1402) for the treatment of NSCLC, MBC, and CRC.

[0011] ADC drugs are composed of antibodies, bioactive molecules and linkers. The bioactive molecules are covalently coupled to the antibodies through linkers; antibodies (such as monoclonal antibodies) can specifically recognize specific targets on the surface of tumor cells, and then guide ADC to the surface of cancer cells, and allow ADC to enter cancer cells through cellular endocytosis; then the bioactive molecules are released inside the cancer cells, killing cancer cells while minimizing damage to normal tissue cells.

[0012] U3-1402 was developed by Daiichi Sankyo Co., Ltd., using GGFG tetrapeptide as the enzyme cleavage linker and Dxd as the cytotoxic drug. According to reports, in the first-phase dose expansion (5.6 mg / kg, Q3W) of NSCLC with EGFR mutations, ORR was 39%, DCR was 72%, and mPFS was 8.2 months. It has similar efficacy in patients with brain metastases; in terms of safety, it is mainly blood toxicity (thrombocytopenia, neutropenia, etc.) and interstitial pneumonia (5-7%). Summary of the invention

[0013] In one aspect, the present application provides an antibody drug conjugate having the formula Ab-[MLED] x The structure shown, wherein:

[0014] 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);

[0015] M is a linker site that binds to an antibody or its antigen-binding fragment;

[0016] L is a linker between the linker sites M and E;

[0017] E is a structural fragment connecting L and D;

[0018] D is the cytotoxic drug fragment;

[0019] x is selected from 1 to 10.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0021] (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0022] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 3 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 6 or a variant thereof; or,

[0023] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 21 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 24 or a variant thereof; or,

[0024] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 36 or a variant thereof, CDR-H2 of SEQ ID NO: 37 or a variant thereof, and CDR-H3 of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 45 or a variant thereof, CDR-L2 of SEQ ID NO: 23 or a variant thereof, and CDR-L3 of SEQ ID NO: 52 or a variant thereof;

[0025] wherein the variant described in any one of (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 more 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 with the amino acid sequence of the corresponding CDR of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3;

[0026] or,

[0027] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0028] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 6 or a variant thereof; or,

[0029] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 25 or a variant thereof, CDR-H2 of SEQ ID NO: 26 or a variant thereof, and CDR-H3 of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 22 or a variant thereof, CDR-L2 of SEQ ID NO: 23 or a variant thereof, and CDR-L3 of SEQ ID NO: 24 or a variant thereof; or,

[0030] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 39 or a variant thereof, CDR-H2 of SEQ ID NO: 40 or a variant thereof, and CDR-H3 of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 45 or a variant thereof, CDR-L2 of SEQ ID NO: 23 or a variant thereof, and CDR-L3 of SEQ ID NO: 52 or a variant thereof;

[0031] wherein the variant described in any one of (2a), (2b), and (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 sequence from which it is derived, or the variant has one or more 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 with the amino acid sequence of the corresponding CDR of VH and VL of (2a), (2b), and (2c), and the variant binds to HER3;

[0032] or,

[0033] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0034] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 6 or a variant thereof; or,

[0035] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 27 or a variant thereof, CDR-H2 of SEQ ID NO: 28 or a variant thereof, and CDR-H3 of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 30 or a variant thereof, CDR-L2 of SEQ ID NO: 31 or a variant thereof, and CDR-L3 of SEQ ID NO: 24 or a variant thereof; or,

[0036] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:41 or a variant thereof, CDR-H2 of SEQ ID NO:42 or a variant thereof, and CDR-H3 of SEQ ID NO:43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:44 or a variant thereof, CDR-L2 of SEQ ID NO:31 or a variant thereof, and CDR-L3 of SEQ ID NO:52 or a variant thereof;

[0037] wherein the variant described in any one of (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 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

[0038] In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or antigen-binding fragment thereof via a linker (such as the "MLE" fragment shown in this application).

[0039] In some embodiments, M is Wherein ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 The alkyl group is substituted; M1 is selected from a single bond and C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene;

[0040] L is selected from the following one or more structures: C 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), 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-G ly-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,

[0041] Where R' represents hydrogen, C 1-6 Alkyl or alkyl containing -(CH2CH2O)r-; r is an integer selected from 1-10; s is an integer selected from 1-20;

[0042] E is a single bond, -NH-CH2-, -NH-CH2-O-CH2-CO- or selected from the following structures:

[0043]

[0044] In some embodiments, Select from the following structures:

[0045]

[0046]

[0047]

[0048]

[0049] 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 or a maytansine. 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, belotecan, 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.

[0050] In some embodiments, the cytotoxic drug is selected from the following Formula I and Formula II:

[0051]

[0052] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0053] R3 is selected from H and -CO-CH2OH;

[0054] R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;

[0055] R6 is selected from hydrogen and -C 1-4 Alkylene-NR a R b ;

[0056] R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0057] Where R a , R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6alkyl.

[0058] In a further embodiment, the cytotoxic drug is selected from the following structures:

[0059]

[0060] In some embodiments, the antibody drug conjugate is selected from ADC A-01 to ADC A-25, ADCB-01 to ADC B-05 shown below:

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] wherein HA is any of the antibodies disclosed in the present application and S is the sulfur atom of the side chain of the cysteine ​​residue of the antibody.

[0071] In yet another embodiment, the HA is an antibody comprising:

[0072] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1, CDR-H2 with an amino acid sequence of SEQ ID NO: 2, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3; and, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4, CDR-L2 with an amino acid sequence of SEQ ID NO: 5, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6; or,

[0073] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19, CDR-H2 with an amino acid sequence of SEQ ID NO: 20, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24; or,

[0074] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36, CDR-H2 with an amino acid sequence of SEQ ID NO: 37, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52;

[0075] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7, CDR-H2 with an amino acid sequence of SEQ ID NO: 8, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4, CDR-L2 with an amino acid sequence of SEQ ID NO: 5, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6; or,

[0076] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25, CDR-H2 with an amino acid sequence of SEQ ID NO: 26, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24; or,

[0077] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39, CDR-H2 with an amino acid sequence of SEQ ID NO: 40, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52;

[0078] or,

[0079] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10, CDR-H2 with an amino acid sequence of SEQ ID NO: 11, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13, CDR-L2 with an amino acid sequence of SEQ ID NO: 14, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6; or,

[0080] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27, CDR-H2 with an amino acid sequence of SEQ ID NO: 28, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30, CDR-L2 with an amino acid sequence of SEQ ID NO: 31, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24; or,

[0081] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:41, CDR-H2 with an amino acid sequence of SEQ ID NO:42, and CDR-H3 with an amino acid sequence of SEQ ID NO:43; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:44, CDR-L2 with an amino acid sequence of SEQ ID NO:31, and CDR-L3 with an amino acid sequence of SEQ ID NO:52.

[0082] In yet another embodiment, the HA comprises: a VH comprising the amino acid sequence shown in SEQ ID NO:15, and a VL comprising the amino acid sequence shown in SEQ ID NO:16.

[0083] In yet another embodiment, the HA comprises: a VH comprising the amino acid sequence shown in SEQ ID NO:32, and a VL comprising the amino acid sequence shown in SEQ ID NO:33.

[0084] In yet another embodiment, the HA comprises: a VH comprising the amino acid sequence shown in SEQ ID NO:46, and a VL comprising the amino acid sequence shown in SEQ ID NO:47.

[0085] In another embodiment, the HA comprises: (1) a heavy chain comprising a VH having an amino acid sequence as shown in SEQ ID NO: 15 and a heavy chain constant region (CH) as shown in SEQ ID NO: 50, and a light chain comprising a VL having an amino acid sequence as shown in SEQ ID NO: 16 and a light chain constant region (CL) as shown in SEQ ID NO: 51.

[0086] In another embodiment, the HA comprises: (2) a heavy chain comprising a VH having an amino acid sequence as shown in SEQ ID NO: 32 and a heavy chain constant region (CH) as shown in SEQ ID NO: 50, and a light chain comprising a VL having an amino acid sequence as shown in SEQ ID NO: 33 and a light chain constant region (CL) as shown in SEQ ID NO: 51; or

[0087] In another embodiment, the HA comprises: (3) a heavy chain comprising a VH with an amino acid sequence as shown in SEQ ID NO: 46 and a heavy chain constant region (CH) as shown in SEQ ID NO: 50, and a light chain comprising a VL with an amino acid sequence as shown in SEQ ID NO: 47 and a light chain constant region (CL) as shown in SEQ ID NO: 51.

[0088] In yet another embodiment, the HA comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO:17, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO:18.

[0089] In yet another embodiment, the HA comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO:34, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO:35.

[0090] In yet another embodiment, the HA comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO:48, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO:49.

[0091] In specific embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

[0092] Thus, the composition may comprise a population of multiple antibody drug conjugate species, wherein each species may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid or the N-terminal amino acid cyclized to pyroglutamic acid.

[0093] Therefore, in specific embodiments, the present invention further provides a composition comprising the ADC disclosed herein, wherein the major ADC species in the composition comprises (i) an antibody lacking a lysine residue at the C-terminus of the heavy chain; (ii) an antibody with glutamine, glutamic acid or pyroglutamate 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 with glutamine, glutamic acid or pyroglutamate at the N-terminus of the heavy chain.

[0094] The present application also provides a pharmaceutical composition, which contains the antibody-drug conjugate described in any of the above items, and one or more pharmaceutical excipients.

[0095] The present application also provides a method for treating a HER3-overexpressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody drug conjugate (ADC) as described herein or a pharmaceutical composition thereof. In a specific embodiment, the cancer comprises a solid tumor or a hematological malignancy. In another embodiment, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer or lymphoma. In another embodiment, the lung cancer is non-small cell lung cancer.

[0096] The application also provides the use of an antibody drug conjugate (ADC) or a pharmaceutical composition thereof as described herein in the preparation of a drug for treating a HER3-overexpressing cancer. In a specific embodiment, the cancer comprises a solid tumor or a hematological malignancy. In another embodiment, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer or lymphoma. In another embodiment, the lung cancer is non-small cell lung cancer.

[0097] The present application also provides use of the antibody-drug conjugate or a pharmaceutical composition thereof described herein in treating cancers with high HER3 expression.

[0098] In certain embodiments, the cancer comprises a solid tumor or a hematological malignancy. In yet another embodiment, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In yet another embodiment, the lung cancer is non-small cell lung cancer.

[0099] The application also provides an antibody drug conjugate or pharmaceutical composition as described herein for use in treating HER3 high-expressing cancers. In a specific embodiment, the cancer comprises a solid tumor or a hematological malignancy. In another embodiment, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer or lymphoma. In another embodiment, the lung cancer is non-small cell lung cancer.

[0100] In one embodiment, the HER3-high expressing cancer includes solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0101] The present invention further provides a pharmaceutically acceptable salt or solvate of any of the aforementioned antibody-drug conjugates.

[0102] The present invention further provides a composition comprising any of the aforementioned antibody-drug conjugates and a pharmaceutically acceptable carrier or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1A The results of affinity test between anti-human HER3 antibody-drug conjugate and MDA-MB-453 cells are shown.

[0104] Figure 1B The results of affinity testing of anti-human HER3 antibody-drug conjugates with NCI-H358 cells are shown.

[0105] Figure 1C The results of affinity test between anti-human HER3 antibody-drug conjugate and KPL-4 cells are shown.

[0106] Figure 1D The results of affinity test between anti-human HER3 antibody-drug conjugate and A431 cells are shown.

[0107] Figure 2A The results of in vitro cytotoxicity assay of anti-human HER3 antibody-drug conjugates on MDA-MB-453 cells are shown.

[0108] Figure 2B The results of in vitro cytotoxicity assay of anti-human HER3 antibody-drug conjugates on HCC1569 cells are shown.

[0109] Figure 2CThe results of in vitro cytotoxicity assay of anti-human HER3 antibody-drug conjugates on HEK293T-h HER3 cells are shown.

[0110] Figures 3A to 3C The results of bystander effect detection of anti-human HER3 antibody-drug conjugates on HEK293T and HEK293T-h HER3 cells are shown.

[0111] Figure 4 The results of plasma stability test of anti-human HER3 antibody-drug conjugates are shown.

[0112] Figure 5A The results show the changes in tumor volume of each group of mice in the NCI-H358 CDX model with anti-human HER3 antibody-drug conjugates.

[0113] Figure 5B The results show the changes in body weight of each group of mice in the NCI-H358 CDX model with anti-human HER3 antibody-drug conjugates.

[0114] Fig. 6A The results show the changes in tumor volume of each group of mice in the NCI-N87 CDX model with anti-human HER3 antibody-drug conjugates.

[0115] Figure 6B The results show the changes in body weight of each group of mice in the NCI-N87 CDX model with anti-human HER3 antibody-drug conjugates.

[0116] Fig. 7A The results of the changes in tumor volume of each group of mice in the MDA-MB-453CDX model are shown.

[0117] Figure 7B The results show the changes in body weight of each group of mice in the MDA-MB-453CDX model with anti-human HER3 antibody-drug conjugates.

[0118] Fig. 8A The HPLC spectra of compounds A-07-A and A-07-B prepared in Example 3 are shown.

[0119] Figure 8B The HPLC spectra of compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9 are shown.

[0120] Fig. 9 The results of the affinity test of the anti-human HER3 antibody-drug conjugate to MDA-MB-453 cells are shown.

[0121] Fig.10Shows the results of the endocytic activity test of anti-human HER3 antibody-drug conjugates in MDA-MB-453 cells

[0122] Fig.11 The results of the endocytosis activity test of anti-human HER3 antibody-drug conjugates in HCC1569 cells are shown.

[0123] Fig.12 The results of the cell killing activity test of the anti-human HER3 antibody-drug conjugate are shown. DETAILED DESCRIPTION OF THE INVENTION

[0125] definition

[0126] Unless otherwise defined below, the meaning of all technical terms and scientific terms used herein is the same as that commonly understood by those skilled in the art. Reference to the technology used herein means the technology commonly understood in the art, including those changes in technology that are obvious to those skilled in the art or the replacement of equivalent technology. In addition, the laboratory operation steps such as genomics, nucleic acid chemistry, molecular biology used herein are all conventional steps widely used in the corresponding fields. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0127] The term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the isotype of the antibody 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 consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists 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 domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins 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 regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

[0128] The term "antibody" further includes embodiments in which the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable domain has undergone cyclization to pyroglutamic acid. Thus, in a composition comprising an antibody disclosed herein, each antibody therein may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid or the N-terminal amino acid is cyclized to pyroglutamic acid.

[0129] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable region of the heavy chain and light chain each contains three CDRs, designated CDR1, CDR2, and CDR3. The precise 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 USA86:9268-9272). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0130] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined according to various numbering systems known in the art, such as Kabat, Chothia, IMGT or AbM numbering systems. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof are determined by the Chothia numbering system.

[0131] www.bioinf.org.uk: The following general rules, published in Prof. Andrew CR Martin's Group and reproduced below, can be used to define CDRs in antibody sequences that include those amino acids that specifically interact with the amino acids that make up the epitope of the antigen to which the antibody binds. In rare cases, these normally constant features do not occur. However, Cys residues are the most conserved feature.

[0132] In rare cases, these generally constant features do not occur; however, the Cys residue is the most conserved feature.

[0133]

[0134]

[0135] V H The entire amino acid sequence of is generally numbered according to Kabat, and the three CDRs within the variable region may be defined according to any of the above numbering systems. In a specific embodiment, V H The amino acid positions in the sequence may be numbered sequentially starting from amino acid position 1 to the end of the sequence, or may be numbered according to Kabat. H and V L The amino acid positions in are defined according to sequential numbering.

[0136] The numbering of amino acid positions in the heavy chain constant region can be sequentially numbered starting from amino acid position 1 to the end of the sequence, or can be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, which are numbered from 1 to 330. The corresponding sequence numbered according to Eu starts at position 118 and ends at position 447. Unless otherwise specified, the amino acid positions of the heavy and light chains described herein are defined according to sequential numbering.

[0137] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0138] The term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by 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-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAb, nanobodies), and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0139] The term "Fd" means an antibody fragment consisting of a VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in a F(ab')2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.

[0140] The term "Fv" means an antibody fragment consisting 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 that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a single variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0141] The term "Fc" means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via disulfide bonds. The Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.

[0142] The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., 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, Vol. 113, Roseburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention 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 also 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, containing NH2-VH-VH-COOH, NH 2- scFv of VL-VL-COOH.

[0143] The term "single-domain antibody (sdAb)" has a 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 as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.

[0144] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0145] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0146] Antibody antigen-binding fragments (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

[0147] The terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, and generally contain at least two or more different antibodies, which generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.

[0148] The term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen. For example, the term "chimeric antibody" may include antibodies in which the heavy chain and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy chain and light chain variable regions of the antibody are derived from a second antibody (e.g., a human antibody). For example, an antibody produced by immunizing a fully human transgenic mouse can be called a chimeric antibody, which consists of a fully human variable region and a murine constant region.

[0149] The term "murine antibody" refers to antibodies obtained by the following method: fusing B cells of immunized mice with myeloma cells, screening out mouse hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, and then screening, antibody preparation and antibody purification; or refers to antibodies secreted by plasma cells formed by differentiation and proliferation of B cells after antigens invade the mouse body.

[0150] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes 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) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0151] The term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, two sequences are compared when they are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0152] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions 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).

[0153] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0154] 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, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators 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, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but 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), etc.

[0155] As used herein, the term "solvate" refers to the physical association of an ADC disclosed herein with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonds, including hydrogen bonds. In some cases, the solvate will be able to separate, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses solution phase and separable solvates. Non-limiting examples of solvates include ethanolates, methanolates, etc. "Hydrate" is a solvate in which the solvent molecule is water.

[0156] One or more ADCs disclosed herein may be optionally converted into solvates. The preparation of solvates is well known. Thus, for example, M. Caira et al., J. Pharmaceutical Sci., 93 (3), 601-611 (2004) describes the preparation of solvates of the antifungal fluconazole in ethyl acetate and water. Similar preparations of solvates, hemisolvates, hydrates, etc. are described in EC van Tonder et al., AAPS Pharm Sci Techours., 5 (1), article 12, (2004); and AL Bingham et al., Chem. Commun., 603-604 (2001). A typical non-limiting method includes dissolving the compound of the invention in a desired amount of a desired solvent (organic solvent or water or a mixture thereof) at a temperature above room temperature, and cooling the solution at a rate sufficient to form crystals, and then isolating the crystals by standard methods. Analytical techniques, such as infrared spectroscopy, show that the solvent (or water) in the crystals is present in the form of a solvate (or hydrate).

