Bispecific antibodies and antibody-drug conjugates targeting B7-H3 and PSMA

Abstract

The present disclosure provides bispecific antibodies and antibody-drug conjugates that target B7-H3 and PSMA, and their use for the treatment of tumors.

Description

TECHNICAL FIELD

[0001] The present disclosure provides bispecific antibodies and antibody-drug conjugates targeting B7-H3 and PSMA, and their use for treating tumors. BACKGROUND

[0002] Prostate cancer is a common malignant tumor in men, which often occurs in men over 60 years old. Globally, the prevalence of prostate cancer ranks second among male malignant tumors, with about 1.4 million new cases each year. Most patients with advanced prostate cancer will progress to metastatic castration-resistant prostate cancer (mCRPC), and these patients have limited treatment options, especially after new endocrine therapy and chemotherapy.

[0003] B7-H3 (CD276) is an immune checkpoint molecule, which is one of the members of the B7 family. It is expressed on a variety of immune cells, including dendritic cells, macrophages, T cells and B cells, etc. (Nature immunology, 2001, 2(3): 269-274.). In normal cells, the expression level of B7-H3 is usually low, but it is highly expressed in a variety of types of cancer cells, including prostate cancer, breast cancer, gastric cancer, lung cancer, liver cancer, cervical cancer and many other tumors. In addition to high expression on cancer cells, B7-H3 is also highly expressed on stromal fibroblasts and tumor-associated vessels (TAV) in the tumor microenvironment (Cancer cell, 2017, 31(4): 501-515.e8; Clinical cancer research, 2012, 18(14): 3834-3845).

[0004] PSMA (Prostate-specific membrane antigen) is a type II transmembrane protein specifically expressed in normal prostate secretory epithelium, but upregulated in prostate cancer. PSMA is a non-secreting membrane enzyme with carboxypeptidase and folic acid hydrolase activities. It acts on glutamate receptors and activates the Pi3K and Akt growth pathways, playing a carcinogenic signaling role in prostate cancer cells (Proceedings of the National Academy of Sciences, 2005, 102(17):5981-5986). The expression level of PSMA in prostate cancer patients is closely related to the severity of the disease, making it an ideal diagnostic and therapeutic target for prostate cancer. Over the past decade, numerous therapies targeting PSMA have been developed, and clinical studies have demonstrated the tolerability and efficacy of PSMA-targeted radioligand therapy (PRLT). Pluvicto, a PSMA-targeting radiopharmaceutical, has significantly improved survival in PSMA-positive mCRPC patients (New England Journal of Medicine, 2021, 385(12):1091-1103). However, radiopharmaceuticals are expensive and poorly accessible. New treatment strategies are urgently needed for PSMA-negative patients and some patients who cannot receive Pluvicto.

[0005] There is a huge unmet clinical need for the treatment of prostate cancer. Currently, there are no antibody drugs targeting B7-H3 or PSMA on the market. Therefore, it is necessary to develop more drugs targeting B7-H3 and / or PSMA to meet clinical needs. Summary of the Invention

[0006] This disclosure provides antibodies that specifically bind to B7-H3, bispecific antibodies against B7-H3 and PSMA, and B7-H3×PSMA bispecific antibody-drug conjugate (ADC) molecules. The B7-H3×PSMA bispecific antibody-drug conjugate (BsADC) consists of two parts: a bispecific antibody capable of simultaneously binding to both B7-H3 and PSMA proteins, and a cytotoxic drug linked to the bispecific antibody. The mechanism of action of the B7-H3×PSMA BsADC is primarily through the binding of the bispecific antibody to cancer cells overexpressing B7-H3 and / or PSMA, followed by the release of the cytotoxic drug to kill the cancer cells. This mechanism allows the B7-H3×PSMA BsADC to precisely deliver the cytotoxic drug to cancer cells, thereby improving therapeutic efficacy and specificity while reducing toxicity to normal cells. B7-H3 and PSMA are both highly expressed targets in prostate cancer. Bispecific antibody-drug conjugates (ADCs) targeting both B7-H3 and PSMA can cover the vast majority of mCRPC patients, improve tumor targeting, and increase efficacy, potentially providing a new treatment strategy for mCRPC patients. This leads to the following aspects.

[0007] Antibody-drug conjugates and compositions

[0008] In one aspect, this disclosure provides antibody-drug conjugates comprising: a bispecific antibody that specifically binds to B7-H3 and PSMA, or an antigen-binding fragment thereof, and a bioactive molecule linked thereto.

[0009] In some embodiments, the bioactive molecule is a therapeutic agent. In some embodiments, the bioactive molecule is a cytotoxic drug.

[0010] In some embodiments, the cytotoxic drug is a camptothecin derivative. The applicant's prior patent applications PCT / CN2023 / 142848, PCT / CN2024 / 085983, and PCT / CN2025 / 075956 each disclose a class of camptothecin derivatives, and all contents relating to those three patent applications are incorporated herein by reference.

[0011] In some embodiments, the bispecific antibody that specifically binds to B7-H3 and PSMA, or its antigen-binding fragment, is optionally conjugated to the bioactive molecule via a linker.

[0012] In some embodiments, the antibody-drug conjugate has the structure shown in Formula I.

[0013] Ab-[LD] β (Formula I)

[0014] Wherein, Ab is a bispecific antibody that specifically binds to B7-H3 and PSMA or its antigen-binding fragment, D is a structural fragment of a bioactive molecule, L is a linker connecting Ab and D, and β is selected from integers or decimals between 1 and 10.

[0015] In some embodiments, D is selected from the compound represented by Formula II or Formula III, its racemate, stereoisomer, isotopic label, or structural fragment after dehydrogenation of a pharmaceutically acceptable salt, wherein:

[0016] (1) The structure of the compound represented by formula II is shown below:

[0017]

[0018] Among them, R1 and R2 may be the same or different, and are independently selected from H, OH, CN, halogens, and C. 1-10 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Alkoxy, halogenated C 1-10 Alkyl, Halogenated C 1-10 Alkoxy, cyano C 1-10 Alkyl, cyano C 1-10 Alkoxy, C 3-10 cycloalkyl;

[0019] Y1 is selected from O or CH2;

[0020] R 51 R 52 They are either the same or different, and are independently selected from H and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkyl-NH-, (C 1-6 Alkyl)2N-, C 1-6 Alkyl-NH-C 1-6 Alkyl, (C 1-6 Alkyl)2N-C 1-6 Alkyl, C 1-6 Alkoxyalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl and 5-14 heteroaryl groups;

[0021] n is selected from 0, 1, or 2;

[0022] (2) The structure of the compound represented by Formula III is shown below:

[0023]

[0024] In some embodiments, R1 is selected from H, OH, CN, halogen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl or halogenated C 1-6 Alkyl group. In some embodiments, R1 is selected from H, OH, Br, methyl, difluoromethoxy, 2,2,2-trifluoroethoxy, vinyl, cyclopropyl, or ethynyl. In some embodiments, R1 is selected from methyl or cyclopropyl.

[0025] In some implementations, R2 is selected from H, halogen, CN, or C. 1-6 Alkyl group. In some embodiments, R2 is selected from H or F.

[0026] In some implementations, R 51 Selected from H, methyl, ethyl, isopropyl or cyclopropyl.

[0027] In some implementations, R 52 Selected from H or methyl.

[0028] In some embodiments, the compound structure represented by Formula II is as follows:

[0029]

[0030] Among them, R1, R2, R 51 R 52 Y1 and n have the definitions described in any of the above embodiments. In some embodiments, the structure of the compound of formula II is as follows:

[0031]

[0032]

[0033] In some implementations, the structure of D is as follows:

[0034]

[0035]

[0036]

[0037] In some implementations, L is selected from the connector shown in Formula IV or Formula V:

[0038]

[0039] Where Z is N or CR 22 R 21 R 22 and R23 Each is independently selected from H, halogen, cyano, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, 3-8 membered heterocyclic groups, C 1-6 Alkyl-OC 1-6 Alkylene; condition is that when Z is N, R 21 R 23 They are not both H.

[0040] In some embodiments, n1 is selected from an integer from 1 to 36; more preferably, n1 is selected from an integer from 3 to 12, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; most preferably, n1 is selected from an integer from 4 to 10.