[0157] The term "pharmaceutically acceptable salt" as used herein includes acid addition salts and basic salts.

[0158] Exemplary acid addition salts include acetate, ammonium, ascorbate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, fumarate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, methanesulfonate (also known as mesylate), naphthylate, nitrate, oxalate, phosphate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate (also known as tosylate), and the like. In addition, acids generally considered suitable for forming pharmaceutically useful salts from the basic pharmaceutical compound NAI-1536717630v1-36- are discussed, for example, P. Stahl et al., Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. 2nd 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 website, Washington, DC). Such disclosures are incorporated herein by reference. In one embodiment, the acid addition salt is an ammonium salt or a diammonium salt.

[0159] Exemplary basic salts include ammonium salts, alkali metal salts such as sodium salts, lithium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts with organic bases (e.g., organic amines) such as dicyclohexylamine, tert-butylamine, choline, and salts with amino acids such as arginine and lysine. Basic nitrogen-containing groups can be replaced with compounds such as lower alkyl halides (e.g., methyl, ethyl and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl sulfate, diethyl and dibutyl sulfate), long chain halides (e.g., decyl, lauryl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and the like.

[0160] All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the disclosure and all acid salts and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the disclosure.

[0161] The terms "comprises," "comprising," "having," "containing," or "involving" and other variations thereof herein are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0162] The term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc. The therapeutically effective amount of an ADC may vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0163] The term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., not worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0164] The term "subject" as used herein refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, a human) suffers from a tumor, or has a risk of suffering from the above-mentioned disease.

[0165] The terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade neighboring tissues and may metastasize to distant parts of 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 blood tumors, especially hematologic malignancies.

[0166] The term "hematologic malignancies" includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphomas. Hematologic malignancies also include leukemias, such as secondary leukemias or acute lymphocytic leukemias. Hematologic malignancies also include myeloma (e.g., multiple myeloma) and other blood and / or B-cell or T-cell related cancers.

[0167] The term "alkyl" refers to a straight chain or branched hydrocarbon radical with one hydrogen atom removed, for example, "C 1-20 Alkyl", "C 1-10 Alkyl", "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 Specific examples include, but are not limited to, 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, and the like.

[0168] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight or branched hydrocarbon group, for example, "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 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.

[0169] The term "alkenylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon double bond losing two hydrogen atoms, including, for example, "C 2-20 Alkenylene", "C 3-10 Alkenylene", "C 5-8Examples include, but are not limited to, vinylene, 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, and the like.

[0170] The term "alkynylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond losing two hydrogen atoms. 2-20 Alkynylidene", "C 3-10 Alkynylidene", "C 5-8 Examples include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, and the like.

[0171] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one ring member selected from N, O and S. Specific examples include, but are not limited to, 5-6 membered aliphatic heterocycles, 5-6 membered nitrogen-containing aliphatic heterocycles, 5-6 membered oxygen-containing aliphatic heterocycles, and the like, such as tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, and the like.

[0172] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O and S. Specific examples include, but are not limited to, 5-6 membered aromatic heterocycles, 5-6 membered nitrogen-containing aromatic heterocycles, 5-6 membered oxygen-containing aromatic heterocycles, and the like, 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, and the like.

[0173] The term "aromatic ring system" refers to a monocyclic or polycyclic system comprising at least one aromatic ring (e.g., benzene ring, etc.) or heteroaromatic ring (e.g., pyrimidine ring, etc.), two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be connected by a single bond (e.g., dipyrimidinylphenyl, etc.), and the aromatic ring system may be divalent or higher valent (e.g., trivalent or tetravalent), for example, a 5-20 membered aromatic ring system.

[0174] 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 range of experimental error (e.g., within a 95% confidence interval for the mean) or within 10% or greater.

[0175] It should be noted that if there is a discrepancy between a described structure and the name of that structure, the described structure will be given greater weight.

[0176] The present application relates to an antibody-drug conjugate (ADC) for treating HER3-positive cancers, and exemplarily discloses an antibody-drug conjugate having a structure as shown in the general formula Ab-[MLED]x, and using the fully human antibody 22B6D2-hIgG1 as a targeting portion. The ADC (or conjugate) of the present invention has an average drug-to-antibody ratio (DAR) of up to 7.99 and exhibits a targeted killing effect on HER3-positive cancers such as colon cancer, gastric cancer, and breast cancer. Cancer, lung cancer (e.g., non-small cell lung cancer, especially lung adenocarcinoma). The results of the present invention show that the conjugate has a good drug-antibody ratio, and the conjugate has excellent binding activity to HER3-positive cells, and has a good targeted killing effect on HER3-positive cancers such as colon cancer, gastric cancer, etc. Cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, especially lung adenocarcinoma). Therefore, the present application provides an antibody-drug conjugate for treating cancers with high HER3 expression, a pharmaceutical composition comprising the antibody-drug conjugate, and its use in treating cancers with high HER3 expression.

[0177] Antibody Drug Conjugates

[0178] In one aspect, the present application provides an antibody drug conjugate having the formula Ab-[MLED] x The structure shown, wherein:

[0179] 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);

[0180] M is a linker site that binds to an antibody or its antigen-binding fragment;

[0181] L is a linker between the linker sites M and E;

[0182] E is a structural fragment connecting L and D;

[0183] D is the cytotoxic drug fragment;

[0184] x is selected from 1 to 10.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0186] (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0187] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 3 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 6 or a variant thereof; or,

[0188] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 21 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 24 or a variant thereof; or,

[0189] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 36 or a variant thereof, CDR-H2 of SEQ ID NO: 37 or a variant thereof, and CDR-H3 of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 45 or a variant thereof, CDR-L2 of SEQ ID NO: 23 or a variant thereof, and CDR-L3 of SEQ ID NO: 52 or a variant thereof;

[0190] wherein the variant described in any one of (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 more 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 with the amino acid sequence of the corresponding CDR of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3;

[0191] or,

[0192] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0193] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 6 or a variant thereof; or,

[0194] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 25 or a variant thereof, CDR-H2 of SEQ ID NO: 26 or a variant thereof, and CDR-H3 of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 22 or a variant thereof, CDR-L2 of SEQ ID NO: 23 or a variant thereof, and CDR-L3 of SEQ ID NO: 24 or a variant thereof; or,

[0195] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 39 or a variant thereof, CDR-H2 of SEQ ID NO: 40 or a variant thereof, and CDR-H3 of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 45 or a variant thereof, CDR-L2 of SEQ ID NO: 23 or a variant thereof, and CDR-L3 of SEQ ID NO: 52 or a variant thereof;

[0196] wherein the variant described in any one of (2a), (2b), and (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 sequence from which it is derived, or the variant has one or more 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 with the amino acid sequence of the corresponding CDR of VH and VL of (2a), (2b), and (2c), and the variant binds to HER3;

[0197] or,

[0198] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0199] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 6 or a variant thereof; or,

[0200] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 27 or a variant thereof, CDR-H2 of SEQ ID NO: 28 or a variant thereof, and CDR-H3 of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 30 or a variant thereof, CDR-L2 of SEQ ID NO: 31 or a variant thereof, and CDR-L3 of SEQ ID NO: 24 or a variant thereof; or,

[0201] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:41 or a variant thereof, CDR-H2 of SEQ ID NO:42 or a variant thereof, and CDR-H3 of SEQ ID NO:43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:44 or a variant thereof, CDR-L2 of SEQ ID NO:31 or a variant thereof, and CDR-L3 of SEQ ID NO:52 or a variant thereof;

[0202] wherein the variant described in any one of (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 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

[0203] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0204] (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0205] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0206] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0207] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0208] wherein the variant described in any one of (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 more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0209] or,

[0210] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0211] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0212] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0213] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 40 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0214] wherein the variant described in any one of (2a), (2b), and (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 sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0215] or,

[0216] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0217] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0218] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0219] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 41 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 42 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 44 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0220] Among them, the variant described in any one of (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 with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions, or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0221] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0222] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0223] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0224] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0225] wherein the variant described in any one of (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 more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0226] or,

[0227] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0228] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0229] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 40 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0230] wherein the variant described in any one of (2a), (2b), and (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 sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0231] or,

[0232] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0233] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0234] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 41 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 42 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 44 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0235] Among them, the variant described in any one of (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 with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions, or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0236] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0237] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1, CDR-H2 with an amino acid sequence of SEQ ID NO: 2, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4, CDR-L2 with an amino acid sequence of SEQ ID NO: 5, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6; or,

[0238] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19, CDR-H2 with an amino acid sequence of SEQ ID NO: 20, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24; or,

[0239] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36, CDR-H2 with an amino acid sequence of SEQ ID NO: 37, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52;

[0240] or,

[0241] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7, CDR-H2 with an amino acid sequence of SEQ ID NO: 8, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4, CDR-L2 with an amino acid sequence of SEQ ID NO: 5, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6; or,

[0242] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25, CDR-H2 with an amino acid sequence of SEQ ID NO: 26, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24; or,

[0243] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39, CDR-H2 with an amino acid sequence of SEQ ID NO: 40, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52;

[0244] or,

[0245] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10, CDR-H2 with an amino acid sequence of SEQ ID NO: 11, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13, CDR-L2 with an amino acid sequence of SEQ ID NO: 14, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6; or,

[0246] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27, CDR-H2 with an amino acid sequence of SEQ ID NO: 28, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30, CDR-L2 with an amino acid sequence of SEQ ID NO: 31, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24; or,

[0247] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:41, CDR-H2 with an amino acid sequence of SEQ ID NO:42, and CDR-H3 with an amino acid sequence of SEQ ID NO:43; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:44, CDR-L2 with an amino acid sequence of SEQ ID NO:31, and CDR-L3 with an amino acid sequence of SEQ ID NO:52.

[0248] In some embodiments, the antibody or its antigen-binding fragment comprises: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1, CDR-H2 with an amino acid sequence of SEQ ID NO: 2, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4, CDR-L2 with an amino acid sequence of SEQ ID NO: 5, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6.

[0249] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19, CDR-H2 with an amino acid sequence of SEQ ID NO: 20, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, CDR-L3 with an amino acid sequence of SEQ ID NO: 24.

[0250] The CDR-L2 of SEQ ID NO:23 and the CDR-L3 of SEQ ID NO:24 have an amino acid sequence.

[0251] In some embodiments, the antibody or its antigen-binding fragment comprises: (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:36, CDR-H2 with an amino acid sequence of SEQ ID NO:37, and CDR-H3 with an amino acid sequence of SEQ ID NO:38; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:45, CDR-L2 with an amino acid sequence of SEQ ID NO:23, and CDR-L3 with an amino acid sequence of SEQID NO:52.

[0252] In some embodiments, the antibody or its antigen-binding fragment comprises: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:7, CDR-H2 with an amino acid sequence of SEQ ID NO:8, and CDR-H3 with an amino acid sequence of SEQ ID NO:9; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:4, CDR-L2 with an amino acid sequence of SEQ ID NO:5, and CDR-L3 with an amino acid sequence of SEQ ID NO:6.

[0253] In some embodiments, the antibody or its antigen-binding fragment comprises: (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:25, CDR-H2 with an amino acid sequence of SEQ ID NO:26, and CDR-H3 with an amino acid sequence of SEQ ID NO:21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:22, CDR-L2 with an amino acid sequence of SEQ ID NO:23, and CDR-L3 with an amino acid sequence of SEQID NO:24.

[0254] In some embodiments, the antibody or its antigen-binding fragment comprises: (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:39, CDR-H2 with an amino acid sequence of SEQ ID NO:40, and CDR-H3 with an amino acid sequence of SEQ ID NO:38; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:45, CDR-L2 with an amino acid sequence of SEQ ID NO:23, and CDR-L3 with an amino acid sequence of SEQID NO:52.

[0255] In some embodiments, the antibody or its antigen-binding fragment comprises: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10, CDR-H2 with an amino acid sequence of SEQ ID NO: 11, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13, CDR-L2 with an amino acid sequence of SEQ ID NO: 14, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6.

[0256] In some embodiments, the antibody or its antigen-binding fragment comprises: (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:27, CDR-H2 with an amino acid sequence of SEQ ID NO:28, and CDR-H3 with an amino acid sequence of SEQ ID NO:29; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:30, CDR-L2 with an amino acid sequence of SEQ ID NO:31, and CDR-L3 with an amino acid sequence of SEQID NO:24.

[0257] In some embodiments, the antibody or its antigen-binding fragment comprises: (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:41, CDR-H2 with an amino acid sequence of SEQ ID NO:42, and CDR-H3 with an amino acid sequence of SEQ ID NO:43; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:44, CDR-L2 with an amino acid sequence of SEQ ID NO:31, and CDR-L3 with an amino acid sequence of SEQID NO:52.

[0258] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0259] (a) VH or a variant thereof as shown in SEQ ID NO: 15, and / or VL or a variant thereof as shown in SEQ ID NO: 16;

[0260] (b) VH or a variant thereof as shown in SEQ ID NO: 32, and / or VL or a variant thereof as shown in SEQ ID NO: 33; or

[0261] (c) VH or a variant thereof as shown in SEQ ID NO: 46, and / or VL or a variant thereof as shown in SEQ ID NO: 47;

[0262] 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% 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 substitutions are conservative substitutions, provided that the amino acid sequence of the CDR of the variant has 100% sequence identity with the amino acid sequence of the corresponding CDRs of VH and VL of (a), (b) and (c), and the variant binds to HER3.

[0263] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0264] (a) comprising a VH having an amino acid sequence as shown in SEQ ID NO: 15 or a variant thereof, and a VL having an amino acid sequence as shown in SEQ ID NO: 16 or a variant thereof;

[0265] (b) a VH comprising the amino acid sequence shown in SEQ ID NO: 32 or a variant thereof, and a VL comprising the amino acid sequence shown in SEQ ID NO: 33 or a variant thereof; or

[0266] (c) a VH comprising the amino acid sequence shown in SEQ ID NO: 46 or a variant thereof, and a VL comprising the amino acid sequence shown in SEQ ID NO: 47 or a variant thereof;

[0267] 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% sequence identity compared to the sequence from which it is derived, or the variant has one or more 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 substitutions are conservative substitutions.

[0268] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0269] (a) a VH comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising the amino acid sequence of SEQ ID NO: 16;

[0270] (b) a VH comprising the amino acid sequence of SEQ ID NO: 32, and a VL comprising the amino acid sequence of SEQ ID NO: 33; or

[0271] (c) VH comprising the amino acid sequence shown in SEQ ID NO:46, and VL comprising the amino acid sequence shown in SEQ ID NO:47.

[0272] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0273] (a) VH comprising the amino acid sequence shown in SEQ ID NO: 15, and, VL comprising the amino acid sequence shown in SEQ ID NO: 16.

[0274] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0275] (b) VH comprising the amino acid sequence shown in SEQ ID NO:32, and VL comprising the amino acid sequence shown in SEQ ID NO:33.

[0276] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0277] (c) VH comprising the amino acid sequence shown in SEQ ID NO:46, and VL comprising the amino acid sequence shown in SEQ ID NO:47.

[0278] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH), which comprises CDR-H1, CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 described in VH, respectively comprising the amino acid sequence of SEQ ID NO: 15; and a light chain variable region (VL), which comprises CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 described in VL, respectively comprising the amino acid sequence of SEQ ID NO: 16.

[0279] 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, wherein the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 described in VH comprising the amino acid sequence of SEQ ID NO: 32, respectively;

[0280] and a light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 described in VL respectively comprising the amino acid sequence of SEQ ID NO: 33.

[0281] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH), which comprises CDR-H1, CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 described in VH, which respectively comprise the amino acid sequence of SEQ ID NO:46; and a light chain variable region (VL), which comprises CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 described in VL, which respectively comprise the amino acid sequence of SEQ ID NO:47.

[0282] In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0283] (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the wild-type sequence from which it is derived; and

[0284] (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof, which 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.

[0285] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0286] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 50 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 50 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0287] In some embodiments, the light chain constant region is a kappa light chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 51 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 51 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0288] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:50 and a light chain constant region (CL) as shown in SEQ ID NO:51.

[0289] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0290] (1) a heavy chain comprising a VH sequence of SEQ ID NO: 15 and a heavy chain constant region (CH) of SEQ ID NO: 50, and a light chain comprising a VL sequence of SEQ ID NO: 16 and a light chain constant region (CL) of SEQ ID NO: 51;

[0291] (2) a heavy chain comprising a VH 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 sequence set forth in SEQ ID NO: 33 and a light chain constant region (CL) set forth in SEQ ID NO: 51; or

[0292] (3) A heavy chain comprising the VH of SEQ ID NO:46 and the heavy chain constant region (CH) of SEQ ID NO:50, and a light chain comprising the VL of SEQ ID NO:47 and the light chain constant region (CL) of SEQ ID NO:51.

[0293] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0294] (1) A heavy chain comprising VH of the amino acid sequence shown in SEQ ID NO: 15 and the heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising VL of the amino acid sequence shown in SEQ ID NO: 16 and the light chain constant region (CL) shown in SEQ ID NO: 51.

[0295] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0296] (2) A heavy chain comprising VH of the amino acid sequence shown in SEQ ID NO:32 and the heavy chain constant region (CH) shown in SEQ ID NO:50, and a light chain comprising VL of the amino acid sequence shown in SEQ ID NO:33 and the light chain constant region (CL) shown in SEQ ID NO:51.

[0297] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0298] (3) A heavy chain comprising VH of the amino acid sequence shown in SEQ ID NO:46 and the heavy chain constant region (CH) shown in SEQ ID NO:50, and a light chain comprising VL of the amino acid sequence shown in SEQ ID NO:47 and the light chain constant region (CL) shown in SEQ ID NO:51.

[0299] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO:17, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO:18.

[0300] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO:34, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO:35.

[0301] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO:48, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO:49.

[0302] In specific embodiments of antibodies, the heavy chain constant domain disclosed herein may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid. Thus, in a composition comprising a specific antibody as described herein, the composition may comprise a population of antibody species, wherein each species may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid or an N-terminal amino acid cyclized to pyroglutamic acid.

[0303] In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or antigen-binding fragment thereof via a linker (such as the "MLE" fragment shown in this application).