[0041] In some implementations, Z is N or C-CN.

[0042] In some implementations, R 21 Selected from H, halogen, cyano, C 1-6 Alkoxy or C 1-6 Alkyl-OC 1-6 Alkylene.

[0043] In some implementations, Z is N, R 21 Selected from halogen, cyano, C 1-3 Alkoxy or C 1-3 Alkyl-OC 1-3 Alkylene.

[0044] In some implementations, Z is N, R 21 Selected from methoxy or CH3-O-CH2-.

[0045] In some implementations, Z is C-CN, R 21 Selected from H.

[0046] In some implementations, R 23 Selected from H.

[0047] In some implementations, bit 1 is connected to Ab, and bit 2 is connected to D.

[0048] In some implementations, L is selected from:

[0049]

[0050]

[0051] In this configuration, bit 1 is connected to Ab, and bit 2 is connected to D. In some implementations, LD is selected from:

[0052]

[0053]

[0054]

[0055] In some implementations, β is 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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.

[0056] In some embodiments, β is 4-9, for example, 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, 4.0-8.5, 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, 4.5-8.5, 4.5-9.0, 5.0-5.5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5.0-8.0, 5.0-8.5, 5.0-9. 0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 5.5~8.5, 5.5~9.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.0, 6.0~8.5, 6.0~9.0, 6.5~7.0, 6.5~7.5, 6.5~8.0, 6.5~8.5, 6.5~9.0, 7.0~7.5, 7.0~8.0, 7.0~8.5, 7.0~9.0, 7.5~8.0, 7.5~8.5, 7.5~9.0, 8.0~8.5, 8.0~9.0 or 8.5~9.0.

[0057] In some embodiments, β is 5-9. In some embodiments, β is 5.5-8.5. In some embodiments, β is 7.0-8.5. In some embodiments, β is 5.5-6.0.

[0058] In some embodiments, the antibody-drug conjugate of this disclosure comprises a bispecific antibody or antigen-binding fragment thereof that specifically binds to B7-H3 and PSMA, having the following characteristics: it comprises a first antigen-binding domain that specifically binds to one of B7-H3 and PSMA and a second antigen-binding domain that specifically binds to the other of B7-H3 and PSMA.

[0059] I. B7-H3 binding domain

[0060] In some embodiments, the bispecific antibody or antigen-binding fragment that specifically binds to B7-H3 and PSMA includes an antigen-binding domain that specifically binds to B7-H3 as one of the first antigen-binding domain and the second antigen-binding domain.

[0061] In some embodiments, the antigen-binding domain that specifically binds to B7-H3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL respectively comprise three heavy chain CDR sequences (i.e., heavy chain CDR1-CDR3) or heavy chain variable region sequences and three light chain CDR sequences (i.e., light chain CDR1-CDR3) or light chain variable region sequences derived from clones 1.2.3, 10.15.3, 3.17.1, 10.7.1 or their humanized antibodies or P13738.

[0062] In some embodiments, the antigen-binding domain that specifically binds to B7-H3 includes:

[0063] (a) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:45 or 11; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:49 or 12;

[0064] (b) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46 or 13; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:50 or 14;

[0065] (c) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:47 or 15; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:51 or 16;

[0066] (d) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof, and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:48 or 17; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof, and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:52 or 18; or,

[0067] (e) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62 or 53; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:63 or 54;

[0068] The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions). In some embodiments, the substitutions are conservative substitutions.

[0069] In some embodiments, the antigen-binding domain that specifically binds to B7-H3 includes:

[0070] (a) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:45 or 11; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:49 or 12;

[0071] (b) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46 or 13; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:50 or 14;

[0072] (c) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:47 or 15; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:51 or 16.

[0073] (d) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:48 or 17; and / or, three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:52 or 18; or,

[0074] (e) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62 or 53; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:63 or 54.

[0075] In some implementations, the three heavy chain CDRs contained in the VH and / or the three light chain CDRs contained in the VL may be defined by the Kabat, AbM, IMGT or Chothia numbering system, or by a combination of the above numbering systems.

[0076] In some embodiments, the antigen-binding domain that specifically binds to B7-H3 includes:

[0077] (1) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:19, the heavy chain CDR2 shown in SEQ ID NO:108, and the heavy chain CDR3 shown in SEQ ID NO:21, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:22, the light chain CDR2 shown in SEQ ID NO:23, and the light chain CDR3 shown in SEQ ID NO:24;

[0078] (2) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:25, the heavy chain CDR2 shown in SEQ ID NO:109, and the heavy chain CDR3 shown in SEQ ID NO:27, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:28, the light chain CDR2 shown in SEQ ID NO:29, and the light chain CDR3 shown in SEQ ID NO:30;

[0079] (3) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:31, the heavy chain CDR2 shown in SEQ ID NO:110, and the heavy chain CDR3 shown in SEQ ID NO:33, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:34, the light chain CDR2 shown in SEQ ID NO:35, and the light chain CDR3 shown in SEQ ID NO:36;

[0080] (4) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:37, the heavy chain CDR2 shown in SEQ ID NO:111, and the heavy chain CDR3 shown in SEQ ID NO:39, and / or, a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:40, the light chain CDR2 shown in SEQ ID NO:41, and the light chain CDR3 shown in SEQ ID NO:42; or

[0081] (5) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:56, the heavy chain CDR2 shown in SEQ ID NO:112, and the heavy chain CDR3 shown in SEQ ID NO:58, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:59, the light chain CDR2 shown in SEQ ID NO:60, and the light chain CDR3 shown in SEQ ID NO:61;

[0082] The CDR is defined by the AbM numbering system.

[0083] In certain specific embodiments, the antigen-binding domain that specifically binds to B7-H3 includes:

[0084] (1) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:19, the heavy chain CDR2 shown in SEQ ID NO:20, and the heavy chain CDR3 shown in SEQ ID NO:21, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:22, the light chain CDR2 shown in SEQ ID NO:23, and the light chain CDR3 shown in SEQ ID NO:24;

[0085] (2) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:25, the heavy chain CDR2 shown in SEQ ID NO:26 or SEQ ID NO:105, and the heavy chain CDR3 shown in SEQ ID NO:27, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:28, the light chain CDR2 shown in SEQ ID NO:29, and the light chain CDR3 shown in SEQ ID NO:30;

[0086] (3) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:31, the heavy chain CDR2 shown in SEQ ID NO:32 or SEQ ID NO:106, and the heavy chain CDR3 shown in SEQ ID NO:33, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:34, the light chain CDR2 shown in SEQ ID NO:35, and the light chain CDR3 shown in SEQ ID NO:36;

[0087] (4) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:37, the heavy chain CDR2 shown in SEQ ID NO:38 or SEQ ID NO:107, and the heavy chain CDR3 shown in SEQ ID NO:39, and / or, a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:40, the light chain CDR2 shown in SEQ ID NO:41, and the light chain CDR3 shown in SEQ ID NO:42; or

[0088] (5) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:56, the heavy chain CDR2 shown in SEQ ID NO:57, and the heavy chain CDR3 shown in SEQ ID NO:58, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:59, the light chain CDR2 shown in SEQ ID NO:60, and the light chain CDR3 shown in SEQ ID NO:61;

[0089] The CDR is defined by a hybrid Kabat / AbM framework.

[0090] In some embodiments, the antigen-binding domain that specifically binds to B7-H3 includes:

[0091] (1a) VH containing the sequence shown in SEQ ID NO:45 or a variant thereof and VL containing the sequence shown in SEQ ID NO:49 or a variant thereof;

[0092] (1b) VH containing the sequence shown in SEQ ID NO:11 or a variant thereof and VL containing the sequence shown in SEQ ID NO:12 or a variant thereof;

[0093] (2a) VH containing the sequence shown in SEQ ID NO:46 or a variant thereof and VL containing the sequence shown in SEQ ID NO:50 or a variant thereof;

[0094] (2b) VH containing the sequence shown in SEQ ID NO:13 or a variant thereof and VL containing the sequence shown in SEQ ID NO:14 or a variant thereof;

[0095] (3a) VH containing the sequence shown in SEQ ID NO:47 or a variant thereof and VL containing the sequence shown in SEQ ID NO:51 or a variant thereof;

[0096] (3b) VH containing the sequence shown in SEQ ID NO:15 or a variant thereof and VL containing the sequence shown in SEQ ID NO:16 or a variant thereof;

[0097] (4a) VH containing the sequence shown in SEQ ID NO:48 or a variant thereof and VL containing the sequence shown in SEQ ID NO:52 or a variant thereof;

[0098] (4b) VH containing the sequence shown in SEQ ID NO:17 or a variant thereof and VL containing the sequence shown in SEQ ID NO:18 or a variant thereof;

[0099] (5a) A VH comprising the sequence shown in SEQ ID NO:62 or a variant thereof and a VL comprising the sequence shown in SEQ ID NO:63 or a variant thereof; or

[0100] (5b) VH containing the sequence shown in SEQ ID NO:53 or a variant thereof and VL containing the sequence shown in SEQ ID NO:54 or a variant thereof;

[0101] The variant has 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 with the sequence from which it originates, or 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 original sequence; preferably, the substitution is a conservative substitution.

[0102] In some embodiments, the antigen-binding domain that specifically binds to B7-H3 includes:

[0103] (1a) VH containing the sequence shown in SEQ ID NO:45 and VL containing the sequence shown in SEQ ID NO:49;

[0104] (1b) VH containing the sequence shown in SEQ ID NO:11 and VL containing the sequence shown in SEQ ID NO:12;

[0105] (2a) VH containing the sequence shown in SEQ ID NO:46 and VL containing the sequence shown in SEQ ID NO:50;

[0106] (2b) VH containing the sequence shown in SEQ ID NO:13 and VL containing the sequence shown in SEQ ID NO:14;

[0107] (3a) VH containing the sequence shown in SEQ ID NO:47 and VL containing the sequence shown in SEQ ID NO:51;

[0108] (3b) VH containing the sequence shown in SEQ ID NO:15 and VL containing the sequence shown in SEQ ID NO:16;

[0109] (4a) VH containing the sequence shown in SEQ ID NO:48 and VL containing the sequence shown in SEQ ID NO:52;

[0110] (4b) VH containing the sequence shown in SEQ ID NO:17 and VL containing the sequence shown in SEQ ID NO:18;

[0111] (5a) VH comprising the sequence shown in SEQ ID NO:62 and VL comprising the sequence shown in SEQ ID NO:63; or

[0112] (5b) VH containing the sequence shown in SEQ ID NO:53 and VL containing the sequence shown in SEQ ID NO:54.

[0113] II. PSMA binding domain

[0114] In some embodiments, the bispecific antibody that specifically binds to B7-H3 and PSMA, or its antigen-binding fragment, includes an antigen-binding domain that specifically binds to PSMA as the other of the first antigen-binding domain and the second antigen-binding domain.

[0115] In some embodiments, the antigen-binding domain that specifically binds to PSMA includes: heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof, and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:64; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof, and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:65;

[0116] The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions). In some embodiments, the substitutions are conservative substitutions.

[0117] In some embodiments, the antigen-binding domain that specifically binds to PSMA includes: three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:64; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:65.

[0118] In some implementations, the three heavy chain CDRs contained in the VH and / or the three light chain CDRs contained in the VL may be defined by the Kabat, AbM, IMGT or Chothia numbering system, or by a combination of the above numbering systems.

[0119] In some embodiments, the antigen-binding domain that specifically binds to PSMA includes: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:98, heavy chain CDR2 shown in SEQ ID NO:113, and heavy chain CDR3 shown in SEQ ID NO:100, and / or a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:101, light chain CDR2 shown in SEQ ID NO:102, and light chain CDR3 shown in SEQ ID NO:103; wherein the CDRs are defined by the AbM numbering system.

[0120] In certain specific embodiments, the antigen-binding domain that specifically binds to PSMA includes: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:98, heavy chain CDR2 shown in SEQ ID NO:99, and heavy chain CDR3 shown in SEQ ID NO:100, and / or a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:101, light chain CDR2 shown in SEQ ID NO:102, and light chain CDR3 shown in SEQ ID NO:103; wherein the CDR is defined by a mixture of Kabat and AbM.

[0121] In some embodiments, the antigen-binding domain that specifically binds to PSMA comprises: a VH containing the sequence shown in SEQ ID NO:64 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:65 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions); preferably, the substitution is a conservative substitution.

[0122] In some embodiments, the antigen-binding domain that specifically binds to PSMA includes a VH containing the sequence shown in SEQ ID NO:64 and a VL containing the sequence shown in SEQ ID NO:65.

[0123] III. Structure

[0124] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain of the bispecific antibody that specifically binds B7-H3 and PSMA or its antigen-binding fragment includes a Fab structure or an sdAb / VHH structure, and the other includes a Fab structure, an scFv structure, or an sdAb / VHH structure.

[0125] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain is Fab, and the other is scFv or Fab.

[0126] In some embodiments, the bispecific antibody or its antigen-binding fragment further comprises an Fc domain, the Fc domain comprising a first Fc domain monomer and a second Fc domain monomer, wherein the first antigen-binding domain and the second antigen-binding domain are optionally linked to the N-terminus of the first Fc domain monomer and the second Fc domain monomer via a first peptide linker, respectively.

[0127] Fab-Fab

[0128] In some embodiments, the first antigen-binding domain is a Fab, and the second antigen-binding domain is a Fab containing domain exchanges of the form of CrossMab.

[0129] In some implementations, the CrossMab-style domain swaps are selected from:

[0130] (a) CrossMab Fab: In the Fab, VL and VH are interchanged, and CH1 and CL are interchanged;

[0131] (b) CrossMab VH-VL: In the Fab, VL and VH are interchanged; or

[0132] (c)CrossMab CH1-CL: In the Fab, CH1 and CL are interchanged.

[0133] In some embodiments, the bispecific antibody or its antigen-binding fragment comprises:

[0134] (i) Peptide chain IA, which includes the VL of the first antigen-binding domain and the light chain constant region (CL);

[0135] (ii) A peptide chain IB, comprising the VH of the first antigen-binding domain, the CH1 region of the heavy chain, and a monomer of the first Fc domain.

[0136] (iii) A peptide chain IC comprising the VH of the second antigen-binding domain, a light chain constant region (CL), and a second Fc domain monomer.

[0137] (iv) Peptide chain ID, which includes the VL region of the second antigen-binding domain and the CH1 region of the heavy chain.

[0138] In some implementations, the first Fc domain monomer and the second Fc domain monomer form a dimer.

[0139] In some implementations, the CL is the constant region of the kappa light chain.

[0140] In some embodiments, the first Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1. In some embodiments, the first Fc domain monomer includes a hinge region, CH2, and CH3.

[0141] In some embodiments, the second Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1. In some embodiments, the second Fc domain monomer includes a hinge region, CH2, and CH3.

[0142] In some embodiments, the first antigen-binding domain is selected from the antigen-binding domain that specifically binds to B7-H3 as described above, and the second antigen-binding domain is selected from the antigen-binding domain that specifically binds to PSMA as described above.

[0143] In some embodiments, the first antigen-binding domain includes:

[0144] (1) VH containing the sequence shown in SEQ ID NO:45 and VL containing the sequence shown in SEQ ID NO:49;

[0145] (2) VH containing the sequence shown in SEQ ID NO:46 and VL containing the sequence shown in SEQ ID NO:50;

[0146] (3) VH containing the sequence shown in SEQ ID NO:47 and VL containing the sequence shown in SEQ ID NO:51;

[0147] (4) VH containing the sequence shown in SEQ ID NO:48 and VL containing the sequence shown in SEQ ID NO:52;

[0148] (5) VH containing the sequence shown in SEQ ID NO:62 and VL containing the sequence shown in SEQ ID NO:63; or

[0149] (6) VH containing the sequence shown in SEQ ID NO:53 and VL containing the sequence shown in SEQ ID NO:54.

[0150] In some embodiments, the second antigen-binding domain includes a VH comprising the sequence shown in SEQ ID NO:64 and a VL comprising the sequence shown in SEQ ID NO:65.