[0304] In some embodiments, M is Wherein ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl radical substitution; M1 is selected from a single bond, C 1-20 Alkylene, C 2-20 Alkenylene and C 2-20 Alkyne group.

[0305] In some embodiments, M is Wherein ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings to a benzene ring via a single bond, wherein the alicyclic heterocycle is optionally substituted with one or more selected from oxy (=O), halogen and C 1-4 Alkyl radical substitution; M1 is selected from a single bond, C 3-10 Alkylene, C 3-10 Alkenylene and C 3-10 Alkyne group.

[0306] In some embodiments, M is wherein ring A is selected from M1 is selected from a single bond, C 5-8 Alkylene, C 5-8 Alkenylene or C 5-8 Alkyne group.

[0307] In some embodiments, M is selected from the following structures:

[0308]

[0309] In some embodiments, M is selected from the following structures:

[0310]

[0311] In some embodiments, L is selected from the structure consisting of one or more of the following: 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), 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-G ly-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,

[0312] Where R' represents hydrogen, C 1-6 Alkyl or alkyl containing -(CH2CH2O)r-; r is an integer selected from 1-10; s is an integer selected from 1-20.

[0313] In some embodiments, L is selected from the structure consisting of one or more of the following: 1-6 Alkylene, -NH-, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), Gly, Gly-Gly-Phe-Gly,

[0314] wherein s is selected from an integer of 1-20.

[0315] In some embodiments, L is selected from the following structures:

[0316]

[0317]

[0318] wherein s is selected from an integer of 1-20.

[0319] In some embodiments, L is selected from the following structures:

[0320]

[0321] In some embodiments, L is selected from the following structures:

[0322]

[0323] In some embodiments, E is a single bond, -NH-CH2-, -NH-CH2-O-CH2-CO-, or is selected from the following structures:

[0324] In some embodiments, E is a single bond, -NH-CH2-, -NH-CH2-O-CH2-CO-,

[0325] In some embodiments, E is -NH-CH2-O-CH2-CO-, In some embodiments, E is -NH-CH2-O-CH2-CO- or In some embodiments, M is selected from the following structures:

[0326]

[0327] L is selected from the following structures:

[0328]

[0329] E is -NH-CH2-O-CH2-CO-, In some embodiments, Select from the following structures:

[0330]

[0331]

[0332]

[0333] In some embodiments, Select from the following structures:

[0334]

[0335]

[0336] In some embodiments, the cytotoxic drug is selected from a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor. In some embodiments, the microtubule inhibitor is an auristatin or a maytansine. 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, belotecan, 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.

[0337] The cytotoxic drugs disclosed in the present application generally contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amino (-NH2), secondary amine (-NR A H) or tertiary amine (-NR B R C ), where R A , R B , R C Only non-hydrogen substituents on N are represented here, and the cytotoxic drug can be attached to the linker in the conjugate via these functional groups.

[0338] In some embodiments, the cytotoxic drug is linked to E in the antibody drug conjugate via -OH, -SH, primary amino, secondary amine or tertiary amine groups on the cytotoxic drug.

[0339] In some embodiments, the cytotoxic drug is selected from the following Formula I and Formula II:

[0340]

[0341] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0342] R3 is selected from H and -CO-CH2OH;

[0343] R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;

[0344] R6 is selected from hydrogen and -C 1-4 Alkylene-NR a R b ;

[0345] R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0346] Where Ra , R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl.

[0347] In some embodiments, the cytotoxic drug is selected from the following compounds:

[0348]

[0349]

[0350] In some embodiments, the cytotoxic drug is selected from the following compounds:

[0351]

[0352] The corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is the formula Ab-[MLED] x In some embodiments, D is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amine group on the cytotoxic drug.

[0353] In some embodiments, D is selected from the following structures:

[0354]

[0355]

[0356] In some embodiments, the antibody drug conjugate is selected from ADC A-01 to ADC A-25, ADCB-01 to ADC B-05 shown below:

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366] Wherein, HA in each antibody drug conjugate represents an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO: 15 and VL as shown in SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO: 15 and CH as shown in SEQ ID NO: 50, and VL as shown in SEQ ID NO: 16 and CL as shown in SEQ ID NO: 51;

[0367] in, It indicates the specific connection mode between the thiol group in the antibody or its antigen-binding fragment and the linker.

[0368] In some embodiments, the antibody drug conjugate is selected from:

[0369]

[0370]

[0371] wherein x is 3 to 8, or 3 to 4, or 7 to 8, and the HA in each antibody drug conjugate is selected from an antibody or an antigen-binding fragment comprising:

[0372] (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0373] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0374] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0375] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0376] wherein the variant described in any one of (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 more 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3;

[0377] or,

[0378] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0379] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0380] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0381] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 40 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0382] wherein the variant described in any one of (2a), (2b), and (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 more 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (2a), (2b), and (2c), and the variant binds to HER3;

[0383] or,

[0384] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0385] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0386] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0387] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 41 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 42 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 44 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0388] wherein the variant described in any one of (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% 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

[0389] In yet another embodiment, the HA in each antibody drug conjugate is selected from an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0390] (a) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof;

[0391] (b) a VH comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof; or

[0392] (c) a VH comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof;

[0393] wherein the variant of any one of (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., 1, 2, 3, 4 or 5 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (a), (b) and (c), and the variant binds to HER3.

[0394] In yet another embodiment, the HA in each antibody drug conjugate is selected from an antibody or an antigen-binding fragment thereof comprising:

[0395] (a) a VH comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising the amino acid sequence of SEQ ID NO: 16;

[0396] (b) a VH comprising the amino acid sequence of SEQ ID NO: 32, and a VL comprising the amino acid sequence of SEQ ID NO: 33; or

[0397] (c) VH comprising the amino acid sequence shown in SEQ ID NO:46, and VL comprising the amino acid sequence shown in SEQ ID NO:47.

[0398] In yet another embodiment, the antibody drug conjugate is selected from:

[0399]

[0400]

[0401] Wherein the HA in each antibody drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises:

[0402] (1) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO: 51;

[0403] (2) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a VL comprising the amino acid sequence of SEQ ID NO:33, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a CH as set forth in SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:33 and a CL as set forth in SEQ ID NO:51; and

[0404] (3) An antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a VL comprising the amino acid sequence of SEQ ID NO:47, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:47 and a CL comprising the amino acid sequence of SEQ ID NO:51.

[0405] In some embodiments, the antibody drug conjugate is selected from:

[0406]

[0407] Wherein the HA in each antibody drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises:

[0408] (1) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO: 51;

[0409] (2) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a VL comprising the amino acid sequence of SEQ ID NO:33, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:33 and a CL comprising the amino acid sequence of SEQ ID NO:51; and

[0410] (3) An antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a VL comprising the amino acid sequence of SEQ ID NO:47, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:47 and a CL comprising the amino acid sequence of SEQ ID NO:51.

[0411] In some embodiments, the antibody drug conjugate is:

[0412]

[0413] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: (1) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO: 51.

[0414] In some embodiments, the antibody drug conjugate is:

[0415]

[0416] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, the antibody or antigen-binding fragment comprising: (2) an antibody or an antigen-binding fragment comprising a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 33, or an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 32 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 33 and a CL comprising the amino acid sequence of SEQ ID NO: 51.

[0417] In some embodiments, the antibody drug conjugate is:

[0418]

[0419] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, the antibody or antigen-binding fragment comprising: (3) an antibody or an antigen-binding fragment comprising a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO: 47, or an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 46 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 47 and a CL comprising the amino acid sequence of SEQ ID NO: 51.

[0420] In some embodiments, the antibody drug conjugate is:

[0421]

[0422] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 18; and x is 7 to 8.

[0423] In some embodiments, the antibody drug conjugate is:

[0424]

[0425] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 34, and a light chain (LC) comprising the sequence shown in SEQ ID NO: 35; and x is 7 to 8.

[0426] In some embodiments, the antibody drug conjugate is:

[0427]

[0428] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 49; and x is 7 to 8.

[0429] In some embodiments, the antibody drug conjugate is:

[0430]

[0431] Wherein the HA in each antibody drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises:

[0432] (1) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO: 51;

[0433] (2) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a VL comprising the amino acid sequence of SEQ ID NO:33, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:33 and a CL comprising the amino acid sequence of SEQ ID NO:51; and

[0434] (3) An antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a VL comprising the amino acid sequence of SEQ ID NO:47, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:47 and a CL comprising the amino acid sequence of SEQ ID NO:51.

[0435] In some embodiments, the antibody drug conjugate is:

[0436]

[0437] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: (1) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO: 51.

[0438] In some embodiments, the antibody drug conjugate is:

[0439]

[0440] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: (2) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 32 and a VL comprising the amino acid sequence shown in SEQ ID NO: 33, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 32 and a CH comprising the amino acid sequence shown in SEQ ID NO: 50, and a VL comprising the amino acid sequence shown in SEQ ID NO: 33 and a CL comprising the amino acid sequence shown in SEQ ID NO: 51.

[0441] In some embodiments, the antibody drug conjugate is:

[0442]

[0443] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, the antibody or antigen-binding fragment comprising: (3) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO: 47, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 46 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 47 and a CL comprising the amino acid sequence of SEQ ID NO: 51.

[0444] In some embodiments, the antibody drug conjugate is:

[0445]

[0446] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 18; and x is 7 to 8.

[0447] In some embodiments, the antibody drug conjugate is:

[0448]

[0449] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 34, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 35; and x is 7 to 8.

[0450] In some embodiments, the antibody drug conjugate is:

[0451]

[0452] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 49; and x is 7 to 8.

[0453] In some embodiments, the antibody drug conjugate is:

[0454]

[0455] Wherein the HA in each antibody drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises:

[0456] (1) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO: 51;

[0457] (2) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a VL comprising the amino acid sequence of SEQ ID NO:33, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:33 and a CL comprising the amino acid sequence of SEQ ID NO:51; and

[0458] (3) An antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a VL comprising the amino acid sequence of SEQ ID NO:47, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:47 and a CL comprising the amino acid sequence of SEQ ID NO:51.

[0459] In some embodiments, the antibody drug conjugate is:

[0460]

[0461] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: (1) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO: 51.

[0462] In some embodiments, the antibody drug conjugate is:

[0463]

[0464] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: (2) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 32 and a VL comprising the amino acid sequence shown in SEQ ID NO: 33, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 32 and a CH comprising the amino acid sequence shown in SEQ ID NO: 50, and a VL comprising the amino acid sequence shown in SEQ ID NO: 33 and a CL comprising the amino acid sequence shown in SEQ ID NO: 51.

[0465] In some embodiments, the antibody drug conjugate is:

[0466]

[0467] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, the antibody or antigen-binding fragment comprising: (3) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO: 47, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 46 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 47 and a CL comprising the amino acid sequence of SEQ ID NO: 51.

[0468] In some embodiments, the antibody drug conjugate is:

[0469]

[0470] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 18; and x is 7 to 8.

[0471] In some embodiments, the antibody drug conjugate is:

[0472]

[0473] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 34, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 35; and x is 7 to 8.

[0474] In some embodiments, the antibody drug conjugate is:

[0475]

[0476] wherein HA is an antibody or antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 49; and x is 7 to 8. In some embodiments, the antibody drug conjugate is:

[0477]

[0478] Wherein the HA in each antibody drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises:

[0479] (1) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO: 51;

[0480] (2) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a VL comprising the amino acid sequence of SEQ ID NO:33, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:33 and a CL comprising the amino acid sequence of SEQ ID NO:51; and

[0481] (3) An antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a VL comprising the amino acid sequence of SEQ ID NO:47, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:47 and a CL comprising the amino acid sequence of SEQ ID NO:51.

[0482] In some embodiments, the antibody drug conjugate is:

[0483]

[0484] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: (1) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO: 51.

[0485] In some embodiments, the antibody drug conjugate is:

[0486]

[0487] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: (2) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 32 and a VL comprising the amino acid sequence shown in SEQ ID NO: 33, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 32 and a CH comprising the amino acid sequence shown in SEQ ID NO: 50, and a VL comprising the amino acid sequence shown in SEQ ID NO: 33 and a CL comprising the amino acid sequence shown in SEQ ID NO: 51.

[0488] In some embodiments, the antibody drug conjugate is:

[0489]

[0490] The HA in each antibody-drug conjugate is selected from an antibody or an antigen-binding fragment, the antibody or antigen-binding fragment comprising: (3) an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO: 47, for example, an antibody or an antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 46 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 47 and a CL comprising the amino acid sequence of SEQ ID NO: 51.

[0491] In some embodiments, the antibody drug conjugate is:

[0492]

[0493] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 18; and x is 7 to 8.

[0494] In some embodiments, the antibody drug conjugate is:

[0495]

[0496] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 34, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 35; and x is 7 to 8.

[0497] In some embodiments, the antibody drug conjugate is:

[0498]

[0499] Wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 49; and x is 7 to 8.

[0500] In some embodiments, x in the antibody drug conjugate is 1-10, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10.

[0501] In some embodiments, x in the antibody drug conjugate is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0502] In some embodiments, the antibodies disclosed herein are genetically engineered to include one or more cysteine ​​or atypical amino acid substitutions of amino acids at defined positions within the antibody. These cysteine ​​residues or atypical amino acid residues can then be coupled to a drug linker through the sulfhydryl group of the cysteine ​​residue or the reactive group of the atypical amino acid. Therefore, the antibody-drug conjugates of the present invention can include one or more substitutions of amino acids in the heavy or light chain of the antibody with cysteine ​​residues or atypical amino acid residues, which are then conjugated to the drug linker disclosed herein. In a specific embodiment, the amino acid positions that can be substituted are selected from positions 152, 153, 171, 172, 173 and 375 (numbered according to the Eu numbering scheme) of the heavy chain constant domain and positions 165 and 168 (numbering starts from the amino acid 1 at the N-terminus) of the light chain constant region. In certain embodiments, cysteine ​​may replace one or more of the amino acids at positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain (numbered according to the Eu numbering scheme) and positions 165 and 168 of the light chain constant region (numbered starting from amino acid 1 at the N-terminus). In certain embodiments, the antibody-drug conjugate comprises an S375C amino acid substitution conjugated to a drug linker disclosed herein. In certain embodiments, the antibody comprises an S375C amino acid substitution and an E152C amino acid substitution, each conjugated to a drug linker disclosed herein. In certain embodiments, the antibody comprises an S375C amino acid substitution and an S168C amino acid substitution, each conjugated to a drug linker disclosed herein.

[0503] In some embodiments, the DAR value (drug-antibody coupling ratio) of the antibody drug conjugate is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4- 10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-9, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5 ~6.0,3.5~6.5,3.5~7.0,3.5~7.5,3.5~8.0,4.0~4.5,4.0~5.0,4.0~5.5,4.0~6.0,4.0~6.5,4.0~7.0,4.0~7.5,4.0~8.0,4.5~5.0,4.5~5.5,4.5~6.0,4.5~6.5,4.5~7.0,4.5~7.5,4.5~8.0,5.0~5 .5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0504] Drug-Linker

[0505] Those skilled in the art will appreciate that the antibody-drug conjugates described in the present application can be prepared modularly. For example, a free form of a "drug-linker" (which can be understood as M'-LED, where M' is the structural form before M is covalently linked to an antibody or its antigen-binding fragment) is first obtained, and then covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in the present application. Accordingly, one or more thiol groups (-SH), amino groups (-NH2) or carboxyl groups (-COOH) on the antibody or its antigen-binding fragment are connected to M' in the free form of the "drug-linker" by a substitution reaction (e.g., removing structures such as -SO2Me or -Br thereon) or by an addition reaction.

[0506] In another aspect, the present invention provides a drug-linker having a structure shown in formula M'-LED, wherein:

[0507] M' is Lg is a leaving group for a nucleophilic substitution reaction (e.g.,

[0508] Halogen, methylsulfonyl, fluorophenol or ), or is hydroxyl (-OH), thiol (-SH) or amino (-NH2); or, Lg forms an unsaturated double bond with the adjacent atom on ring A; ring A is a 5-6 membered alicyclic ring, or a 5-20 membered aromatic ring system, wherein the alicyclic ring and the aromatic ring system are optionally substituted with one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 The alkyl group is substituted; M1 is selected from a single bond and C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene;

[0509] The L, E and D structures are as defined in any of the aforementioned antibody drug conjugates.

[0510] In some embodiments, M' is Lg is a methylsulfonyl group, or Lg forms a carbon-carbon double bond with the adjacent atom on ring A; ring A is a 5-membered alicyclic heterocyclic ring, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings to a benzene ring via a single bond, wherein the alicyclic heterocyclic ring is optionally substituted with one or more selected from oxy (=O), halogen and C 1-4 The alkyl group is substituted; M1 is selected from a single bond and C 3-10 Alkylene, C 3-10 Alkenylene or C 3-10 Alkyne group.

[0511] In some embodiments, M' is Selected from M1 is selected from a single bond and C 5-8 Alkylene, C 5-8 Alkenylene or C 5-8 Alkyne group.

[0512] In some embodiments, M' is selected from

[0513] In some embodiments, M' is

[0514] In some embodiments, M' is selected from

[0515] And, when M' is When E is or wherein s is selected from an integer of 1-20.

[0516] 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:

[0517] A-01:

[0518] A-02:

[0519] A-03:

[0520] A-04: A-05: A-06: A-07: A-08: A-09: A-10: A-11: A-12: A-13: A-14: A-15: A-16: A-17: A-18: A-19: A-20: A-21: A-22: A-23:

[0521] A-24: A-25: B-01: B-02:

[0522] B-03:

[0523] B-04:

[0524] B-05:

[0525] The present invention also provides an ADC comprising an antibody that binds to HER3, wherein the antibody 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 cysteine.

[0526] In yet another embodiment of the ADC, the antibody or antigen-binding fragment thereof is selected from:

[0527] (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0528] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0529] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0530] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0531] or,

[0532] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0533] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0534] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0535] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 40 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0536] or,

[0537] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0538] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0539] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0540] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:41 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:42 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:44 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:52 or a variant thereof.

[0541] In yet another embodiment of the ADC, the antibody or antigen-binding fragment thereof is selected from:

[0542] (a) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof;

[0543] (b) a VH comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof; or

[0544] (c) VH comprising the amino acid sequence shown in SEQ ID NO: 46 or a variant thereof, and / or VL comprising the amino acid sequence shown in SEQ ID NO: 47 or a variant thereof.

[0545] In yet another embodiment of the ADC, the antibody or antigen-binding fragment is selected from:

[0546] (1) an antibody or an antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO: 15 and a VL as set forth in SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO: 15 and a CH as set forth in SEQ ID NO: 50, and a VL as set forth in SEQ ID NO: 16 and a CL as set forth in SEQ ID NO: 51;

[0547] (2) an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a VL comprising the amino acid sequence of SEQ ID NO:33, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:32 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:33 and a CL comprising the amino acid sequence of SEQ ID NO:51; and

[0548] (3) An antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a VL comprising the amino acid sequence of SEQ ID NO:47, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:46 and a CH comprising the amino acid sequence of SEQ ID NO:50, and a VL comprising the amino acid sequence of SEQ ID NO:47 and a CL comprising the amino acid sequence of SEQ ID NO:51.

[0549] In another embodiment of the ADC, the antibody or antigen-binding fragment comprises: an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 15 and a VL comprising the amino acid sequence shown in SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 15 and a CH comprising the amino acid sequence shown in SEQ ID NO: 50, and a VL comprising the amino acid sequence shown in SEQ ID NO: 16 and a CL comprising the amino acid sequence shown in SEQ ID NO: 51.

[0550] In another embodiment of the ADC, the antibody or antigen-binding fragment comprises: an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 32 and a VL comprising the amino acid sequence shown in SEQ ID NO: 33, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 32 and a CH comprising the amino acid sequence shown in SEQ ID NO: 50, and a VL comprising the amino acid sequence shown in SEQ ID NO: 33 and a CL comprising the amino acid sequence shown in SEQ ID NO: 51.

[0551] In another embodiment of the ADC, the antibody or antigen-binding fragment comprises: an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 46 and a VL comprising the amino acid sequence shown in SEQ ID NO: 47, for example, an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 46 and a CH comprising the amino acid sequence shown in SEQ ID NO: 50, and a VL comprising the amino acid sequence shown in SEQ ID NO: 47 and a CL comprising the amino acid sequence shown in SEQ ID NO: 51.

[0552] In yet another embodiment, the antibody comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO:17, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO:18.

[0553] In yet another embodiment, the antibody comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO:34, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO:35.

[0554] In yet another embodiment, the antibody comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 49. Preparation of Antibodies

[0555] 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 chains of the antibodies described herein are obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions and express the antibodies of the present invention.

[0556] Conjugation

[0557] In another aspect, the present application provides a method of conjugating the drug-linker described herein to the antibody described herein to prepare the antibody drug conjugate (ADC) described herein.

[0558] In certain embodiments, an antibody described herein is conjugated to a drug-linker described herein via a lysine in the antibody.

[0559] In certain embodiments, the antibodies described herein are conjugated to the drug-linkers described herein via cysteine ​​in the antibody. In certain embodiments, the cysteine ​​is from a reduced intrachain disulfide bond in the antibody. In certain embodiments, the cysteine ​​is from a reduced interchain disulfide bond in the antibody.

[0560] In some embodiments, the antibody is conjugated to a drug-linker via reduced interchain disulfide bonds in the antibody. For example, an IgG1 antibody consists of four polypeptide chains, two heavy chains comprising VH, CH1 and Fc (e.g., hinge, CH2 and CH3) domains, and two light chains comprising VL and CL domains, connected by interchain cysteine ​​disulfide bonds (-SS-) (e.g., two heavy chain-light chain interchain disulfide bonds and two hinge heavy chain-heavy chain interchain disulfide bonds). In certain embodiments, when these disulfide bonds are broken under reducing conditions, eight (8) reactive cysteine ​​thiol moieties are generated. In a specific embodiment, each of the eight reactive cysteine ​​thiol moieties is a site of attachment for a drug-linker, such that a maximum of eight (x=8) drug-linkers can be attached to a reduced antibody. In a specific embodiment, any one of the four disulfide bonds is broken under reducing conditions, resulting in two (2) reactive cysteine ​​thiol moieties. In further embodiments, each of the two reactive cysteine ​​thiol moieties is a site of attachment for a drug-linker, such that two (x=2) drug-linkers can be attached to the reduced antibody. In specific embodiments, any two of the four disulfide bonds are cleaved under reducing conditions, resulting in four (4) reactive cysteine ​​thiol moieties. In further embodiments, each of the four reactive cysteine ​​thiol moieties is a site of attachment for a drug-linker, such that four (x=4) drug-linkers can be attached to the reduced antibody. In specific embodiments, any three of the four disulfide bonds are cleaved under reducing conditions, resulting in six (6) reactive cysteine ​​thiol moieties. In further embodiments, each of the six reactive cysteine ​​thiol moieties is a site of attachment for a drug-linker, such that six (x=6) drug-linkers can be attached to the reduced antibody.

[0561] In some embodiments, the interchain disulfide bond is located between two cysteine ​​residues, which break under reducing conditions to produce two reactive cysteine ​​sulfhydryl moieties. In a further embodiment, the interchain disulfide bond in the antibody is located between the heavy chain and the light chain, for example, between C220 of the heavy chain according to EU numbering and C214 of the κ light chain, or between C220 of the heavy chain according to EU numbering and C214 of the λ light chain according to Kabat numbering. In addition, optionally, the interchain disulfide bond in the antibody is located between two heavy chains, for example, between C226 and / or C229 of the first heavy chain according to EU numbering and C226 and / or C229 of the second heavy chain. In some embodiments, the cysteine ​​residue is located in the hinge region of the antibody. In some embodiments, according to EU numbering, the cysteine ​​residue is located in any one or more of positions 220, 226 or 229 in the heavy chain (also referred to herein as C220, C226 or C229, respectively). In some embodiments, according to EU and / or Kabat numbering, the cysteine ​​residue is located at position 214 of the light chain (also referred to herein as C214, e.g., according to EU and Kabat numbering, at position 214 of a kappa light chain, or according to Kabat numbering, at position 214 of a lambda light chain). In one embodiment, according to EU numbering, the cysteine ​​residue is located at position 220, 226, and 229 of the heavy chain, and according to EU or Kabat numbering, at position 214 of the light chain. In one embodiment, according to EU numbering, the cysteine ​​residue is located at position 220, 226, and 229 of the heavy chain, and according to EU and Kabat numbering, at position 214 of a kappa light chain. In one embodiment, according to EU numbering, the cysteine ​​residue is located at position 220, 226, and 229 of the heavy chain, and according to Kabat numbering, at position 214 of a lambda light chain. In one embodiment, the cysteine ​​residue is located at any one or more of the following positions:

[0562] (i) any one, any two, any three, or all four of positions 220, 226, and 229 in the first heavy chain according to EU numbering;

[0563] (ii) any one, any two, any three, or all four of positions 220, 226, and 229 in the second heavy chain, according to EU numbering;

[0564] (iii) position 214 in the first light chain according to Kabat numbering; and / or

[0565] (iv) position 214 in the second light chain according to Kabat numbering.

[0566] As used herein, C220, C226 and C229 refer to the amino acid residues of immunoglobulins identified according to EU numbering (cysteine, Cys, C). As will be appreciated by those skilled in the art, such numbering corresponds to amino acid residues of polypeptides that are aligned with the amino acid residues defined for immunoglobulins, as shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0567] As used herein, the 214th cysteine ​​residue of the kappa light chain refers to the amino acid residues of immunoglobulins identified according to the Kabat numbering (cysteine, Cys, C). As will be appreciated by those skilled in the art, such numbering corresponds to amino acid residues of polypeptides that are aligned with the amino acid residues defined for immunoglobulins, as shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html.

[0568] As used herein, the cysteine ​​residue at position 214 of the lambda light chain refers to the amino acid residues of immunoglobulins identified according to Kabat numbering (cysteine, Cys, C). As will be appreciated by those skilled in the art, such numbering corresponds to amino acid residues of polypeptides that are aligned with the amino acid residues defined for immunoglobulins, as shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGLCnber.html.

[0569] In certain embodiments, the antibodies described herein comprise four interchain disulfide bonds in the hinge region, which can be reduced, thereby breaking and revealing reactive thiol moieties that can be conjugated to a maleimide moiety on a drug-linker, such as a maleimide moiety on a drug-linker described herein.

[0570] In one embodiment, the present invention provides a method for preparing the ADC described herein, comprising the following steps:

[0571] a) providing a solution comprising an antibody;

[0572] b) contacting the solution of a) with a reducing agent;

[0573] c) contacting the solution of b) with a solution comprising a drug-linker or a salt thereof as described herein to prepare the ADC.

[0574] In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0575] Composition

[0576] On the other hand, the present application provides a composition of an antibody drug conjugate (ADC) as described herein. Such a composition may include a plurality of ADCs as described herein, wherein each ADC includes a drug-linker as described herein, wherein 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. Therefore, the composition is characterized in that the "drug-antibody" ratio (DAR) is in the range of about 1 to about 10. Methods for determining DAR are well known to technicians, including methods using reverse phase chromatography or HPLC-MS.

[0577] For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, e.g., about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10.

[0578] In certain embodiments, the DAR of the ADC compositions described herein is about 3 to 9, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8. .0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5 about 5.0 to 8.0, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.5, about 7.0 to 7.5.

[0579] Pharmaceutical composition

[0580] In another aspect, the present application provides a pharmaceutical composition, which contains the antibody-drug conjugate described in any of the foregoing and optionally the drug-linker described in any of the foregoing, and one or more pharmaceutical excipients.

[0581] Pharmaceutical excipients include, for example, pharmaceutically acceptable carriers and / or excipients. Pharmaceutical excipients further include salts and solvates.

[0582] The antibody drug conjugates described herein are usually formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumor injection, etc. Optionally, the antibody drug conjugate having a desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16 th The antibody drug conjugates described herein or pharmaceutical compositions containing the same may be administered by any route appropriate to the individual to be treated.

[0583] The ADC 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, lozenges, suppositories, injections (including injections, 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 composition of the present invention should be sterile and stable under production and storage conditions. The preferred dosage form is an injection. This injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: the necessary dose of the antibody of the present invention is incorporated into a suitable solvent, and other required ingredients (including but not limited to pH regulators, surfactants, adjuvants, ionic strength enhancers, etc., permeants, preservatives, diluents or any combination thereof) are optionally added, and then filtered and sterilized. In addition, for ease of storage and use, the sterile injection can be prepared into a sterile lyophilized powder (e.g., by vacuum drying or freeze drying). This sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0584] In addition, the ADC described herein can be present in a pharmaceutical composition in a unit dosage form for administration by any suitable method known in the art, including but not limited to oral, oral, sublingual, ophthalmic, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment or drops) or intranasal routes. However, for many therapeutic uses, the preferred route of administration / mode is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The skilled person will understand that the route and / or mode of administration will vary according to the intended purpose. In some preferred embodiments, the ADC and pharmaceutical compositions described herein are administered by intravenous infusion or injection.

[0585] In certain embodiments, the pharmaceutical composition may also include other pharmaceutically active agents. In certain embodiments, the other pharmaceutically active agents are drugs with anti-tumor activity. In certain embodiments, the other pharmaceutically active agents are selected from EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic drugs or any combination thereof. In certain embodiments, the ADC described herein and other pharmaceutically active agents are provided as separate components or as mixed components. Therefore, the ADC described herein and other pharmaceutically active agents can be administered simultaneously, separately or sequentially.

[0586] How to use

[0587] The antibody drug conjugates described herein, the drug-linkers described herein, or pharmaceutical compositions thereof can be used to treat various diseases or conditions, such as cancers with high HER3 expression, including solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), or lymphoma.

[0588] Therefore, the present application provides use of an antibody drug conjugate (ADC), a drug-linker, or a pharmaceutical composition comprising the same as described in any of the aforementioned embodiments in the preparation of a drug for treating cancers with high HER3 expression.

[0589] At the same time, the present application also provides a method for treating HER3-overexpressing cancer, which comprises the step of administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate (ADC), a drug-linker, or a pharmaceutical composition comprising the same as described in any of the aforementioned embodiments.

[0590] In certain embodiments, the antibody drug conjugate (ADC), drug-linker or pharmaceutical composition is sufficient (e.g., in a subject):

[0591] (1) Inhibit the proliferation of cells (such as tumor cells);

[0592] (2) Inhibit tumor growth;

[0593] (3) Induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0594] (4) Inhibit HER3-mediated signal transduction;

[0595] (5) preventing and / or treating HER3-mediated diseases / disorders; or

[0596] (6) Any combination of the above (1) to (5).

[0597] In certain embodiments, the HER3-mediated disease / disorder is a tumor, such as a tumor expressing HER3. In specific embodiments, the tumor is selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, pancreatic cancer, head and neck squamous cell carcinoma, melanoma, ovarian cancer, prostate cancer, liver cancer, kidney cancer, bladder cancer, or any combination thereof. Example

[0598] The present invention is further described by the following examples, which are not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the principles and scope of the present invention.

[0599] The information of the sequences involved in the present invention is described in the following Tables A and B:

[0600] Table A

[0601]

[0602] Table B

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611] The abbreviations used in this document have the following meanings:

[0612] Table C

[0613]

[0614] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0615] Nuclear magnetic resonance (NMR) 1 H NMR) was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard substance was tetramethylsilane (TMS).

[0616] The abbreviations in the nuclear magnetic resonance (NMR) spectra used in the Examples are shown below.

[0617] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0618] Mass spectrometry (MS) was measured using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0619] Example 1 N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-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-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1-H-pyrrol-1-yl)hexanamide (A-01)

[0620]

[0621] Compound A-01-1 (0.40 g, 640.59 μmol, its synthesis reference patent application CN 111936169A), exotecan 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 mixture was reacted 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, which was purified by preparative high performance liquid chromatography (conditions as follows) to obtain 273 mg of the title compound.

[0622] Chromatographic column: Waters XBridge Prep C18 OBD 45mm×450mm×8.0μm

[0623] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0624] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 70 70 5.00 30 70 70 60.00 70 30 70

[0625] The structural characterization data are as follows:

[0626] ESI-MS (m / z): 1034.4 [M+H] + .

[0627] 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)

[0628]

[0629] Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene (1-8-2)

[0630] 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 in batches at 50°C, and the mixture was kept at 50°C for 2 hours. The reaction was detected by thin layer chromatography (ethyl acetate: petroleum ether = 1:10). The reaction solution was cooled to room temperature, and then added dropwise to ice water, extracted with toluene, and the organic phases were combined, washed with sodium sulfite solution, water, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative high performance liquid chromatography (conditions as follows), and the preparative solution was freeze-dried to obtain 4.88 g of the title compound.

[0631] Chromatographic column: C18 ODS 45mm×450mm×8.0μm

[0632] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0633] Time [min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL / min] 0 60 40 60 10 60 40 60 40 100 0 60

[0634] Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline (1-8-3)

[0635] 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. After hydrogen replacement, the mixture was reacted at 60°C for 4 h under hydrogen protection, and the reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and concentrated to obtain 3.68 g of crude title compound, which was directly used in the next step without further purification.

[0636] Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide (1-8-4)

[0637] 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 reaction was maintained at 20°C for 20 h, and the reaction was monitored by HPLC-MS. Water was added to the reaction solution, 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 with a mixed solvent of ethyl acetate: petroleum ether = 1:5 to obtain 2.86 g of the title compound.

[0638] Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid (1-8-5)

[0639] At 20°C, compound 1-8-4 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL), and vinylacetic acid (708.31 mg, 8.23 ​​mmol), DIPEA (1.95 g, 15.08 mmol), tri(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added. The reaction system was replaced with nitrogen and heated to 70°C for 5 h. The reaction was monitored by HPLC-MS. 1N sodium hydroxide solution was added to the reaction solution to adjust pH=8, and ethyl acetate was added for extraction. The aqueous phase was adjusted to pH=3 with 1N hydrochloric acid, extracted with ethyl acetate, and 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 directly used in the next step.

[0640] Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butyric acid (1-8-6)

[0641] At 20°C, compound 1-8-5 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL), Pd / C (0.52 g, content 10%) was added, the system was replaced with hydrogen and reacted at 40°C for 2 h under the protection of a hydrogen balloon, and the reaction was monitored by HPLC-MS / 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 step without further purification.

[0642] Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-7)

[0643] Compound 1-8-6 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5°C, trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise, and the reaction was maintained at 5°C for 4 h. The reaction was monitored by HPLC-MS. The reaction solution was added to water, the pH was adjusted to 9 with 10N sodium hydroxide solution, 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.

[0644] 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)

[0645] 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. The reaction was maintained at 5°C for 1 hour, and the reaction was monitored by HPLC-MS. The reaction solution was adjusted to pH = 5 with 1N hydrochloric acid, extracted with ethyl acetate, the combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was beaten with methyl tert-butyl ether to obtain 1.20 g of the title compound.

[0646] Step 8: Synthesis of N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-9)

[0647] 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, 10% content) was added, the system was replaced with hydrogen and kept at 5°C for 2 h under the protection of a hydrogen balloon, and the reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and concentrated to obtain 0.52 g of the hydrochloride of the title compound, which was directly used in the next step without further purification.

[0648] Step 9: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-8-10)

[0649] 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 maintained at 20°C for 2 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was added to water, 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.

[0650] Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-8-11)

[0651] 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, and the mixture was heated to 60°C for 2 h, and the reaction was monitored by HPLC-MS. Saturated sodium bicarbonate solution was added to the cooled reaction solution to adjust the pH to 8, 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 230.00 mg of the title compound, which was used directly in the next step without further purification.

[0652] 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-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-8-12)

[0653] 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, and the mixture was heated to 140°C for 5 h, and the reaction was monitored by HPLC-MS / 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.

[0654] Step 12: Synthesis of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-2)

[0655] Compound 1-8-12 (40.00 mg, 0.081 mmol) was added to concentrated hydrochloric acid (1 mL), heated to 100 ° C for 5 h, and the reaction was monitored by HPLC-MS. The reaction solution was filtered, and the filtrate was purified by preparative HPLC (conditions as follows), and the preparative solution was freeze-dried to obtain the hydrochloride of the title compound 1-2. The hydrochloride of 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 min) and 1-2-B (trifluoroacetate 7.00 mg, retention time 10.62 min) according to the retention time.

[0656] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0657] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0658] Time [min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL / min] 0 5 95 28 2 5 95 28 18 50 50 28

[0659] The structural characterization data are as follows:

[0660] 1-2-A:

[0661] 1 H NMR(400MHz,DMSO-d6)δ8.42(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=7.2Hz,3H).