[0151] In some embodiments, in any of the above Fab-Fab structures, the first and second Fc domain monomers are wild-type IgG Fc domains or each independently contains one or more amino acid modifications that can promote the dimerization of the first and second Fc domain monomers.

[0152] In some embodiments, the first and second Fc domain monomers may or may not contain disulfide bonds.

[0153] In some embodiments, the dimerization-promoting modification comprises a "knob" modification in one of the two Fc domain monomers and a "hole" modification in the other of the two Fc domain monomers, forming a "knobs-into-holes" modification. In some embodiments, the "knob" modification is selected from the following mutations with EU numbers: T366W, T366W / S354C, or T366W / S354C / K409A, for example, T366W or T366W / S354C. In some implementations, the “hole” modification is selected from the following mutations with EU numbers: T366S / L368A / Y407V, T366S / L368A / Y407V / Y349C, or T366S / L368A / Y407V / Y349C / F405K, such as T366S / L368A / Y407V or T366S / L368A / Y407V / Y349C.

[0154] In some embodiments, the knocks-into-holes modification includes: T366W in one of the two Fc domain monomers and T366S / L368A / Y407V in the other of the two Fc domain monomers. In some embodiments, the knocks-into-holes modification includes: T366W / S354C in one of the two Fc domain monomers and T366S / L368A / Y407V / Y349C in the other of the two Fc domain monomers.

[0155] In some embodiments, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:97 and 97, SEQ ID NO:94 and 78, SEQ ID NO:78 and 94.

[0156] In some embodiments, in any of the above Fab-Fab structures, a flexible amino acid sequence is further included between the VL region of the second antigen-binding domain and the CH1 region of the heavy chain of the peptide chain ID. In some embodiments, the flexible amino acid sequence consists of one or more glycine (G) and / or serine (S). In some embodiments, the flexible amino acid sequence is SS. In some embodiments, the peptide chain ID includes the VL region of the second antigen-binding domain and the SS-heavy chain CH1 region sequence shown in SEQ ID NO: 80.

[0157] In some embodiments, in any of the above Fab-Fab structures, the CL of the peptide chain IC contains the following mutation with EU number: R108A / T109S. In some embodiments, the peptide chain IC contains the CL sequence shown in SEQ ID NO:77.

[0158] In some embodiments, the bispecific antibody or its antigen-binding fragment with any of the above Fab-Fab structures comprises:

[0159] (1) Peptide chain IA containing the sequence shown in SEQ ID NO:55, peptide chain IB containing the sequence shown in SEQ ID NO:104, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:81.

[0160] (2) Peptide chain IA containing the sequence shown in SEQ ID NO:66, peptide chain IB containing the sequence shown in SEQ ID NO:72, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:81.

[0161] (3) Peptide chain IA containing the sequence shown in SEQ ID NO:67, peptide chain IB containing the sequence shown in SEQ ID NO:73, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:81.

[0162] (4) Peptide chain IA containing the sequence shown in SEQ ID NO:68, peptide chain IB containing the sequence shown in SEQ ID NO:74, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:81.

[0163] (5) Peptide chain IA containing the sequence shown in SEQ ID NO:69, peptide chain IB containing the sequence shown in SEQ ID NO:75, peptide chain IC containing the sequence shown in SEQ ID NO:79, peptide chain ID containing the sequence shown in SEQ ID NO:81; or,

[0164] (6) Peptide chain IA containing the sequence shown in SEQ ID NO:70, peptide chain IB containing the sequence shown in SEQ ID NO:76, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:81.

[0165] Fab-scFv

[0166] In some embodiments, the first antigen-binding domain is Fab, the second antigen-binding domain is scFv, and the bispecific antibody or its antigen-binding fragment comprises:

[0167] (i) Peptide chain II-A, which includes the VL of the first antigen-binding domain and the light chain constant region (CL);

[0168] (ii) Peptide chain II-B, which includes the VH of the first antigen-binding domain, the CH1 region of the heavy chain, and a monomer of the first Fc domain.

[0169] and

[0170] (iii) Peptide chain II-C, which includes the second antigen-binding domain and the second Fc domain monomer.

[0171] In some implementations, the second Fc domain monomer forms a dimer with the first Fc domain monomer.

[0172] In some embodiments, the second antigen-binding domain is linked to the N-terminus of the second Fc domain monomer via a first peptide linker.

[0173] In some embodiments, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a second peptide linker.

[0174] In some embodiments, the first peptide linker and the second peptide linker are each independently the same or different peptide linkers.

[0175] In some embodiments, the first peptide linker and the second peptide linker are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), such as flexible peptides containing (G4S)n, where n is an integer not less than 0, such as 1, 2, 3 or 4.

[0176] In some embodiments, the first peptide linker comprises the flexible peptide sequence shown in SEQ ID NO:83.

[0177] In some embodiments, the second peptide linker comprises the flexible peptide sequence shown in SEQ ID NO:82.

[0178] In some implementations, the scFv may or may not contain disulfide bonds between VH and VL.

[0179] In some implementations, the CL is the constant region of the kappa light chain.

[0180] In some embodiments, the first Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1. In some embodiments, the first Fc domain monomer includes a hinge region, CH2, and CH3.

[0181] In some embodiments, the first Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1. In some embodiments, the first Fc domain monomer includes a hinge region, CH2, and CH3.

[0182] In some embodiments, the first antigen-binding domain is selected from the antigen-binding domains that specifically bind PSMA as described herein, and the second antigen-binding domain is selected from the antigen-binding domains that specifically bind B7-H3 as described herein.

[0183] In some embodiments, the first antigen-binding domain includes a VH comprising the sequence shown in SEQ ID NO:64 and a VL comprising the sequence shown in SEQ ID NO:65.

[0184] In some embodiments, the second antigen-binding domain includes a VH containing the sequence shown in SEQ ID NO:47 and a VL containing the sequence shown in SEQ ID NO:51.

[0185] In some embodiments, in any of the above Fab-scFv structures, the first and second Fc domain monomers are wild-type IgG Fc domains or each independently contains one or more amino acid modifications that can promote the dimerization of the first and second Fc domain monomers.

[0186] In some embodiments, the first and second Fc domain monomers may or may not contain disulfide bonds.

[0187] In some embodiments, the dimerization-promoting modification comprises a "knob" modification in one of the two Fc domain monomers and a "hole" modification in the other of the two Fc domain monomers, forming a "knobs-into-holes" modification. In some embodiments, the "knob" modification is selected from the following mutations with EU numbers: T366W, T366W / S354C, or T366W / S354C / K409A, for example, T366W or T366W / S354C. In some implementations, the “hole” modification is selected from the following mutations with EU numbers: T366S / L368A / Y407V, T366S / L368A / Y407V / Y349C, or T366S / L368A / Y407V / Y349C / F405K, such as T366S / L368A / Y407V or T366S / L368A / Y407V / Y349C.

[0188] In some embodiments, the knots-into-holes modification includes: T366W in one of the two Fc domain monomers and T366S / L368A / Y407V in the other of the two Fc domain monomers; or, T366W / S354C in one of the two Fc domain monomers and T366S / L368A / Y407V / Y349C in the other of the two Fc domain monomers.

[0189] In some implementations, in any of the Fab-scFv structures described above, the second Fc domain monomer contains the following mutation with an EU number: C220S.

[0190] In some embodiments, in any of the Fab-scFv structures described above, the first and second Fc domain monomers each independently contain modifications capable of reducing or eliminating effector functionality (e.g., ADCC activity). In some embodiments, the modifications are selected from the following mutations with EU numbers: L234A and / or L235A.

[0191] In some embodiments, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:97 and 97, SEQ ID NO:95 and 88, SEQ ID NO:88 and 95, SEQ ID NO:96 and 92, and SEQ ID NO:92 and 96.

[0192] In some embodiments, the bispecific antibody or its antigen-binding fragment with the above-described Fab-scFv structure comprises:

[0193] (1) Peptide chain II-A containing the sequence shown in SEQ ID NO:84, peptide chain II-B containing the sequence shown in SEQ ID NO:86, and peptide chain II-C containing the sequence shown in SEQ ID NO:89; or

[0194] (2) Peptide chain II-A containing the sequence shown in SEQ ID NO:84, peptide chain II-B containing the sequence shown in SEQ ID NO:91, and peptide chain II-C containing the sequence shown in SEQ ID NO:93.