[0662] ESI-MS (m / z): 452.1 [M+H] + .

[0663] 1-2-B:

[0664] 1 H NMR(400MHz,DMSO-d6)δ8.42(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=7.2Hz,3H).

[0665] ESI-MS (m / z): 452.0 [M+H] + .

[0666] Step 13: 2-((tert-butyldiphenylsilyl)oxy)-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)acetamide and 2-((tert-butyldiphenylsilyl)oxy)-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)acetamide Synthesis of ((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)

[0667] At 25°C, the hydrochloride 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 maintained at 25°C for 0.5 hour and the reaction was monitored by HPLC-MS / 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, and concentrated under reduced pressure. The crude product was purified by preparative thin layer chromatography (dichloromethane:methanol=20:1) to separate 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) according to their Rf values.

[0668] 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 (1-8-A and 1-8-B)

[0669] At 25°C, 1-8-13-A (15.00 mg) and 1-8-13-B (12.00 mg) were dissolved in tetrahydrofuran (1 mL) in two reaction bottles, and a mixture of tetrabutylammonium fluoride (1M tetrahydrofuran solution) / glacial acetic acid (v / v=13 / 1) (50 μL) was added dropwise, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC, and the preparative solution was lyophilized to obtain the title compounds 1-8-A (6.94 mg) and 1-8-B (4.00 mg).

[0670] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0671] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0672] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 3 20 80 28 18 90 10 28

[0673] The structural characterization data of 1-8-A are as follows:

[0674] 1H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.8Hz,1H),8.16(s,1H),7.31(s,1H),6.55(s,1H),5.65-5.36(m,4H),5.21(q,J=19.0H z,2H),3.95(d,J=5.7Hz,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=7.3Hz,3H).

[0675] ESI-MS (m / z): 510.1 [M+H] + .

[0676] The structural characterization data of 1-8-B are as follows:

[0677] 1H NMR (400MHz, DMSO-d6) δ8.45(d,J=8.9Hz,1H),8.15(s,1H),7.31(s,1H),6.54(s,1H),5.64-5.35(m,4H),5.19(q,J=19.0H z,2H),3.97(d,J=5.2Hz,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=7.3Hz,3H).

[0678] ESI-MS (m / z): 510.1 [M+H] + .

[0679] Example 3 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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide 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-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-07-A and A-07-B)

[0680]

[0681] Step 1: Synthesis of (S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosane-22-ynecarboxylic acid (A-07-3)

[0682] 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)hex-5-ynoate (A-07-1, 28.00 mg, 0.08 mmol) was added, and the mixture was reacted at 30°C for 1 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified by preparative HPLC (conditions as follows), and the preparative solution was freeze-dried to obtain 20.00 mg of the title compound.

[0683] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0684] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0685] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0686] The structural characterization data are as follows:

[0687] ESI-MS (m / z): 691.0 [M+H2O] + .

[0688] Step 2: N-((10S)-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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide and N-((10S)-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-tetraazahexadecane-16-yl) Synthesis of (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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-07-A and A-07-B)

[0689] 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, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC (conditions as follows), and the preparative solution was 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 7.5 min) and A-07-B (19.42 mg, retention time 8.0 min) according to the retention time.

[0690] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0691] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0692] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[0693] The structural characterization data are as follows:

[0694] A-07-A:

[0695] ESI-MS (m / z): 1107.3 [M+H] + .

[0696] A-07-B:

[0697] ESI-MS (m / z): 1107.3 [M+H] + .

[0698] Example 4 (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

[0699]

[0700] Step 1: Synthesis of 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxin-5-yl)propan-1-one (2-2-2)

[0701] Compound 2-2-1 (1.90 g, 8.08 mmol) was slowly added to nitric acid (8 mL) at 0°C, and the temperature was slowly raised to 25°C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (acetonitrile / 0.5% formic acid aqueous solution) to obtain 1.50 g of formate salt of the title compound.

[0702] The structural characterization data are as follows:

[0703] ESI-MS (m / z): 281.1 [M+H] + .

[0704] Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-(isopropylamino)propan-1-one (2-2-3)

[0705] The formate of compound 2-2-2 (1.25 g, 4.46 mmol) was added to tetrahydrofuran (20 mL), and 10% palladium carbon (125.00 mg) was added. After hydrogen replacement three times, the mixture was reacted at 25° C. for 16 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to dryness to obtain 895.00 mg of the crude title compound, which was used directly in the next step without purification.

[0706] The structural characterization data are as follows:

[0707] ESI-MS (m / z): 251.1 [M+H] + .

[0708] Step 3: Synthesis of ((S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

[0709] Compound 2-2-3 (23.00 mg, 0.09 mmol) and (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-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. The solvent was removed under reduced pressure to obtain a crude product of the title compound, which was purified by HPLC (mobile phase A: acetonitrile, mobile phase B: 0.05% formic acid aqueous solution), and 3 drops of 3M hydrochloric acid were added to the prepared solution, followed by freeze drying to obtain 8.70 mg of the hydrochloride of the title compound.

[0710] The structural characterization data are as follows:

[0711] ESI-MS (m / z): 478.2 [M+H] + .

[0712] 1 H-NMR (400MHz, DMSO-d6): δ9.38(brs,2H),7.85(s,1H),7.52(s,1H),7.25(s,1H),6.30(d,J=2.0Hz,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=6.4Hz,6H),0.87(t,J=7.6Hz,3H).

[0713] Example 5 Synthesis of 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-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropyl-N-methylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-01)

[0714]

[0715] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropyl-N-methylsulfonylamino)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)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxy-3,9-diazapentatriacontamido)benzyl)carbonate (B-01-2)

[0716] At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis reference patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and 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. The reaction was stirred for 1 hour and then filtered. The filtrate was purified by preparative HPLC (conditions as follows) to obtain 30.00 mg of the title compound.

[0717] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0718] Mobile phase A: acetonitrile; Mobile phase B: water

[0719] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 40 60 28 8.00 40 60 28 50.00 90 10 28

[0720] The structural characterization data are as follows:

[0721] ESI-MS(m / z):815.9[(M-273) / 2+H] + .

[0722] Step 2: Synthesis of 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-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-01)

[0723] Compound 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 reaction was carried out at room temperature for 30 min. The reaction solution was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (conditions as follows) to obtain 20.00 mg of trifluoroacetate salt of the title compound.

[0724] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0725] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0726] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 15 85 28 8.00 15 85 28 50.00 60 40 28

[0727] The structural characterization data are as follows: ESI-MS (m / z): 816.0 [M / 2+H] + .

[0728] 1H NMR (400MHz, DMSO-d6) δ10.18(s,1H),9.10(s,2H),8.38(t,J=5.56Hz,1H),8.32(d,J=8.40Hz,1H),8.22- 8.20(m,2H),8.09(t,J=5.68Hz,1H),7.91-7.87(m,2H),7.82-7.78(m,1H),7.69(brs,3H),7.61(d,J=8.5 6Hz,2H),7.32(d,J=8.56Hz,2H),7.06(s,1H),5.56(d,J=16.96Hz,1H),5.51(d,J=16.96Hz,1H),5.47(d, J=19.28Hz,1H),5.42(d,J=19.28Hz,1H),5.14(d,J=12.20Hz,1H),5.07(d,J=12.16Hz,1H),4.48(t,J=5. 24Hz,2H),4.46-4.43(m,1H),4.29(d,J=5.60Hz,2H),4.08-3.95(m,5H),3.79(t,J=5.28Hz,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 =7.08Hz,2H),2.29(t,J=7.36Hz,2H),2.23-2.13(m,2H),1.82(p,J=7.24Hz,2H),1.78-1.63(m,2H),1.61 -1.49(m,2H),1.42-1.27(m,2H),1.15(d,J=6.80Hz,3H),1.13(d,J=6.76Hz,3H),0.90(t,J=7.32Hz,3H).

[0729] Example 6 (2S, 3S, 4S, 5R, 6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[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-ynamido)ethoxy)ethoxy)acetamido))phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03)

[0730]

[0731] Step 1: Synthesis of (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-3)

[0732] Compound (2R, 3R, 4S, 5S, 6S) -2-bromo-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triacetic acid triester (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), and silver oxide (27.40 g, 118.25 mmol) was added under stirring. After nitrogen replacement, the mixture was reacted at room temperature in the dark for 12 hours. The reaction was monitored by HPLC-MS / MS; the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:3) to obtain 12.80 g of the title compound.

[0733] The structural characterization data are as follows:

[0734] ESI-MS (m / z): 503 [M+18] + .

[0735] Step 2: Synthesis of (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-4)

[0736] Compound B-03-3 (2.20 g, 4.53 mmol) was dissolved in ethyl acetate and tetrahydrofuran (50 mL each), PtO2 (0.20 g) was added, and then the reaction system was replaced with a hydrogen balloon three times, and the reaction was carried out under a hydrogen atmosphere for 2 hours. The reaction was monitored by HPLC-MS / MS; the reaction solution was directly filtered, the filter cake was washed with ethyl acetate, and the filtrate was evaporated to dryness under reduced pressure to obtain 2.02 g of the crude title compound, which was directly used in the next step.

[0737] The structural characterization data are as follows:

[0738] ESI-MS (m / z): 456.1 [M+1] + .

[0739] Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxy-2,7,10-trioxa-4-azadodecane-12-amide)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-5)

[0740] 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 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (495.22 mg, 2.00 mmol) was added under stirring, and the mixture was stirred for 2 hours. The reaction was monitored by HPLC-MS / MS; the reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (methanol: dichloromethane = 1:20) to obtain 507.00 mg of the title compound.

[0741] The structural characterization data are as follows:

[0742] ESI-MS (m / z): 823.3 [M+1] + .

[0743] Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxy-2,7,10-trioxa-4-azadodecane-12-amide)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-6)

[0744] Compound B-03-5 (507.00 mg, 616.18 μmol) and diisopropylethylamine (238.91 mg, 1.85 mmol) were dissolved in dichloromethane (20 mL), and then p-nitrophenyl chloroformate (372.60 mg, 1.85 mmol) was dissolved in dichloromethane (1 mL) and slowly added dropwise to the reaction solution. After addition, the mixture was reacted at room temperature for 15 h. The reaction was monitored by HPLC-MS / MS; the reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (methanol: dichloromethane = 1:20) to obtain 496.00 mg of the title compound.

[0745] The structural characterization data are as follows:

[0746] ESI-MS (m / z): 988.5 [M+1] + .

[0747] Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxy-2,7,10-trioxa-4-azadodecane-12-amide)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[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-triacetate (B-03-7)

[0748] 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, and the mixture was stirred for 12 h, and the reaction was monitored by HPLC-MS. Water and ethyl acetate were added and stirred, and the mixture was separated at rest. The organic phase was washed with saturated brine and dried, and concentrated under reduced pressure to obtain 100.00 mg of the crude title compound, which was directly used for the next step.

[0749] The structural characterization data are as follows:

[0750] ESI-MS (m / z): 1326.2 [M+1] + .

[0751] 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-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[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)

[0752] Compound B-03-7 (100.00 mg, 0.08 mmol) was dissolved in MeOH (5 mL), 1 drop of dichloromethane was added, and an aqueous solution (1 mL) of lithium hydroxide monohydrate (15.82 mg, 0.377 mmol) was added, and the mixture was stirred for 2 hours. The reaction was monitored by HPLC-MS / MS; 3N hydrochloric acid aqueous solution was added to adjust the pH of the reaction solution to 4, and the solution was concentrated under reduced pressure and purified by preparative HPLC (conditions are as follows), and the preparative solution was freeze-dried to obtain 27.00 mg of the title compound.

[0753] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0754] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0755] Time [min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL / min] 0.00 20 80 28 2.00 20 80 28 18.00 80 20 28

[0756] The structural characterization data are as follows:

[0757] ESI-MS (m / z): 964.2 [M+1] + .

[0758] 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]dioxolane[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-ynamido)ethoxy)ethoxy)acetamido))phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03)

[0759] 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 under stirring. The mixture was reacted at room temperature for 4 hours, and the reaction was monitored by HPLC-MS. The reaction solution was purified by preparative HPLC (conditions are as follows), and the preparative solution was freeze-dried to obtain 11.70 mg of the title compound.

[0760] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0761] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0762]

[0763]

[0764] The structural characterization data are as follows:

[0765] ESI-MS (m / z): 1214.4 [M+1] + .

[0766] Example 7 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)

[0767]

[0768] Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline (1-5-02)

[0769] 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 was completed, the mixture was reacted at room temperature for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. 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.

[0770] The structural characterization data are as follows:

[0771] 1 H NMR (400MHz, DMSO-d6) δ6.77 (dd, J=2.5, 1.4Hz, 1H), 6.51 (dd, J=11.7, 2.5Hz, 1H), 5.84 (s, 2H).

[0772] Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide (1-5-03)

[0773] 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 was completed, the temperature was raised to 50°C for 15 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was quenched with methanol (5 mL), it was directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 1.01 g of the title compound.

[0774] The structural characterization data are as follows:

[0775] ESI-MS (m / z): 265.9 [M+H] + .

[0776] Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)-3-butenoic acid (1-5-04)

[0777] 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), and then N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tri(o-methylphenyl)phosphine (114.21 mg, 375.24 μmol) and palladium acetate (42.12 mg, 187.62 μmol) were added. After the addition, the reaction system was replaced with nitrogen three times, and the temperature was raised to 100 ° C under a nitrogen atmosphere for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1N sodium hydroxide aqueous solution (60 mL) and ethyl acetate (50 mL) were added to shake and layer. After separating the lower aqueous phase, the pH was adjusted to about 3 with a 4 mol / L hydrochloric acid aqueous solution, then extracted with ethyl acetate, the combined organic phases were 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 a crude product of the title compound.

[0778] The structural characterization data are as follows:

[0779] ESI-MS (m / z): 272.0 [M+H] + .

[0780] Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid (1-5-05)

[0781] The crude product of compound 1-5-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL), and then 10% palladium carbon (0.10 g) was added. After the addition was complete, the reaction system was replaced three times with a hydrogen balloon, and the reaction was carried out under a hydrogen atmosphere for 4 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain 1.00 g of the crude product of the title compound.

[0782] The structural characterization data are as follows:

[0783] ESI-MS (m / z): 274.0 [M+H] + .

[0784] Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-06)

[0785] 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 trifluoroacetic anhydride (3.84 g, 18.27 mmol, 2.54 mL) was slowly added. After the addition was completed, the temperature was kept at 5 ° C for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by rapid silica gel column to obtain 0.43 g of the title compound.

[0786] The structural characterization data are as follows:

[0787] ESI-MS (m / z): 256.1 [M+H] + .

[0788] Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-07)

[0789] Tetrahydrofuran (16 mL) and tert-butanol (4 mL) were added to the reaction bottle, cooled to 5 ° C in an ice bath, potassium tert-butoxide (415.18 mg, 3.70 mmol) was added, and compound 1-5-06 (0.43 mg, 1.68 mmol) was dissolved in tetrahydrofuran (1 mL) and slowly added dropwise to the reaction solution. After 10 minutes, isoamyl nitrite (315.24 mg, 2.69 mmol) was added. After the addition was completed, the reaction was maintained at 5 ° C for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was quenched with saturated ammonium chloride aqueous solution, it was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to obtain 455.00 mg of the crude product of the title compound.

[0790] The structural characterization data are as follows:

[0791] ESI-MS (m / z): 285.0 [M+H] + .

[0792] Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-08)

[0793] The crude product of compound 1-5-07 (0.40 g, 1.41 mmol) was dissolved in methanol (10 mL), and then 3 mol / L aqueous hydrochloric acid solution (1 mL) and 10% palladium carbon (40.00 mg) were added. After the addition, the reaction system was replaced with hydrogen three times, and the reaction was carried out at room temperature for 1 hour under a hydrogen atmosphere. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 0.43 g of the crude hydrochloride of the title compound.

[0794] The structural characterization data are as follows:

[0795] ESI-MS (m / z): 271.0 [M+H] + .

[0796] Step 8: Synthesis of (9H-fluoren-9-yl)methyl)(8-acetamide-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-09)

[0797] The crude hydrochloride 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 was completed, the reaction was stirred at room temperature for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. 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 C18 reverse phase column (acetonitrile: 0.05% formic acid water = 20%-100%) to obtain 301.00 mg of the title compound.

[0798] The structural characterization data are as follows:

[0799] ESI-MS (m / z): 493.2 [M+H] + .

[0800] 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)

[0801] Compound 1-5-09 (300.00 mg, 608.61 μmol) was dissolved in dioxane (5 mL), and 12 mol / L concentrated hydrochloric acid (1 mL) was added. After the addition was completed, the temperature was raised to 60°C for reaction for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. 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 rapid silica gel column (ethyl acetate: petroleum ether = 0-50%) to obtain 198.00 mg of the title compound.

[0802] The structural characterization data are as follows:

[0803] ESI-MS (m / z): 451.1 [M+H] + .

[0804] Step 10: Synthesis of (9H-fluoren-9-yl)methyl)((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (1-5-11)

[0805] (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), and then p-toluenesulfonic acid (75.53 mg, 439.13 μmol) was added. After the addition was completed, the temperature was raised to 140 ° C and reacted for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140 ° C to obtain a crude product. The crude product was purified by rapid silica gel column (methanol: dichloromethane = 0-5%) to obtain 256.00 mg of the title compound.

[0806] The structural characterization data are as follows:

[0807] ESI-MS (m / z): 678.1 [M+H] + .

[0808] Step 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)

[0809] 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 was completed, the mixture was reacted at room temperature for 0.5 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the ethylenediamine was evaporated under reduced pressure, the pH of the reaction solution was adjusted to 2-3 with 1 mol / L hydrochloric acid aqueous 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).

[0810] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0811] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0812] Time [min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL / min] 0 10 90 28 3 10 90 28 18 70 30 28

[0813] 1-5-A (6min LCMS peak at the front, retention time: 1.276min)

[0814] The structural characterization data are as follows:

[0815] 1 H NMR (400MHz, DMSO-d6) δ8.00(d,J=10.3Hz,1H),7.33(s,1H),6.54(s,1H),5.62(d,J=19.3Hz,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=7.3Hz,3H).

[0816] ESI-MS (m / z): 456.1 [M+H] +.

[0817] The structural characterization data of 1-5-B (6min LCMS peak later, retention time: 1.300min) are as follows:

[0818] 1 H NMR (400MHz, DMSO-d6) δ7.98(d,J=10.3Hz,1H),7.32(s,1H),5.61(d,J=19.4Hz,1H),5.44(s,2H),5.32(d,J=19 .4Hz,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=7.3Hz,3H).

[0819] ESI-MS (m / z): 456.1 [M+H] + .

[0820] 6min LCMS conditions:

[0821] Chromatographic column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

[0822] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0823] Time [min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL / min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

[0824] Step 12: 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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide and N-((S)- Synthesis of 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-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (1-5-12-A and 1-5-12-B)

[0825] The single-configuration compound 1-5-A (36.00 mg, 79.70 μmol) and compound A-07-3 (64.43 mg, 95.64 μmol) were dissolved in N, N-dimethylformamide (2 mL), and then 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After addition, the mixture was reacted at room temperature for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-A (51.00 mg).

[0826] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0827] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0828]

[0829]

[0830] The structural characterization data are as follows:

[0831] ESI-MS (m / z): 1111.0 [M+H] + .

[0832] The single-configuration compound 1-5-B (36.00 mg, 79.70 μmol) and compound A-07-3 (64.43 mg, 95.64 μmol) were dissolved in N, N-dimethylformamide (2 mL), and then 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After addition, the mixture was reacted at room temperature for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-B (52.00 mg).

[0833] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0834] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0835] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[0836] The structural characterization data are as follows:

[0837] ESI-MS (m / z): 1111.0 [M+H] + .

[0838] Step 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)

[0839] Weigh compound 1-5-12-A (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then add 4 mol / L hydrochloric acid in ethyl acetate (1 mL), react at room temperature for 0.5 hours, and detect the reaction by HPLC-MS / MS. The reaction solution is directly concentrated under reduced pressure to obtain a crude product, which is purified by HPLC to obtain the title compound 1-11-A (4.75 mg) of a single configuration.

[0840] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0841] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0842] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 3 15 85 28 18 90 10 28

[0843] The structural characterization data are as follows:

[0844] 1 H NMR (400MHz, DMSO-d6) δ8.50(d,J=8.9Hz,1H),8.05(d,J=10.3Hz,1H),7.33(s,1H),6.55(s,1H),5.67-5.60(m,1H),5.49(t,J=5.8Hz, 1H),5.43(s,2H),5.21(s,2H),3.96(d,J=5.8Hz,2H),3.32-3.22(m,2H),2.28-2.15(m,2H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).

[0845] ESI-MS (m / z): 514.0 [M+H] + .

[0846] Weigh compound 1-5-12-B (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then add 4 mol / L hydrochloric acid ethyl acetate (1 mL), react at room temperature for 0.5 hours, and detect the reaction by HPLC-MS / MS chromatography. The reaction solution is directly concentrated under reduced pressure to obtain a crude product, which is purified by HPLC to obtain the title compound 1-11-B (8.24 mg) of a single configuration.

[0847] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0848] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0849] Time [min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 3 15 85 28 18 90 10 28

[0850] The structural characterization data are as follows:

[0851] 1H NMR (400MHz, DMSO-d6) δ8.52(d,J=9.0Hz,1H),8.05(d,J=10.3Hz,1H),7.34(s,1H),6.55(s,1H),5.68-5.58(m,1H),5.53(t,J=5.8Hz,1H),5.43 (d,J=2.9Hz,2H),5.20(d,J=7.3Hz,2H),3.97(d,J=5.7Hz,2H),3.31-3.21(m,2H),2.26-2.15(m,2H),1.92-1.82(m,2H),0.87(t,J=7.3Hz,3H).

[0852] ESI-MS (m / z): 514.0 [M+H] + .

[0853] Example 8 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)-3 0-(2-(methylsulfonyl)pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontane-29-yn-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triacontane-38-amide or 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-hexaazatriacontane-29-yn-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triacontane-38-amide (A-17-A)

[0854]

[0855] Step 1: N 6Synthesis of (((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triacontane-38-yl)-D-lysine (2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triacontane-38-yl)-6-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanoic acid (A-17-03)

[0856] The hydrochloride of compound A-17-02 (389.68 mg, 962.45 μmol) was dissolved in dichloromethane (8 mL), and 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 drained under reduced pressure. The concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-03 (450.00 mg).

[0857] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0858] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0859] Time [min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL / min] 0 10 90 28 2 10 90 28 18 80 20 28

[0860] ESI-MS (m / z): 939.3 [M+H] + .

[0861] Step 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-tridecahydro-39,42,45,48,51,54-hexaazapentaoctadecane-58-oic acid (A-17-04)

[0862] Compound A-17-03 (50.00 mg, 118.09 μmol) was dissolved in DMF (2.5 mL), 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, and the mixture was reacted at 25° C. for 1 hour. The solvent was removed 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).

[0863] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0864] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0865] Time [min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL / min] 0 10 90 28 2 10 90 28 18 80 20 28

[0866] ESI-MS (m / z): 1344.4 [M+H] + .

[0867] Step 3: (9H-fluoren-9-ylmethyl)((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 Synthesis of 1,6,9,12,15-pentaoxy-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecano-39-aza-44-yl)carbamate (A-17-05)

[0868] Compound A-17-04 (29.86 mg, 59.50 μmol) was dissolved in DMF (3 mL), 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 mixture was reacted at 25° C. for 1 hour. The solvent was removed 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).

[0869] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0870] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0871]

[0872]

[0873] ESI-MS (m / z): 1781.6 [M+H] + .

[0874] Step 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-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecatrioxane-38-amide Synthesis of 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-pentaaza230alkane-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triacontane-38-amide (A-17-06)

[0875] 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 removed under reduced pressure and the concentrate was purified by preparative HPLC to obtain the formate salt of the title compound A-17-06 (10.00 mg).

[0876] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0877] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0878] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 2 15 85 28 18 80 20 28

[0879] ESI-MS (m / z): 1559.7 [M+H]+.

[0880] Step 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)-3 0-(2-(methylsulfonyl)pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontane-29-yn-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecatrioctadecane-38-amide or 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-(methyl) Synthesis of (A-17-A)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontane-29-yn-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoctatridecane-38-amide

[0881] The formate salt 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), and DIPEA (1.74 mg, 13.46 μmol, 2.40 μL) was added, and the mixture was reacted at 25° C. for 2 hours. The solvent was removed 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).

[0882] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0883] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0884] Time [min] Mobile phase A [%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[0885] ESI-MS (m / z): 1809.8 [M+H] + .

[0886] Example 9 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]indolizine[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amide (A-05)

[0887]

[0888] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (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), heated to 35 ° C for 16 hours, and (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]indolizine[1,2-b]quinoline-10,13-dione (1-4 (i.e. 1-2-A), 1.00 g, 1.77 mmol), cooled to 5-15°C with ice water, added DMTMM (0.98 g, 3.53 mmol), then added DIPEA (1.14 g, 8.84 mmol), reacted at 25°C for 16 hours. The reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes, the DCM phase was separated, washed with brine (100 ml), concentrated to obtain a crude product, purified by preparative high performance liquid chromatography, and freeze-dried to obtain 0.98 g of compound A-05 (i.e. A-07-A in Example 3).

[0889] A-05 separation and purification method is as follows:

[0890] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0891] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0892] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 70 24 2.00 30 70 24 18.00 80 20 24

[0893] A-05 structural characterization data are as follows:

[0894] MS m / z(ESI):1107.3[M+H]+ .

[0895] 1 H NMR (400MHz, DMSO) δ9.10 (s, 2H), 8.66-8.63 (m, 1H), 8.51 (d, J = 8.8Hz, 1H), 8.34-8.31 (m, 1H), 8.21-8.19 (m, 1H), 8.17-8.09 (m, 2 H),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),4.6 4(d,J=6.4Hz,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=7.2Hz,3H).

[0896] The following HPLC conditions were used to detect the compounds A-07-A and A-07-B prepared in Example 3; as well as the compound A-07-A prepared in Example 3 and the compound A-05 prepared in Example 9:

[0897] Instrument: Agilent 1260 HPLC VWD detector

[0898] Chromatographic column: Waters Xbridge C18 4.6*100mm*3.5μm

[0899] Mobile phase A: 0.01M ammonium phosphate aqueous solution / acetonitrile = 90 / 10; Mobile phase B: acetonitrile

[0900] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 6 22 78 11 22 78

[0901] As attached Fig. 8A As shown, the retention times of compound A-07-A and compound A-07-B are 6.6 min and 6.8 min, respectively, indicating that the above HPLC conditions have good separation of isomers;

[0902] As attached Figure 8BAs shown, compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9 were shown as one peak in HPLC with a retention time of 6.6 min, indicating that compound A-07-A was compound A-05. Example 10 (1S, 9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]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)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatriacontamido)phenyl)acetate (B-04)

[0903]

[0904] Step 1: Preparation of 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]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (B-04-1)

[0905] Dissolve (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (2g, 3.65mmol) in DMF (50mL), add DIPEA (1.18g, 9.12mmol, 1.59mL), add acetoxyacetyl chloride (548.12mg, 4.01mmol, 431.59μL) under ice-cooling and stirring, continue stirring and reacting for 1 hour. Add the reaction solution into 0.1M dilute hydrochloric acid aqueous solution to precipitate solid, and filter. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column (methanol / dichloromethane = 0% to 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol)

[0906] Its structural characterization data are as follows:

[0907] ESI-MS (m / z): 552.2 [M+1] +.

[0908] 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]indolizine[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-nonyloxy-3,9-diazapentatricarboxamido)phenyl)acetate (B-04-2)

[0909] 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]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetic acid ethyl ester (500 mg,

[0910] 0.905mmol) and DMAP (885.33mg, 7.25mmol) were dissolved in dry dichloromethane (5mL), cooled to 0℃ under nitrogen protection, and triphosgene (268.81mg, 0.905mmol) in dichloromethane solution (5mL) was added dropwise, and the mixture was stirred and reacted for 0.5 hours. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatrinitroamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44g, 1.36mmol) in dichloromethane solution was slowly added dropwise, and the mixture was naturally allowed to react at room temperature for 4 hours. Water was added to quench the reaction, and the organic phases were combined after extraction with dichloromethane for 3 times (100ml x 3), washed with saturated brine, and dried and concentrated. Purification on a silica gel column (MeOH / DCM=0% to 5%) gave the title compound (498 mg, 0.304 mmol)

[0911] Its structural characterization data are as follows:

[0912] ESI-MS (m / z): 1352.8 [M+1] + .

[0913] Step 3: Preparation 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)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentacontriacontamido)phenyl)acetate (B-04-3)

[0914] (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]indolizine[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-nonyloxy-3,9-diazapentacontriacontamido)phenyl)acetate (B-04-2) (200 mg, 0.122 mmol) was dissolved in THF (3 mL) and MeOH (3 mL), and sodium carbonate (25.88 mg, 0.224 mmol) aqueous solution (1 mL) was added dropwise under stirring. After the addition was completed, stirring was continued for 1 hour. Dilute hydrochloric acid was added dropwise to the reaction solution to neutralize the reaction, and the next step was directly carried out after reduced pressure concentration.

[0915] Its structural characterization data are as follows:

[0916] ESI-MS (m / z): 1596.7 [M+1] + .

[0917] Step 4: Preparation 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 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentacontriacontamido)phenyl)acetate (B-04-4)

[0918] (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-methyl) 4-(4-(4-(4-(4-(4-(4-(4-(4-(4-(4-oxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentacontriacontamido)phenyl)acetate (B-04-3) (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (0.5 mL) was added and the reaction was continued for 1 hour. Saturated sodium bicarbonate aqueous solution was added dropwise to the reaction solution for neutralization, and then the liquids were separated. The organic phase was concentrated to obtain a crude product. Purification by reverse phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and freeze-dried to obtain the title compound (95 mg, 69.35 μmol).

[0919] Its structural characterization data are as follows:

[0920] ESI-MS (m / z): 1323.6 [M+1] + .

[0921] Step 5: Preparation of (1S, 9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]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)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentacontriacontamido)phenyl)acetate (B-04)

[0922] (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 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27, 30,33-Nonyloxy-3,9-diazapentacontriacontamido)phenyl)acetate (B-04-4) (90 mg, 0.066 mmol) and N-ethynyl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL), and copper bromide (9.42 mg, 0.066 mmol) was added and stirred for 2 hours. The reaction solution was directly filtered, and the concentrated crude product was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

[0923] Its structural characterization data are as follows:

[0924] ESI-MS (m / z): 1628.7 [M+1] + .

[0925] The preparation high performance liquid chromatography method is as follows:

[0926] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0927] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0928] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0929] Example 11 Preparation of Antibodies

[0930] By immunizing H2L2 mice (provided by Hebo Pharmaceuticals, the antibody produced by this mouse is a chimeric antibody consisting of a fully human variable region and a mouse constant region), 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 hybridoma screening, and the above heavy chain variable region sequences were combined with the human IgG1 heavy chain constant region (SEQ ID NO: 50), and the above light chain variable region sequences were combined with the human IgG1 light chain constant region (SEQ ID NO: 51), respectively. NO:51) were combined to form three complete fully human antibodies (see Table 1), and the codons were optimized and constructed into the pTT5 plasmid. The pTT5 plasmids corresponding to each antibody heavy chain and light chain were simultaneously transfected into CHO-S cells, and the expressed antibodies in the supernatant were purified by protein A to obtain the corresponding antibodies.

[0931] The HER3 control antibody was derived from U1-59 in patent application CN200680049887. After codon optimization, the heavy and light chain nucleotide sequences of the antibody were synthesized and cloned into the pTT5 vector, respectively, and expressed and purified according to the above method.

[0932] Table 1: Sequence information of 22B6D2-hIgG1, 47A3E3-hIgG1, 100H7D3-hIgG1

[0933]

[0934]

[0935] Example 12: Coupling of a compound comprising a cell bioactive molecule and a linker to an antibody

[0936] The antibodies 22B6, 47A3, and 100H7 involved in the antibody-drug conjugates prepared in the following examples are the 22B6D2-hIgG1, 47A3E3-hIgG1, and 100H7D3-hIgG1 antibodies described in the second part above, respectively.

[0937] The sample coupling preparation is as follows:

[0938] 0.46 ml of 22B6, 47A3, 100H7, U1-59, and hIgG1 antibodies (all adjusted to 11.0 mg / mL) were taken respectively, diluted with 0.1 M edetate disodium solution (pH 7.7), then adjusted to pH 7.7 with 1 M Na2HPO4 solution, added with 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution, mixed, and left at room temperature for 90 min. 4.0-10 times the amount of the substance of the "drug-linker" compound dissolved in dimethyl sulfoxide was added to the above solution system, mixed, and left at room temperature for 2 h. After completion, the buffer was replaced with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), and then sucrose and Tween 20 were added and mixed to obtain an antibody drug conjugate (i.e., ADC), as shown in Table 2.

[0939] The ADC drug / antibody ratio (DAR value) is determined as follows:

[0940] LC-MS determination of ADC sample molecular weight and calculation of drug / antibody ratio DAR value

[0941] ADC samples were subjected to LC-MS molecular weight analysis.

[0942] Chromatographic conditions:

[0943] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0944] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[0945] Flow rate: 0.25 ml / min; Sample chamber temperature: 8 °C; Column temperature: 60 °C; Injection volume: 2 μl;

[0946] Time (minutes) 2 20 22 25 26 30 Mobile phase A (volume %) 75 60 5 5 75 75 Mobile phase B (volume %) 25 40 95 95 25 25

[0947] Mass spectrometry conditions:

[0948] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0949] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5s;

[0950] m / z 600-4000; Time bins to sum 40.

[0951] Table 2 ADC number and DAR

[0952] Drug-Linker Number ADC Number Drug-linker to antibody molar ratio DAR A-07-A 22B6-A-07-A-1 10：1 7.99 A-07-A 22B6-A-07-A-2 5：1 3.56 A-07-A 47A3-A-07-A 10：1 6.27 A-07-A 100H7-A-07-A 10：1 8.01 A-07-A U1-59-A-07-A 10：1 8.02 A-07-A hIgG1-A-07-A 10：1 8.03 A-01 22B6-A-01 10：1 7.93 A-01 U1-59-A-01 10：1 7.89 B-03 22B6-B-03-1 10：1 8.0 B-03 22B6-B-03-2 5：1 3.14 B-03 hIgG1-B-03-1 10：1 7.90 B-03 hIgG1-B-03-2 5：1 4.20 B-01 22B6-B-01 10：1 8.07 B-01 hIgG1-B-01 10：1 8.06 A-01 hIgG1-A-01 10：1 7.98

[0953] 1. LC-MS determination of the molecular weight of ADC 22B6-A-07-A-1 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-A-07-A-1 is shown in Table 3.

[0954]

[0955] Table 3: Measured molecular weight of ADC 22B6-A-07-A-1

[0956]

[0957] Table 4: DAR values ​​for ADC 22B6-A-07-A-1

[0958]

[0959] The calculated drug / antibody ratio of ADC 22B6-A-07-A-1 was DAR=7.99 (see Table 4).

[0960] 2. LC-MS determination of the molecular weight of ADC 22B6-A-07-A-2 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-A-07-A-2 is shown in Table 5.

[0961]

[0962] Table 5: Measured molecular weight of ADC 22B6-A-07-A-1

[0963]

[0964] Table 6: DAR values ​​for ADC 22B6-A-07-A-2

[0965]

[0966]

[0967] The drug / antibody ratio of ADC 22B6-A-07-A-2 was calculated to be DAR=3.56 (see Table 6).

[0968] 3. LC-MS determination of ADC 47A3-A-07-A molecular weight and calculation of drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 47A3-A-07-A is shown in Table 7.

[0969]

[0970] Table 7: ADC 47A3-A-07-A measured molecular weight

[0971]

[0972] Table 8: DAR values ​​for ADC 47A3-A-07-A

[0973]

[0974] The drug / antibody ratio of ADC 47A3-A-07-A was calculated to be DAR=6.27 (see Table 8).

[0975] 4. LC-MS determination of the molecular weight of ADC 100H7-A-07-A and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 100H7-A-07-A is shown in Table 9.

[0976] Table 9: ADC 100H7-A-07-A measured molecular weight

[0977]

[0978] Table 10: DAR values ​​for ADC 100H7-A-07-A

[0979]

[0980] The drug / antibody ratio of ADC 100H7-A-07-A was calculated to be DAR=8.01 (see Table 10).