[0195] On the other hand, this disclosure provides an antibody-drug conjugate selected from:

[0196]

[0197]

[0198]

[0199]

[0200]

[0201] in:

[0202] C040 is a bispecific antibody comprising the following peptide chains: peptide chain IA comprising the sequence shown in SEQ ID NO:55, peptide chain IB comprising the sequence shown in SEQ ID NO:104, peptide chain IC comprising the sequence shown in SEQ ID NO:79, and peptide chain ID comprising the sequence shown in SEQ ID NO:81.

[0203] C097 is a bispecific antibody containing the following peptide chains: peptide chain IA containing the sequence shown in SEQ ID NO:67, peptide chain IB containing the sequence shown in SEQ ID NO:73, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:81.

[0204] C101 is a bispecific antibody containing the following peptide chains: peptide chain IA containing the sequence shown in SEQ ID NO:68, peptide chain IB containing the sequence shown in SEQ ID NO:74, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:81.

[0205] C115 is a bispecific antibody comprising the following peptide chains: peptide chain IA comprising the sequence shown in SEQ ID NO:69, peptide chain IB comprising the sequence shown in SEQ ID NO:75, peptide chain IC comprising the sequence shown in SEQ ID NO:79, and peptide chain ID comprising the sequence shown in SEQ ID NO:81.

[0206] C116 is a bispecific antibody comprising the following peptide chains: peptide chain IA comprising the sequence shown in SEQ ID NO:70, peptide chain IB comprising the sequence shown in SEQ ID NO:76, peptide chain IC comprising the sequence shown in SEQ ID NO:79, and peptide chain ID comprising the sequence shown in SEQ ID NO:81.

[0207] C123 is a bispecific antibody containing the following peptide chains: peptide chain II-A containing the sequence shown in SEQ ID NO:84, peptide chain II-B containing the sequence shown in SEQ ID NO:86, and peptide chain II-C containing the sequence shown in SEQ ID NO:89.

[0208] C123(LALA) is a bispecific antibody containing the following peptide chains: peptide chain II-A containing the sequence shown in SEQ ID NO:84, peptide chain II-B containing the sequence shown in SEQ ID NO:91, and peptide chain II-C containing the sequence shown in SEQ ID NO:93.

[0209] In another aspect, this disclosure provides a composition comprising one or more antibody-drug conjugates as described in any of the foregoing aspects.

[0210] In some embodiments, the composition comprises a group of multiple antibody-drug conjugates. In some embodiments, the multiple antibody-drug conjugates have the same Ab, L, and D moieties.

[0211] In some embodiments, the DAR value (drug-antibody conjugate ratio) of the composition is 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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.

[0212] In some embodiments, the DAR value of the composition is 4-9, for example, 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, 4.0-8.5, 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, 4.5-8.5, 4.5-9.0, 5.0-5.5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5.0-8.0, 5.0-8.5, 5. 0~9.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 5.5~8.5, 5.5~9.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.0, 6.0~8.5, 6.0~9.0, 6.5~7.0, 6.5~7.5, 6.5~8.0, 6.5~8.5, 6.5~9.0, 7.0~7.5, 7.0~8.0, 7.0~8.5, 7.0~9.0, 7.5~8.0, 7.5~8.5, 7.5~9.0, 8.0~8.5, 8.0~9.0 or 8.5~9.0.

[0213] In some embodiments, the DAR value of the composition is 5-9. In some embodiments, the DAR value of the composition is 5.5-8.5. In some embodiments, the DAR value of the composition is 7.0-8.5. In some embodiments, the DAR value of the composition is 5.5-6.0.

[0214] B7-H3 x PSMA bispecific antibody

[0215] On the other hand, this disclosure also provides a bispecific antibody or antigen-binding fragment thereof that specifically binds to B7-H3 and PSMA, comprising a first antigen-binding domain that specifically binds to one of B7-H3 and PSMA and a second antigen-binding domain that specifically binds to the other of B7-H3 and PSMA.

[0216] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain includes Fab or sdAb / VHH, and the other includes Fab or scFv or sdAb / VHH.

[0217] In some embodiments, the bispecific antibody or its antigen-binding fragment is selected from the bispecific antibodies or their antigen-binding fragments that specifically bind B7-H3 and PSMA contained in the antibody-drug conjugates of this disclosure as described in any of the foregoing aspects.

[0218] The bispecific antibodies or antigen-binding fragments of this disclosure can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization of the antibody does not adversely affect its binding to B7-H3 and PSMA. Therefore, the bispecific antibodies or antigen-binding fragments of this disclosure are also intended to include such derivatized forms. For example, the bispecific antibodies or antigen-binding fragments of this disclosure can be functionally linked (through chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody, a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., avidin or a multihistidine tag) capable of mediating the binding of the bispecific antibody or its antigen-binding fragment to another molecule.

[0219] The bispecific antibodies or antigen-binding fragments thereof disclosed herein can be prepared by various methods known in the art, such as recombinant genetic engineering techniques. For example, the bispecific antibodies or antigen-binding fragments thereof disclosed herein can be generated by co-expressing multiple polynucleotides encoding various polypeptide chains of the bispecific antibody or antigen-binding fragment. The polypeptide chains generated by co-expression can be linked via, for example, disulfide bonds or other means to form a functional bispecific antibody or antigen-binding fragment. For example, the light chain portion of the Fab fragment can be encoded by separate polynucleotides with a portion of the bispecific antibody or antigen-binding fragment containing the heavy chain portion of the Fab fragment (which may further contain an Fc domain monomer and optionally other antigen-binding domains). When co-expressed, the polypeptide containing the heavy chain portion of the Fab fragment is linked with the polypeptide containing the light chain portion of the Fab fragment to form the Fab fragment. As another example, a portion of the bispecific antibody or antigen-binding fragment provided herein containing one of two Fc domain monomers (which may further contain an antigen-binding domain) can be encoded by separate polynucleotides with a portion of the other of the two Fc domain monomers (which may further contain an antigen-binding domain). When co-expressed, the two Fc domain monomers will combine to form an Fc domain.

[0220] On the other hand, this disclosure provides isolated nucleic acid molecules containing nucleotide sequences encoding the bispecific antibody of this disclosure or its antigen-binding fragment or at least one peptide chain thereof.

[0221] In some embodiments, the isolated nucleic acid molecule comprises nucleotide sequences encoding peptide chains of the bispecific antibody or antigen-binding fragment thereof disclosed herein, and the nucleotide sequences encoding the peptide chains are present on the same or different isolated nucleic acid molecules.

[0222] On the other hand, this disclosure provides vectors (e.g., expression vectors) that contain nucleic acid molecules encoding the isolated samples described above.

[0223] In some embodiments, the vector comprises nucleotide sequences encoding each peptide chain of the bispecific antibody or antigen-binding fragment thereof disclosed herein, and the nucleotide sequences encoding each peptide chain are present on the same or different vectors. For example, the vectors of this disclosure comprise: a first vector comprising a nucleotide sequence encoding peptide chain IA, a second vector comprising a nucleotide sequence encoding peptide chain IB, a third vector comprising a nucleotide sequence encoding peptide chain IC, and a fourth vector comprising a nucleotide sequence encoding peptide chain ID. For example, the vectors of this disclosure comprise: a first vector comprising a nucleotide sequence encoding peptide chain II-A, a second vector comprising a nucleotide sequence encoding peptide chain II-B, and a third vector comprising a nucleotide sequence encoding peptide chain II-C.

[0224] On the other hand, this disclosure provides host cells that contain nucleic acid molecules or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., Escherichia coli cells), eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0225] On the other hand, this disclosure provides a method for preparing the bispecific antibody or antigen-binding fragment thereof as described herein, comprising culturing host cells as described above under conditions that allow protein expression, and recovering the bispecific antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0226] Antibodies that bind B7-H3

[0227] On the other hand, this disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind to B7-H3.