[0981] 5. LC-MS determination of ADC U1-59-A-07-A molecular weight and calculation of drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC U1-59-A-07-A is shown in Table 11.

[0982]

[0983] Table 11: ADC U1-59-A-07-A measured molecular weight

[0984]

[0985] Table 12: DAR values ​​for ADC U1-59-A-07-A

[0986]

[0987]

[0988] The calculated drug / antibody ratio of ADC U1-59-A-07-A was DAR=8.02 (see Table 12).

[0989] 6. LC-MS determination of ADC hIgG1-A-07-A molecular weight and calculation of drug / antibody ratio DAR value The LC-MS molecular weight analysis of ADC hIgG1-A-07-A is shown in Table 13.

[0990]

[0991] Table 13: Measured molecular weight of ADC hIgG1-A-07-A

[0992]

[0993] Table 14: DAR values ​​of ADC hIgG1-A-07-A

[0994]

[0995] The drug / antibody ratio of ADC hIgG1-A-07-A(8) was calculated to be DAR=8.03 (see Table 14).

[0996] 7. LC-MS determination of ADC 22B6-A-01 molecular weight and calculation of drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-A-01 is shown in Table 15.

[0997]

[0998] Table 15: Measured molecular weight of ADC 22B6-A-01

[0999]

[1000] Table 16: DAR values ​​for ADC 22B6-A-01

[1001]

[1002] The drug / antibody ratio of ADC 22B6-A-01 was calculated to be DAR=7.93 (see Table 16).

[1003] 8. LC-MS determination of ADC U1-59-A-01 molecular weight and calculation of drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC U1-59-A-01 is shown in Table 17.

[1004]

[1005] Table 17: ADC U1-59-A-01 measured molecular weight

[1006]

[1007]

[1008] Table 18: DAR values ​​for ADC U1-59-A-01

[1009]

[1010] The calculated drug / antibody ratio of ADC U1-59-A-01 is DAR=7.89 (see Table 18).

[1011] 9. LC-MS determination of the molecular weight of ADC 22B6-B-03-1 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-B-03-1 is shown in Table 19.

[1012]

[1013] Table 19: Measured molecular weight of ADC 22B6-B-03-1

[1014]

[1015] Table 20: DAR values ​​for ADC 22B6-B-03-1

[1016]

[1017]

[1018] The calculated drug / antibody ratio of ADC 22B6-B-03-1 was DAR=8.0 (see Table 20).

[1019] 10. LC-MS determination of the molecular weight of ADC 22B6-B-03-2 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-B-03-2 is shown in Table 21.

[1020]

[1021] Table 21: Measured molecular weight of ADC 22B6-B-03-2

[1022]

[1023] Table 22: DAR values ​​for ADC 22B6-B-03-2

[1024]

[1025] The drug / antibody ratio of ADC 22B6-B-03-2 was calculated to be DAR=3.14, see Table 22.

[1026] 11. LC-MS determination of the molecular weight of ADC hIgG1-B-03-1 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC hIgG1-B-03-1 is shown in Table 23.

[1027]

[1028] Table 23: Measured molecular weight of ADC hIgG1-B-03-1

[1029]

[1030] Table 24: DAR values ​​of ADC hIgG1-B-03-1

[1031]

[1032] The calculated drug / antibody ratio of ADC hIgG1-B-03-1 was DAR=7.90 (see Table 24).

[1033] 12. LC-MS determination of the molecular weight of ADC hIgG1-B-03-2 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC hIgG1-B-03-2 is shown in Table 25.

[1034]

[1035] Table 25: Measured molecular weight of ADC hIgG1-B-03-2

[1036]

[1037]

[1038] Table 26: DAR values ​​of ADC hIgG1-B-03-2

[1039]

[1040] The calculated drug / antibody ratio of ADC hIgG1-B-03-2 was DAR=4.20 (see Table 26).

[1041] 13. LC-MS determination of the molecular weight of ADC 22B6-B-01 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-B-01 is shown in Table 27.

[1042]

[1043] Table 27: ADC 22B6-B-01 measured molecular weight

[1044]

[1045] Table 28: DAR values ​​for ADC 22B6-B-01

[1046]

[1047]

[1048] The calculated drug / antibody ratio of ADC 22B6-B-01 was DAR=8.07 (see Table 28).

[1049] 14. LC-MS determination of the molecular weight of ADC hIgG1-B-01 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC hIgG1-B-01 is shown in Table 29.

[1050]

[1051] Table 29: Measured molecular weight of ADC hIgG1-B-01

[1052]

[1053] Table 30: DAR values ​​of ADC hIgG1-B-01

[1054]

[1055] The calculated drug / antibody ratio of ADC hIgG1-B-01 was DAR=8.06 (see Table 30).

[1056] 15. LC-MS determination of ADC hIgG1-A-01 molecular weight and calculation of drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC hIgG1-A-01 is shown in the table below.

[1057] Table 31: DAR values ​​of ADC hIgG1-A-01

[1058]

[1059]

[1060] Example 13a Conjugation of ADC 22B6-B-04-1

[1061] Take 0.854ml 22B6 antibody (23.43mg / mL), dilute with 42.68uL 20mM PB+0.1M EDTA (pH 7.60), then adjust the pH to 7.57 with 1M Na2HPO4 solution, add 20mM TCEP (tri(2-carboxyethyl)phosphine, 73.68uL, pH7.60) solution and mix well, and leave at room temperature for 1.5 hours. Then slowly add 12 times the amount of substance dissolved in dimethyl sulfoxide B-04 (164uL, 10mM) solution and mix well, let stand at room temperature overnight, and after completion, use NAP-5 gel column (Cytiva) to replace the buffer with 20mM histidine buffer solution at pH 6.0 to obtain ADC 22B6-B-04-1. The DAR value determined by mass spectrometry is 8.62.

[1062] Example 13b. Conjugation of ADC 22B6-B-04 and ADC hIgG1-B-04

[1063] As an alternative, ADC 22B6-B-04 can be prepared by the following method: take 2.667ml 22B6 antibody (18.75mg / mL), dilute with 133.35uL 20mM PB+0.1M EDTA (pH 7.60), then adjust the pH to 7.63 with 1M Na2HPO4 solution, add 10mM TCEP (tris(2-carboxyethyl)phosphine, 184.19uL, pH 7.60) solution, mix well, and let stand at room temperature for 1.5h. Then, 10 times the amount of the substance was slowly added into a solution of B-04 (341.73uL, 10mM) dissolved in dimethyl sulfoxide, and the mixture was allowed to stand at room temperature for 2h. After completion, a NAP-5 gel column (Cytiva) was used to replace the buffer with a 20mM histidine buffer solution at pH 6.0 to obtain ADC 22B6-B-04-2. The DAR value was determined by mass spectrometry to be 6.87.

[1064] Example 13c. Conjugation of ADC hIgG1-B-04

[1065] The antibody was replaced with hIgG1 antibody, and the ADC hIgG1-B-04 sample was prepared by the same method as in Example 13b. The DAR value was determined by mass spectrometry to be 7.02.

[1066] Example 14. Detection of Antibody Drug Conjugate Activity

[1067] 1. Cell affinity detection of anti-human HER3 antibodies and their drug conjugates

[1068] The affinity of anti-human HER3 ADC to MDA-MB-453, NCI-H358, KPL-4, and A431 cells was detected using a flow cytometer (Beckman, model Cytoflex).

[1069] Digest the adherent MDA-MB-453, NCI-H358, KPL-4, and A431 cells with Tryple (Gibco) solution, count and take an appropriate amount of cells, wash twice with 1xPBS, resuspend in PBS solution containing 1% BSA, and then transfer the cells to a 96-well conical bottom plate, 50μl per well; dilute the candidate antibody and ADC with PBS solution containing 1% BSA, starting at 5μg / mL, 3-fold or 2.5-fold gradient dilution, then take 50μl of the diluted antibody or ADC and add it to the conical bottom plate containing cells, incubate at 4 degrees for 40min; wash the cells twice with PBS, then add 50μl of the diluted secondary antibody to each well, mix well, and incubate at 4 degrees for 30min; wash the cells twice with PBS, then resuspend the cells in 400μl PBS, and detect on a flow cytometer. Data processing: Export the average fluorescence signal value, then import it into GraphPad Prism 6 software, calculate EC50, and the results are shown in Table 32 and Figure 1A , 1B , 1C, 1D. The above results show that after the antibodies of the present invention (such as 22B6, 47A3 and 100H7) are coupled to ADC, the cell affinity is significantly higher than the ADC formed by coupling the control antibody U1-59; and the cell affinity of the naked antibody of the present invention (such as 22B6) is significantly higher than the control naked antibody U1-59. In addition, after the naked antibody is coupled to ADC, the cell affinity changes slightly, and the affinity of 22B6 coupled to ADC is still the highest.

[1070] Table 32 Anti-human HER3 antibody and ADC cell affinity determination results

[1071]

[1072] Note: “ / ” means not detected.

[1073] 2. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugates

[1074] MDA-MB-453, HCC1569, and HEK293T-h HER3 cells were digested with TrypLE (manufacturer Gibco) solution, and an appropriate amount of cells were counted and taken. After diluting with growth medium, 5000 cells or 3000 cells (100 μl) / well were plated on a 96-well plate and cultured at 37°C and 5% CO2 overnight; the next day, ADC was diluted with growth medium, starting at 150 μg / ml, and diluted 2.5 times, and then 100 μl of the diluted ADC was added to the 96-well plate containing cells, and the plate was placed in an incubator. After culturing at 37°C and 5% CO2 for 6 days, 20 μl of CCK8 was added to each well, incubated at 37°C for 2-5 hours, and the OD450nm absorbance value was read by an enzyme reader; the raw data was imported into GraphPrism 6 to calculate IC 50 Value. As shown in Table 33 and Figure 2A , 2B As shown in Figure 2C, the ADC formed by the antibodies of the present invention (e.g., 22B6-A-01, 22B6-A-07-A-1) has target-specific killing activity, wherein 22B6-A-07-A-1 has an IC of 1.17 for tumor cells. 50 The difference between hIgG1-A-01 and hIgG1-A-07-A was 6-8 times. hIgG1-A-01 and hIgG1-A-07-A had no obvious killing effect on overexpressing cells (HEK293T-h HER3). The IC values ​​of ADCs such as 22B6-A-01 and 22B6-A-07-A were 6-8 times higher than those of hIgG-A-01 and hIgG-A-07-A. 50 The gap is even bigger.

[1075] Table 33 Anti-human HER3 ADC in vitro cytotoxicity results

[1076]

[1077] Note: “ / ” means not detected.

[1078] 3. Bystander effect of anti-human HER3 antibody-drug conjugates

[1079] HEK293T and HEK293T-h HER3 cells were digested with 0.25% Trypsin-EDTA (manufacturer Gibco), and appropriate amount of cells were taken and washed twice with 1xPBS. The cell density was adjusted to 1*10 6cells / ml, then added Cell Trace Violet at a final concentration of 5 μM for labeling, mixed and placed in an incubator for incubation for 20 min, then washed with growth medium and counted, and plated into 24-well plates (500 μl / well) according to HEK293T-labled: HEK293T=4000:12000 cells / well, HEK293T-labled: HEK293T-hHER3=4000:12000 cells / well, HEK293T-h HER3-labled: HEK293T-h HER3=4000:12000 cells / well, and cultured overnight at 37°C, 5% CO2;

[1080] On the second day, ADC was diluted with growth medium to a concentration of 20 nM, 4 nM, and 0.8 nM, and then 500 μl of the diluted ADC was added to the corresponding 24-well plates containing cells, and the plates were placed in an incubator at 37°C and 5% CO2 for 4 days;

[1081] HEK293T and HEK293T-h HER3 cells were digested with 0.25% Trypsin-EDTA, and growth medium containing PI at a final concentration of 3 μM was added and mixed. After incubation at room temperature for 15 min, the cells were detected by flow cytometry (Thermo, model Attune NxT). Figure 3A , 3B As shown in Figure 3C, 22B6-A-07-A-1 and U1-59-A-01 had basically no killing effect on negative HEK293T cells, but had significant specific killing effect on HEK293T-HER3. In the condition where negative HEK293T cells and positive HEK293T-HER3 cells coexisted, 22B6-A-07-A-1 and U1-59-A-01 had obvious killing effect on negative cells, indicating that 22B6-A-07-A-1 and U1-59-A-01 had bystander activity.

[1082] 4. Anti-human HER3 antibody-drug conjugate plasma stability test

[1083] Accurately measure 3.59μl of 20.9mg / ml 22B6-A-07-A-1ADC sample into an EP tube, add 496μl of blank plasma from humans, cynomolgus monkeys and mice respectively, mix well to obtain human, cynomolgus monkey and mouse plasma samples containing 22B6-A-07-A-1ADC sample. Place the sample in a constant temperature incubator and incubate at 37°C. Take out 50μl of the sample at 1, 3, 6, 24, 48, 96 and 168h, add 200μl of 0.5% formic acid-acetonitrile to precipitate the protein, centrifuge at 4°C, 13,000rpm for 10min, take the supernatant, store at -80°C, and use LC-MS / MS to detect the concentration of cytotoxic drugs after all samples are collected. The results are as follows Figure 4 As shown, the cytotoxic drug shedding does not exceed 5%, indicating that the anti-human HER3 antibody-drug conjugate (eg, 22B6-A-07-A-1) of the present invention is stable in the plasma of different species.

[1084] 5. In vivo efficacy testing of anti-human HER3 antibody-drug conjugate H358 model

[1085] NCI-H358 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-H358 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously in female Balb / c-nu mice to establish a non-small cell lung cancer model. When the average tumor volume was about 130-150mm 3 At 1:10 pm, the mice were randomly divided into 11 groups according to the tumor size, which were: vehicle control group (i.e. negative control group), hIgG1-A-01 1mg / kg group, U1-59-A-011mg / kg group, ADC22B6-A-01 1mg / kg group, hIgG1-B-01 1mg / kg group, ADC 22B6-B-01 1mg / kg group, hIgG1-A-07-A1mg / kg group, ADC 22B6-A-07-A-1 1mg / kg group and 3mg / kg group, hIgG1-B-03-1 1mg / kg group, ADC22B6-B-03-1 1mg / kg group. All the drugs were injected through tail vein on Day0, Day1, Day4, and Day10, for a total of 3 times. After administration, the tumor diameter was measured with a vernier caliper twice a week, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day. The specific results are shown in Table 34. Figure 5A , Figure 5B .

[1086] The tumor growth inhibition rate TGI (%) was calculated using the following formula to evaluate the tumor inhibition efficacy:

[1087] TGI (%) = [1-(VT end-VT beginning) / (VC end-VC beginning)]*100%

[1088] VTend: mean tumor volume at the end of the treatment group experiment

[1089] VTstart: mean tumor volume at the start of drug administration in the treatment group

[1090] VC end: mean tumor volume at the end of the negative control group experiment

[1091] VC Start: Mean tumor volume at the beginning of drug administration in the negative control group

[1092] The experimental results show that the ADC of the present invention has a significant inhibitory effect on tumor growth in the NCI-H358 non-small cell lung cancer transplant model. Compared with the solvent control group, after three doses, the Day32 data showed that ADC 22B6-A-01

[1093] The tumor growth inhibition rates (TGI) of the 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.

[1094] The TGI of the 1 mg / kg group was 121.45%; the TGI of the positive control U1-59-A-01 1 mg / kg group was 44.51%. No animal died or significantly lost weight in each treatment group on Day 32, and no obvious drug toxicity was observed. During the treatment period, the mice tolerated the ADC of the present invention well.

[1095] Table 34 Human non-small cell lung cancer cell NCI-H358 CDX model

[1096]

[1097] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate, the same below. (In this experiment, groups 1 to 9 were grouped and dosed on Day 0, and the tumor volume (TV) ≈ 135 mm 3 Group 10 and Group 11 were grouped and administered on Day 1, and the tumor volume (TV) was ≈149 mm 3 ; Then on Day 4 and Day 10, all groups were given the drug together)

[1098] 6. In vivo efficacy testing of anti-human HER3 antibody-drug conjugates in the N87 model

[1099] NCI-N87 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously in female Balb / c-nu mice to establish a human gastric cancer model. When the average tumor volume was about 120 mm 3 At 14:00, the mice were randomly divided into groups according to the tumor size: vehicle control group, hIgG1-A-07-A 3 mg / kg group, ADC 22B6-A-07-A-1 3 mg / kg group, ADC 47A3-A-07-A 3 mg / kg group, and ADC 100H7-A-07-A 3 mg / kg group. All were injected through the tail vein on Day 0, Day 4, and Day 7, for a total of 3 times. After administration, the tumor diameter was measured with a vernier caliper twice a week, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day. The specific results are shown in Table 35. Fig. 6A , 6B .

[1100] The test results show that compared with the vehicle control group, after three doses, the data of Day32 showed that the tumor growth inhibition rates (TGI) of the ADC 22B6-A-07-A-1 3mg / kg group, the 47A3-A-07-A 3mg / kg group and the 100H7-A-07-A 3mg / kg group were 66.59%, 47.24% and 36.04%, respectively; ADC 22B6-A-07-A-1 and 47A3-A-07-A 3mg / kg had significant inhibitory effects on the tumor growth of the NCI-N87 gastric cancer transplant model. There was no animal death and significant animal weight loss in each treatment group on Day32, and no obvious drug toxicity was observed. During the treatment period, the mice tolerated the ADC of the present invention well.

[1101] Table 35 Human gastric cancer cell NCI-N87 CDX model

[1102]

[1103] 7. In vivo efficacy testing of anti-human HER3 antibody-drug conjugates in the MDA-MB-453 model

[1104] Human breast cancer MDA-MB-453 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5% CO2. MDA-MB-453 cells in the exponential growth phase were collected, resuspended to a suitable concentration by adding PBS and a final concentration of 50% matrix gel, and inoculated subcutaneously in female NCG immunodeficient mice to establish a human breast cancer transplant model. When the average tumor volume was about 150mm3 At the same time, the mice were randomly divided into vehicle control group, hIgG1-A-07-A 3mg / kg, ADC 22B6-A-07-A-1 1mg / kg group, 3mg / kg group and 10mg / kg group, hIgG1-A-01 3mg / kg group, U1-59-A-01 3mg / kg group and 10mg / kg group, hIgG1-B-03-2 3mg / kg group, ADC 22B6-B-03-2 3mg / kg group according to the tumor size. After grouping, the mice were injected into the tail vein, and a single dose was given on Day 0. After administration, the tumor volume and body weight of the mice were observed and measured regularly. The specific results are shown in Table 36. Fig. 7A , 7B .