[0228] In some embodiments, the antibody that specifically binds to B7-H3 or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL respectively comprise three heavy chain CDR sequences (i.e., heavy chain CDR1-CDR3) or heavy chain variable region sequences and three light chain CDR sequences (i.e., light chain CDR1-CDR3) or light chain variable region sequences derived from clones 1.2.3, 10.15.3, 3.17.1 or 10.7.1 or their humanized antibodies or P13738.

[0229] In some embodiments, the antibody or its antigen-binding fragment comprises a CDR sequence derived from clone 1.2.3 or its humanized antibody, comprising: heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof, and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:45 or 11; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof, and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:49 or 12;

[0230] The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions). In some embodiments, the substitutions are conservative substitutions.

[0231] In some embodiments, the antibody or its antigen-binding fragment comprises: three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:45 or 11; and / or, three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:49 or 12.

[0232] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:45; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:49; or (ii) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:11; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:12.

[0233] In some implementations, the three heavy chain CDRs contained in the VH and / or the three light chain CDRs contained in the VL may be defined by the Kabat, AbM, IMGT or Chothia numbering system, or by a combination of the above numbering systems.

[0234] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:19, heavy chain CDR2 shown in SEQ ID NO:108, and heavy chain CDR3 shown in SEQ ID NO:21, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:22, light chain CDR2 shown in SEQ ID NO:23, and light chain CDR3 shown in SEQ ID NO:24; wherein the CDRs are defined by the AbM numbering system.

[0235] In certain specific embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:19, heavy chain CDR2 shown in SEQ ID NO:20, and heavy chain CDR3 shown in SEQ ID NO:21, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:22, light chain CDR2 shown in SEQ ID NO:23, and light chain CDR3 shown in SEQ ID NO:24; wherein the CDR is defined by a mixture of Kabat / AbM.

[0236] In some embodiments, the antibody or its antigen-binding fragment further comprises a framework region sequence derived from mouse immunoglobulin.

[0237] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:11 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:12 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0238] In some embodiments, the antibody or its antigen-binding fragment comprises: VH containing the sequence shown in SEQ ID NO:11 and VL containing the sequence shown in SEQ ID NO:12.

[0239] In some embodiments, the antibody or its antigen-binding fragment is humanized and further comprises a framework region sequence derived from human immunoglobulin.

[0240] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:45 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:49 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0241] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:45 and a VL containing the sequence shown in SEQ ID NO:49.

[0242] In some embodiments, the antibody or its antigen-binding fragment comprises a CDR sequence derived from clone 10.15.3 or its humanized antibody, comprising: heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof, and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46 or 13; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof, and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:50 or 14;

[0243] The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions). In some embodiments, the substitutions are conservative substitutions.

[0244] In some embodiments, the antibody or its antigen-binding fragment comprises: three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46 or 13; and / or, three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:50 or 14.

[0245] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:50; or (ii) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:13; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:14.

[0246] In some implementations, the three heavy chain CDRs contained in the VH and / or the three light chain CDRs contained in the VL may be defined by the Kabat, AbM, IMGT or Chothia numbering system, or by a combination of the above numbering systems.

[0247] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:25, heavy chain CDR2 shown in SEQ ID NO:109, and heavy chain CDR3 shown in SEQ ID NO:27, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:28, light chain CDR2 shown in SEQ ID NO:29, and light chain CDR3 shown in SEQ ID NO:30; wherein the CDRs are defined by the AbM numbering system.

[0248] In certain specific embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:25, heavy chain CDR2 shown in SEQ ID NO:26, and heavy chain CDR3 shown in SEQ ID NO:27, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:28, light chain CDR2 shown in SEQ ID NO:29, and light chain CDR3 shown in SEQ ID NO:30; wherein the CDR is defined by a mixture of Kabat / AbM.

[0249] In some embodiments, the antibody or its antigen-binding fragment further comprises a framework region sequence derived from mouse immunoglobulin.

[0250] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:13 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:14 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0251] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:13 and a VL containing the sequence shown in SEQ ID NO:14.

[0252] In certain specific embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:25, heavy chain CDR2 shown in SEQ ID NO:105, and heavy chain CDR3 shown in SEQ ID NO:27, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:28, light chain CDR2 shown in SEQ ID NO:29, and light chain CDR3 shown in SEQ ID NO:30; wherein the CDR is defined by a mixture of Kabat / AbM.

[0253] In some embodiments, the antibody or its antigen-binding fragment is humanized and further comprises a framework region sequence derived from human immunoglobulin.

[0254] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:46 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:50 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0255] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:46 and a VL containing the sequence shown in SEQ ID NO:50.

[0256] In some embodiments, the antibody or its antigen-binding fragment comprises a CDR sequence derived from clone 3.17.1 or its humanized antibody, comprising: heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof, and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:47 or 15; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof, and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:51 or 16;

[0257] The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions). In some embodiments, the substitutions are conservative substitutions.

[0258] In some embodiments, the antibody or its antigen-binding fragment comprises: three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:47 or 15; and / or, three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:51 or 16.

[0259] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:47; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:51; or (ii) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:15; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:16.

[0260] In some implementations, the three heavy chain CDRs contained in the VH and / or the three light chain CDRs contained in the VL may be defined by the Kabat, AbM, IMGT or Chothia numbering system, or by a combination of the above numbering systems.

[0261] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:31, heavy chain CDR2 shown in SEQ ID NO:110, and heavy chain CDR3 shown in SEQ ID NO:33, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:34, light chain CDR2 shown in SEQ ID NO:35, and light chain CDR3 shown in SEQ ID NO:36; wherein the CDRs are defined by the AbM numbering system.

[0262] In certain specific embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:31, heavy chain CDR2 shown in SEQ ID NO:32, and heavy chain CDR3 shown in SEQ ID NO:33, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:34, light chain CDR2 shown in SEQ ID NO:35, and light chain CDR3 shown in SEQ ID NO:36; wherein the CDR is defined by a mixture of Kabat / AbM.

[0263] In some embodiments, the antibody or its antigen-binding fragment further comprises a framework region sequence derived from mouse immunoglobulin.

[0264] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:15 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:16 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0265] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:15 and a VL containing the sequence shown in SEQ ID NO:16.

[0266] In certain specific embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:31, heavy chain CDR2 shown in SEQ ID NO:106, and heavy chain CDR3 shown in SEQ ID NO:33, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:34, light chain CDR2 shown in SEQ ID NO:35, and light chain CDR3 shown in SEQ ID NO:36; wherein the CDR is defined by a mixture of Kabat / AbM.

[0267] In some embodiments, the antibody or its antigen-binding fragment is humanized and further comprises a framework region sequence derived from human immunoglobulin.

[0268] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:47 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:51 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0269] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:47 and a VL containing the sequence shown in SEQ ID NO:51.

[0270] In some embodiments, the antibody or its antigen-binding fragment comprises a CDR sequence derived from clone 10.7.1 or its humanized antibody, comprising: heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof, and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:48 or 17; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof, and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:52 or 18;

[0271] The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions). In some embodiments, the substitutions are conservative substitutions.

[0272] In some embodiments, the antibody or its antigen-binding fragment comprises: three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:48 or 17; and / or, three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:52 or 18.

[0273] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:48; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:52; or (ii) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:17; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:18.

[0274] In some implementations, the three heavy chain CDRs contained in the VH and / or the three light chain CDRs contained in the VL may be defined by the Kabat, AbM, IMGT or Chothia numbering system, or by a combination of the above numbering systems.

[0275] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:37, heavy chain CDR2 shown in SEQ ID NO:111, and heavy chain CDR3 shown in SEQ ID NO:39, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:40, light chain CDR2 shown in SEQ ID NO:41, and light chain CDR3 shown in SEQ ID NO:42; wherein the CDRs are defined by the AbM numbering system.

[0276] In certain specific embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:37, heavy chain CDR2 shown in SEQ ID NO:38, and heavy chain CDR3 shown in SEQ ID NO:39, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:40, light chain CDR2 shown in SEQ ID NO:41, and light chain CDR3 shown in SEQ ID NO:42; wherein the CDR is defined by a mixture of Kabat / AbM.

[0277] In some embodiments, the antibody or its antigen-binding fragment further comprises a framework region sequence derived from mouse immunoglobulin.

[0278] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:17 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:18 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0279] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:17 and a VL containing the sequence shown in SEQ ID NO:18.

[0280] In certain specific embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:37, heavy chain CDR2 shown in SEQ ID NO:107, and heavy chain CDR3 shown in SEQ ID NO:39, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:40, light chain CDR2 shown in SEQ ID NO:41, and light chain CDR3 shown in SEQ ID NO:42; wherein the CDR is defined by a mixture of Kabat / AbM.

[0281] In some embodiments, the antibody or its antigen-binding fragment is humanized and further comprises a framework region sequence derived from human immunoglobulin.

[0282] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:48 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:52 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0283] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:48 and a VL containing the sequence shown in SEQ ID NO:52.

[0284] In some embodiments, the antibody or its antigen-binding fragment comprises a CDR sequence derived from clone P13738, comprising: heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof, and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62 or 53; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof, and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:63 or 54;

[0285] The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions). In some embodiments, the substitutions are conservative substitutions.

[0286] In some embodiments, the antibody or its antigen-binding fragment comprises: three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62 or 53; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:63 or 54.

[0287] In some embodiments, the antibody or its antigen-binding fragment comprises: (i) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:63; or (ii) three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:53; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:54.

[0288] In some implementations, the three heavy chain CDRs contained in the VH and / or the three light chain CDRs contained in the VL may be defined by the Kabat, AbM, IMGT or Chothia numbering system, or by a combination of the above numbering systems.

[0289] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:56, heavy chain CDR2 shown in SEQ ID NO:112, and heavy chain CDR3 shown in SEQ ID NO:58, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:59, light chain CDR2 shown in SEQ ID NO:60, and light chain CDR3 shown in SEQ ID NO:61; wherein the CDRs are defined by the AbM numbering system.

[0290] In certain specific embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:56, heavy chain CDR2 shown in SEQ ID NO:57, and heavy chain CDR3 shown in SEQ ID NO:58, and a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:59, light chain CDR2 shown in SEQ ID NO:60, and light chain CDR3 shown in SEQ ID NO:61; wherein the CDR is defined by a mixture of Kabat / AbM.

[0291] In some embodiments, the antibody or its antigen-binding fragment is a fully human antibody that further comprises a framework region sequence derived from human immunoglobulins.

[0292] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:53 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:54 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0293] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:53 and a VL containing the sequence shown in SEQ ID NO:54.

[0294] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:62 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:63 or a variant thereof; wherein the variant has 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 with the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). In some embodiments, the substitution is a conserved substitution.

[0295] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO:62 and a VL containing the sequence shown in SEQ ID NO:63.

[0296] In some embodiments, the antibody or its antigen-binding fragment described in any of the above embodiments further comprises a constant region derived from mammalian (e.g., human) immunoglobulins.

[0297] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human immunoglobulins (e.g., IgG, such as IgG1, IgG2, IgG3, or IgG4), and / or, the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from human immunoglobulins (e.g., κ or λ).

[0298] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment contains the heavy chain constant region sequence of wild-type human immunoglobulin.

[0299] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises a heavy chain constant region variant of a human immunoglobulin. This heavy chain constant region variant may contain one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibody: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function. Functional alterations can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue or by chemical modification, for example, altering the antibody's affinity for effector ligands (such as FcR or complement C1q), thereby changing effector function (e.g., reducing or enhancing). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, and CDC.

[0300] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment contains the human IgG1 constant region sequence shown in SEQ ID NO:43, and / or the light chain of the antibody or its antigen-binding fragment contains the human kappa constant region sequence shown in SEQ ID NO:44.

[0301] In some embodiments, in any of the above embodiments, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, diabody; and / or, the antibody is a murine antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or a multispecific antibody.

[0302] On the other hand, this disclosure also provides multispecific antibodies comprising an antibody or antigen-binding fragment thereof that specifically binds to B7-H3 as described above. In some embodiments, the multispecific antibody specifically binds to B7-H3 and additionally specifically binds to one or more other targets. In some embodiments, the multispecific antibody further comprises at least one second antibody or antigen-binding fragment thereof having a second binding specificity against a second target. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0303] The antibodies that specifically bind to B7-H3 or their antigen-binding fragments or multispecific antibodies disclosed herein can be prepared by various methods known in the art, such as by genetic engineering recombination techniques.

[0304] On the other hand, this disclosure provides antibody-drug conjugates comprising an antibody that specifically binds to B7-H3 as described herein, or an antigen-binding fragment thereof, or a multispecific antibody as described herein, and a bioactive molecule linked thereto.

[0305] In some embodiments, the bioactive molecule is a therapeutic agent. In some embodiments, the bioactive molecule is a cytotoxic drug.

[0306] In some embodiments, the cytotoxic drug is a camptothecin derivative.

[0307] In some embodiments, the antibody that specifically binds to B7-H3 or its antigen-binding fragment, or the multispecific antibody, is optionally conjugated to the bioactive molecule via a linker.

[0308] On the other hand, this disclosure provides isolated nucleic acid molecules that encode: (1) an antibody that specifically binds to B7-H3 as described herein, or an antigen-binding fragment thereof, or a variable region of its heavy chain and / or a variable region of its light chain, or its heavy chain and / or light chain; or (2) a multispecific antibody as described herein, or at least one peptide chain thereof.

[0309] In some embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of the antibody or its antigen-binding fragment, and a second nucleotide sequence encoding a light chain or a light chain variable region. The first and second nucleotide sequences are present on the same or different isolated nucleic acid molecules.

[0310] In some embodiments, the isolated nucleic acid molecules contain nucleotide sequences encoding each peptide chain of the multispecific antibody, and the nucleotide sequences encoding each peptide chain are present on the same or different isolated nucleic acid molecules.

[0311] On the other hand, this disclosure provides vectors (e.g., expression vectors) that contain nucleic acid molecules encoding the isolated samples described above.

[0312] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of the antibody or an antigen-binding fragment thereof, and a second nucleotide sequence encoding a light chain or a light chain variable region, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors.

[0313] In some embodiments, the vector contains nucleotide sequences encoding each peptide chain of the multispecific antibody, and the nucleotide sequences encoding each peptide chain are present on the same or different vectors.

[0314] On the other hand, this disclosure provides host cells that contain nucleic acid molecules or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., Escherichia coli cells), eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0315] On the other hand, this disclosure provides a method for preparing the antibody or antigen-binding fragment or multispecific antibody described herein, comprising culturing host cells as described above under conditions that allow protein expression, and recovering the antibody or antigen-binding fragment or multispecific antibody from the cultured host cell culture.

[0316] Pharmaceutical compositions

[0317] On the other hand, this disclosure provides pharmaceutical compositions comprising the antibody-drug conjugate, composition, bispecific antibody or antigen-binding fragment thereof, antibody or antigen-binding fragment thereof, or multispecific antibody, and pharmaceutically acceptable carriers and / or excipients.

[0318] In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate of the present disclosure.

[0319] In some embodiments, the pharmaceutical composition comprises the compositions disclosed herein.

[0320] In some embodiments, the pharmaceutical composition comprises a bispecific antibody or an antigen-binding fragment thereof disclosed herein.

[0321] In some embodiments, the pharmaceutical composition comprises an antibody or an antigen-binding fragment thereof disclosed herein.

[0322] In some embodiments, the pharmaceutical composition comprises a multispecific antibody disclosed herein.

[0323] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In some embodiments, the additional pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide, a radiosensitizer, an antiangiogenic agent, a cytokine, a specific tumor cell-targeting antibody, or an immune checkpoint inhibitor.

[0324] The pharmaceutical compositions disclosed herein are formulated into dosage forms compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or glucose. pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0325] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (wherein which water is soluble) or dispersions and sterile powders for readily preparing sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, polyoxyethylene castor oil (ELTM), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid sufficient for easy injection. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. Carriers can be solvents or dispersion media including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Protection against microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride, will be preferably included in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0326] Therapeutic uses

[0327] On the other hand, this disclosure provides a method for treating tumors, comprising administering to a subject in need of the antibody-drug conjugate, composition, bispecific antibody or antigen-binding fragment thereof, antibody or antigen-binding fragment thereof, multispecific antibody, or pharmaceutical composition of the present disclosure. This disclosure also relates to the use of the antibody-drug conjugate, composition, bispecific antibody or antigen-binding fragment thereof, antibody or antigen-binding fragment thereof, multispecific antibody, or pharmaceutical composition of the present disclosure in the preparation of a medicament for treating tumors.