[1105] The ADC of the present invention has a significant inhibitory effect on the tumor growth of NCG mice with subcutaneous transplantation of human breast cancer MDA-MB-453. The data on Day32 after a single administration showed that compared with the vehicle control group, the TGI of the 22B6-A-07-A-1 1mg / kg, 3mg / kg and 10mg / kg groups were 62.07%, 118.30% and 200.00%, respectively, among which the tumor of the mice in the 10mg / kg group completely disappeared from Day19 and no growth was seen on Day32; the TGI of the 22B6-B-03-2 3mg / kg group was 148.58%; the TGI of the positive control U1-59-A-01 3mg / kg group and the 10mg / kg group were 76.11% and 180.65%, respectively. During the treatment period, the mice tolerated the ADC of the present invention well.

[1106] Table 36 Human breast cancer cell MDA-MB-453CDX model

[1107]

[1108]

[1109] 8. Cell affinity detection of anti-human HER3 antibodies and their drug conjugates

[1110] The affinity of anti-human HER3 ADC to MDA-MB-453 cells was detected using a flow cytometer (Beckman, model Cytoflex).

[1111] Digest the adherent MDA-MB-453 cells with Tryple (Gibco) solution, count and take an appropriate amount of cells, wash twice with 1xPBS, resuspend in 1% BSA (prepared with PBS) solution, and then transfer the cells to a 96-well pointed bottom plate, 50μl per well; dilute the candidate antibody and its drug conjugate with 1% BSA, starting at 15μg / mL, 3-fold gradient dilution, and then take 50μl of the diluted antibody or antibody-conjugated drug and add it to the pointed bottom plate containing the cells, incubate at 4 degrees for 40min; wash the cells twice with PBS, then add 50μl of the diluted secondary antibody to each well, mix well, and incubate at 4 degrees for 30min; wash the cells twice with PBS, then resuspend the cells in 200μl PBS, and detect on a flow cytometer. Data processing: Export the average fluorescence signal value, then import it into GraphPad Prism 6 software, calculate EC50, and the results are shown in Table 37 and Fig. 9 The above results show that after the antibody of the present invention (such as 22B6) is coupled to ADC, the cell affinity is significantly higher than that of the ADC formed by coupling to the control antibody U1-59.

[1112] Table 37 Anti-human HER3 antibody and ADC cell affinity determination results

[1113]

[1114] 9. Anti-human HER3 antibody-drug conjugate endocytosis activity test

[1115] The endocytic activity of anti-human HER3 ADC in MDA-MB-453 and HCC1569 cells was detected using a flow cytometer (Beckman, model Cytoflex).

[1116] The adherent cells were digested with Trypsin-EDTA (0.25%, Shanghai Yuanpei) solution and counted. The cell density was adjusted to 1×10 5 100 μl of cell suspension was added to each well of a 96-well plate (the number of cells was 1×10 4 / well), and place the 96-well plate in a 37°C, CO2 constant temperature incubator for 24 hours. Take out the 96-well plate, discard the culture medium, and add 50μl of fresh complete culture medium to each well; use complete culture medium to gradient dilute the bispecific antibody and control antibody to a final concentration of 0.55, 1.64, 4.94, 14.81, 44.44, 133.33, 400, and 1200ng / ml, for a total of 8 concentration points; dilute PHrodo reagent (Thermo) to 12μg / ml with complete culture medium (the final concentration of PHrodo is 3μg / ml); mix the gradient diluted antibody to be tested with the diluted PHrodo reagent was mixed evenly in a 1:1 ratio (30 μl:30 μl), and incubated at room temperature in the dark for 30 min; 50 μl of the test antibody and PHrodo reagent mixture was added to a 96-well plate, and cultured at 37°C, 5% CO2 for 24 h; the 96-well plate was removed, the culture medium was discarded, and sterile PBS was used to wash once, 100 μl of Trypsin-EDTA (0.25%) was added to each well to digest the cells, and then 100 μl of complete culture medium was added to neutralize; the cells in the wells were dispersed by blowing, and then detected by FACS. Data processing: The average fluorescence signal value was exported, and then imported into GraphPad Prism 6 software to calculate EC 50 , the results are shown in Table 38 and Fig.10 , Fig.11 As shown, the endocytic activity of 22B6 and the ADC after coupling is better than or not inferior to the control antibody U1-59 and the ADC formed by its coupling.

[1117] Table 38 Anti-human HER3 antibody and ADC endocytosis activity assay results

[1118]

[1119] 10. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugates

[1120] Digest MDA-MB-453 cells with TrypLE (Gibco) solution, count and take appropriate amount of cells, dilute with growth medium, and spread 5000 cells (100 μl / well) on 96-well plate, culture at 37°C, 5% CO2 overnight; the next day, dilute ADC with growth medium, starting at 150 μg / ml, 2.5-fold gradient dilution, then take 100 μl of diluted ADC and add it to the 96-well plate containing cells, put the plate into the incubator, culture at 37°C, 5% CO2 for 6 days, add 20 μl CCK8 to each well, incubate at 37°C for 2-5 hours, and read the OD450nm absorbance with a microplate reader; import the raw data into Graph Prism 6 to calculate the IC50 value. As shown in Table 39 and Fig.12As shown, both 22B6-A-07-A-1 and 22B6-B-04-2 exhibited significant cell killing activity on MDA-MB-453 cells.

[1121] Table 39 Anti-human HER3 ADC in vitro cytotoxicity results

[1122] IC50(nM) drug MDA-MB-453 hIgG1-A-07-A 29.4 22B6-A-07-A-1 4.0 hIgG1-B-04-2 0.18 22B6-B-04-2 0.07

[1123] Note: “ / ” means not detected.

[1124] Although the present invention has been described in detail with reference to the embodiments illustrated, it should be understood that the present invention is not limited thereto. Those of ordinary skill in the art and those who understand the teachings herein will appreciate other modifications and embodiments within the scope of the present invention. Therefore, the present invention is limited only by the appended claims.

Claims

1. An antibody drug conjugate having the formula Ab-[MLED] x The structure shown, wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3; M is a linker site that binds to an antibody or its antigen-binding fragment; L is a linker between the linker sites M and E; E is a structural fragment connecting L and D; D is the cytotoxic drug fragment; x is selected from 1 to 10.

2. The antibody-drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or, (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or, (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof; in, The variant described in any one of (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 amino acid sequence from which it is derived, or the variant has one or more 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 with the amino acid sequence of the corresponding CDR of VH and VL of (1a), (1b) and (1c), and the variant binds to HER3; or, (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or, (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or, (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 40 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof; wherein the variant described in any one of (2a), (2b), and (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 amino acid sequence from which it is derived, or the variant has one or more 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (2a), (2b), and (2c), and the variant binds to HER3; or, (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or, (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or, (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:41 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:42 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:44 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:52 or a variant thereof; wherein the variant described in any one of (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% amino acid sequence identity compared to the amino acid sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; provided that the amino acid sequence of the CDR of the variant has 100% sequence identity with the amino acid sequence of the corresponding CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

3. The antibody-drug conjugate of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof; (b) a VH comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof; in, 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 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (a), (b) and (c), and the variant binds to HER3.

4. The antibody-drug conjugate of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin 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; and (b) a light chain constant region (CL) of a human immunoglobulin 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.

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

51.

6. The antibody drug conjugate of any one of claims 1 to 5, wherein M is in, Ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl radical substitution; M1 is selected from a single bond, C 1-20 Alkylene, C 2-20 Alkenylene and C 2-20 Alkyne group.

7. The antibody drug conjugate of any one of claims 1 to 6, wherein M is Wherein ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings to a benzene ring via a single bond, wherein the alicyclic heterocycle is optionally substituted with one or more selected from oxy (=O), halogen and C 1-4 Alkyl radical substitution; M1 is selected from a single bond, C 3-10 Alkylene, C 3-10 Alkenylene and C 3-10 Alkyne group.

8. The antibody drug conjugate of any one of claims 1 to 7, wherein M is wherein ring A is selected from M1 is selected from a single bond, C 5-8 Alkylene, C 5-8 Alkenylene and C 5-8 Alkyne group.

9. The antibody drug conjugate of any one of claims 1 to 7, wherein M is 10. The antibody drug conjugate according to any one of claims 1 to 7, Where M is 11. The antibody drug conjugate of any one of claims 1 to 10, wherein L is selected from one or more of the following structures: C 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), 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-G ly-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, Where R' represents hydrogen, C 1-6 Alkyl or containing -(CH2CH2O) r -alkyl; r is an integer selected from 1-10; s is an integer selected from 1-20.

12. The antibody drug conjugate of any one of claims 1 to 11, wherein L is selected from one or more of the following structures: C 1-6 Alkylene, -NH-, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), Gly, Gly-Gly-Phe-Gly, wherein s is an integer selected from 1-20.

13. The antibody drug conjugate of any one of claims 1 to 11, wherein L is selected from the following structures: Wherein s is an integer selected from 1-20; 14. The antibody drug conjugate of any one of claims 1 to 11, wherein L is selected from the following structures:

15. The antibody drug conjugate of any one of claims 1 to 11, wherein L is selected from the following structures:

16. The antibody drug conjugate of any one of claims 1 to 15, wherein E is a single bond, -NH-CH2-, -NH-CH2-O-CH2-CO- or is selected from the following structures:

17. The antibody drug conjugate of any one of claims 1 to 16, wherein: E is a single bond, -NH-CH2-, -NH-CH2-O-CH2-CO-, 18. The antibody drug conjugate of any one of claims 1 to 16, wherein: E is -NH-CH2-O-CH2-CO-, 19. The antibody drug conjugate of any one of claims 1 to 16, wherein: E is -NH-CH2-O-CH2-CO- or 20. The antibody drug conjugate according to any one of claims 1 to 19, Select from the following structures:

21. The antibody drug conjugate of any one of claims 1-20, wherein the cytotoxic drug is selected from a microtubule 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 of 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 of any one of claims 1 to 21, wherein the DNA intercalator is a pyrrolobenzodiazepine (PBD), or a pharmaceutically acceptable salt, ester or analog thereof.

24. The antibody drug conjugate of any one of claims 1-21, 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 of claim 24, wherein the topoisomerase I inhibitor is camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan, or a pharmaceutically acceptable salt, ester, or analog thereof.

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

28. The antibody drug conjugate of any one of claims 1-21, wherein the cytotoxic drug is selected from the compounds of Formula I and Formula II: in, R1, R2 are each independently selected from C 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring; R6 is selected from hydrogen and -C 1-4 Alkylene-NR a R b ; R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ; Where R a , R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl.

29. The antibody drug conjugate of any one of claims 1 to 21, wherein The cytotoxic drug is selected from the group consisting of the following compounds: The corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula.

30. The antibody drug conjugate of any one of claims 1 to 29, wherein D is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amine group on the cytotoxic drug.

31. The antibody drug conjugate of any one of claims 1 to 29, wherein D is selected from the following structures:

32. The antibody drug conjugate of any one of claims 1 to 31, wherein the antibody drug conjugate is selected from the group consisting of ADC A-01 to ADC A-25, ADC B-01 to ADC B-05 shown below: in, HA in each antibody drug conjugate represents an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 15 and a VL comprising the amino acid sequence shown in SEQ ID NO: 16; in, It indicates the specific connection mode between the thiol group in the antibody or antigen-binding fragment thereof and the linker.

33. The antibody drug conjugate of any one of claims 1-32, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 15 and a CH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 16 and a CL comprising the amino acid sequence of SEQ ID NO:

51.

34. The antibody drug conjugate of any one of claims 1 to 33, wherein the antibody drug conjugate comprises the structural formula: in, The HA in each antibody drug conjugate is selected from: (1) an antibody or an antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO: 15 and a VL as set forth in SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO: 15 and a CH as set forth in SEQ ID NO: 50, and a VL as set forth in SEQ ID NO: 16 and a CL as set forth in SEQ ID NO: 51; (2) an antibody or an antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO:32 and the VL set forth in SEQ ID NO:33, for example, an antibody or an antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO:32 and the CH set forth in SEQ ID NO:50, and the VL set forth in SEQ ID NO:33 and the CL set forth in SEQ ID NO:51; and (3) an antibody or an antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO:46 and a VL as set forth in SEQ ID NO:47, for example, an antibody or an antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO:46 and a CH as set forth in SEQ ID NO:50, and a VL as set forth in SEQ ID NO:47 and a CL as set forth in SEQ ID NO:51; x is 3 to 8.

35. The antibody drug conjugate of any one of claims 1-33, wherein the antibody drug conjugate comprises the structural formula: in, HA is an antibody or an antigen-binding fragment thereof comprising HC shown in SEQ ID NO: 17 and LC shown in SEQ ID NO: 18; and x is 7 to 8.

36. The antibody drug conjugate of any one of claims 1-35, wherein (i) the heavy chain C-terminus lacks a lysine residue; (ii) the heavy chain N-terminus is glutamine, glutamic acid or pyroglutamate; or, (iii) the heavy chain C-terminus lacks a lysine residue and the heavy chain N-terminus is glutamine, glutamic acid or pyroglutamate.

37. A drug-linker compound having a structure represented by formula M'-LED, wherein: M' is Lg is a leaving group of a nucleophilic substitution reaction, or is a hydroxyl (-OH), a thiol (-SH) or an amino (-NH2); Ring A is a 5-6 membered alicyclic ring, or a 5-20 membered aromatic ring system, wherein the alicyclic ring and the aromatic ring system are optionally substituted with one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl radical substitution; M1 is selected from a single bond, C 1-20 Alkylene, C 2-20 Alkenylene and C 2-20 Alkynylidene; L is a linker between the linker sites M and E; E is a structural fragment connecting L and D; D is a cytotoxic drug fragment.

38. The drug-linker compound of claim 37, wherein Lg is halogen, mesyl, fluorophenol or 39. The drug-linker compound of claim 37, wherein Lg forms an unsaturated double bond with the adjacent atom on ring A.

40. The drug-linker compound of claim 37, wherein M' is Lg is a methylsulfonyl group, or Lg forms a carbon-carbon double bond with the adjacent atom on ring A; ring A is a 5-membered alicyclic heterocyclic ring, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings to a benzene ring via a single bond, wherein the alicyclic heterocyclic ring is optionally substituted with one or more selected from oxy (=O), halogen and C 1-4 Alkyl radical substitution; M1 is selected from a single bond, C 3-10 Alkylene, C 3-10 Alkenylene and C 3-10 Alkyne group.

41. The drug-linker compound of claim 37, wherein M' is in Selected from M1 is selected from a single bond, C 5-8 Alkylene, C 5-8 Alkenylene and C 5-8 Alkyne group.

42. The drug-linker compound of claim 37, wherein M' is selected from 43. The drug-linker compound of claim 37, wherein M' is 44. The drug-linker compound according to any one of claims 37 to 43, which is selected from the group consisting of A-01 to A-25, B-01 to B-05 shown below: A-01： A-02： A-03： A-04： A-05: A-06: A-07: A-08: A-09: A-10: A-11: A-12: A-13： A-14: A-15: 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： 45. An antibody drug conjugate comprising an antibody that binds to HER3, said antibody being conjugated to the drug-linker of any one of claims 37 to 44 via one or more cysteine ​​residues of said antibody.

46. ​​An antibody drug conjugate comprising an antibody that binds HER3, wherein the antibody 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 through one or more cysteine ​​residues of the antibody.

47. The antibody drug conjugate of claim 45 or 46, wherein the antibody that binds to HER3 is selected from: (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or, (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or, (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof; in, The variant described in any one of (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 more 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 with the amino acid sequence of the corresponding CDR of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3; or, (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or, (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or, (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 40 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof; wherein the variant described in any one of (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 more 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (2a), (2b), and (2c), and the variant binds to HER3; or, (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or, (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or, (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:41 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:42 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:44 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:52 or a variant thereof; wherein the variant described in any one of (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., 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

48. The antibody drug conjugate of claim 45 or 46, wherein the antibody that binds to HER3 is selected from: (a) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof; (b) a VH comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof; in, 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 amino acid 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 with the amino acid sequence of the corresponding CDRs of VH and VL of (3a), (3b) and (3c), and the variant binds to HER3.

49. The antibody drug conjugate of claim 45 or 46, wherein the antibody that binds to HER3 is selected from: (1) an antibody or an antigen-binding fragment thereof, comprising (i) a heavy chain (HC) comprising a VH having an amino acid sequence as set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) having an amino acid sequence as set forth in SEQ ID NO: 50, and (ii) a light chain (LC) comprising a VL having an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain constant region (CL) having an amino acid sequence as set forth in SEQ ID NO: 51; (2) an antibody or an antigen-binding fragment thereof, comprising (i) a VH comprising the amino acid sequence of SEQ ID NO: 32 and a HC comprising a heavy chain constant region (CH) comprising the amino acid sequence of SEQ ID NO: 50, and (ii) a VL comprising the amino acid sequence of SEQ ID NO: 33 and a light chain constant region (CL) comprising the amino acid sequence of SEQ ID NO: 51; or (3) An antibody or an antigen-binding fragment thereof, comprising (i) a VH comprising the amino acid sequence shown in SEQ ID NO: 46 and a HC comprising a heavy chain constant region (CH) comprising the amino acid sequence shown in SEQ ID NO: 50, and (ii) a VL comprising the amino acid sequence shown in SEQ ID NO: 47 and a light chain constant region (CL) comprising the amino acid sequence shown in SEQ ID NO:

51.

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

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

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

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

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

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

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

57. Use of the antibody drug conjugate of any one of claims 1 to 36 or claims 45 to 49, or the pharmaceutical composition of claim 50, in the preparation of a drug for treating cancers with high HER3 expression.

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

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

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

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

62. Use of the antibody drug conjugate of any one of claims 1-36 or claims 45-49, or the pharmaceutical composition of claim 50 in treating cancers with high HER3 expression.

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

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

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

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

67. The antibody drug conjugate of any one of claims 1-36 or claims 45-49, or the pharmaceutical composition of claim 50, for use in treating HER3-overexpressing cancer.

68. The antibody drug conjugate of claim 67, wherein the cancer comprises a solid tumor or a hematological malignancy.

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

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

71. The antibody drug conjugate of claim 69, wherein the lung cancer is lung adenocarcinoma.

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