[0328] In some implementations, the tumor is B7-H3 and / or PSMA positive.

[0329] In some implementations, the tumor is selected from prostate cancer, lung cancer, breast cancer, stomach cancer, liver cancer, cervical cancer, endometrial cancer, and head and neck cancer.

[0330] In some embodiments, the tumor is prostate cancer. In some embodiments, the tumor is metastatic castration-resistant prostate cancer (mCRPC).

[0331] In some implementations, the tumor is lung cancer, such as small cell lung cancer and non-small cell lung cancer.

[0332] In some implementations, the tumor is breast cancer, such as triple-negative breast cancer.

[0333] In some implementations, the tumor is head and neck cancer, such as squamous cell carcinoma of the head and neck.

[0334] In some embodiments, the method includes administering to a subject the antibody-drug conjugate of this disclosure or a pharmaceutical composition containing thereof.

[0335] In some embodiments, the method includes administering to a subject a composition disclosed herein or a pharmaceutical composition comprising thereunder.

[0336] In some embodiments, the method includes administering to a subject a bispecific antibody of the present disclosure or an antigen-binding fragment thereof, or a pharmaceutical composition comprising the thereof.

[0337] In some embodiments, the method includes administering to a subject an antibody of the present disclosure or an antigen-binding fragment thereof, or a pharmaceutical composition comprising thereunder.

[0338] In some embodiments, the method includes administering to a subject a multispecific antibody of the present disclosure or a pharmaceutical composition comprising thereunder.

[0339] In some embodiments, the antibody-drug conjugate, composition, bispecific antibody or its antigen-binding fragment, antibody or its antigen-binding fragment, multispecific antibody, or pharmaceutical composition is administered in combination with an additional pharmaceutically active agent. In some embodiments, the additional pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, antiangiogenic agent, cytokine, antibody specifically targeting tumor cells, or immune checkpoint inhibitor.

[0340] In some embodiments, the method further includes administering additional antitumor therapy to the subject, such as surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0341] The antibody-drug conjugates, compositions, bispecific antibodies or their antigen-binding fragments, antibodies or their antigen-binding fragments, multispecific antibodies, or pharmaceutical compositions comprising them disclosed 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 injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The antibody-drug conjugates, compositions, bispecific antibodies or their antigen-binding fragments, antibodies or their antigen-binding fragments, multispecific antibodies, or pharmaceutical compositions comprising them disclosed herein shall be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the active ingredient into a suitable solvent, and optionally, by simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0342] The antibody-drug conjugates, compositions, bispecific antibodies or their antigen-binding fragments, antibodies or their antigen-binding fragments, multispecific antibodies, or pharmaceutical compositions comprising them of this disclosure may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route of administration and / or method will vary depending on the intended purpose. In some embodiments, the antibody-drug conjugates, compositions, bispecific antibodies or their antigen-binding fragments, antibodies or their antigen-binding fragments, multispecific antibodies, or pharmaceutical compositions comprising them of this disclosure are administered by intravenous injection or bolus.

[0343] The antibody-drug conjugates, compositions, bispecific antibodies or their antigen-binding fragments, antibodies or their antigen-binding fragments, multispecific antibodies, or pharmaceutical compositions comprising them disclosed herein can be formulated in unit dosage form for ease of administration. Unit dosage form refers to physically discrete units suitable for use as a single dose in a subject to be treated; each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect when combined with the desired drug carrier.

[0344] The antibody-drug conjugates, compositions, bispecific antibodies or their antigen-binding fragments, antibodies or their antigen-binding fragments, multispecific antibodies, or pharmaceutical compositions comprising them disclosed herein may be administered alone or in combination with other pharmaceutically active agents (e.g., antitumor agents) or other therapies (e.g., antitumor therapies).

[0345] The subjects mentioned in this article may be mammals, such as humans.

[0346] Definitions of terms

[0347] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0348] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0349] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0350] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures should be understood as being within the scope of this application specification and / or claims.

[0351] The "linker" or "linking unit" described in this invention refers to a chemical structural fragment or bond that is linked at one end to an antibody and at the other end to a drug (drug compound). It may also be linked to other linkers before being linked to a drug compound. The linker structure of this invention can be synthesized using methods known in the art, or it can be synthesized using the methods described in this invention.

[0352] The "antibody-drug conjugate" (ADC) described in this invention refers to a target portion that is linked to a biologically active drug via a stable linker unit.

[0353] The "bioactive molecules" described in this invention can include any therapeutic agent, which typically includes cytotoxic drugs, which are chemical molecules that can strongly disrupt the normal growth of tumor cells.

[0354] Unless otherwise stated, the numerical ranges described in this specification and claims are equivalent to describing at least each specific integer value therein. For example, the numerical range "1-12" is equivalent to describing each integer value in the numerical range "1-12", namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Furthermore, when certain numerical ranges are defined as "numbers", it should be understood that they describe the two endpoints of the range, each integer within the range, and each decimal within the range.

[0355] The term "integers from 0 to 6" refers to 0, 1, 2, 3, 4, 5, and 6.

[0356] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0357] “C 1-10 "Alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. 1-8 "Alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. 1-6 "Alkyl" means a straight-chain or branched alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers.

[0358] “C 2-10"Alkenyl" should be understood to preferably represent a straight or branched monovalent hydrocarbon group containing one or more double bonds and having 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably "C 2-8 "Alkenyl". "C" 2-10 "Alkenyl" should be understood to preferably represent a straight or branched monovalent hydrocarbon group containing one or more double bonds and having 2, 3, 4, 5, 6, 7 or 8 carbon atoms, for example, having 2, 3, 4, 5 or 6 carbon atoms (i.e., C... 2-6 alkenyl), having 2 or 3 carbon atoms (i.e., C24, C34, C4 ... 2-3 Alkenyl). It should be understood that when the alkenyl group contains more than one double bond, the double bonds may be separable or conjugated. The alkenyl group is, for example, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, pent-4-enyl, (E)-pent-3-enyl, (Z)-pent-3-enyl, (E)-pent-2-enyl, (Z)-pent-2-enyl, (E)- Pentyl-1-enyl, (Z)-pentyl-1-enyl, hex-5-enyl, (E)-hex-4-enyl, (Z)-hex-4-enyl, (E)-hex-3-enyl, (Z)-hex-3-enyl, (E)-hex-2-enyl, (Z)-hex-2-enyl, (E)-hex-1-enyl, (Z)-hex-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl 2-Methylprop-1-enyl, (E)-1-methylprop-1-enyl, (Z)-1-methylprop-1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, (E)-2-methylbut-2-enyl, (Z)-2-methylbut-2-enyl, (E)-1-methylbut-2-enyl, (Z)-1-methyl But-2-enyl, (E)-3-methylbut-1-enyl, (Z)-3-methylbut-1-enyl, (E)-2-methylbut-1-enyl, (Z)-2-methylbut-1-enyl, (E)-1-methylbut-1-enyl, (Z)-1-methylbut-1-enyl, 1,1-dimethylprop-2-enyl, 1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl.

[0359] Term "C" 2-10 "Alkyne" should be understood to preferably represent a straight or branched monovalent hydrocarbon group containing one or more triple bonds and having 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, for example, having 2, 3, 4, 5, 6, 7, or 8 carbon atoms (i.e., "C"). 2-8 "Alkyne group" has 2, 3, 4, 5 or 6 carbon atoms (i.e., "C"). 2-6The alkynyl group ("C") has 2 or 3 carbon atoms ("C") 2-3 The alkynyl group is, for example, ethynyl, prop-1-alkynyl, prop-2-alkynyl, but-1-alkynyl, but-2-alkynyl, but-3-alkynyl, pent-1-alkynyl, pent-2-alkynyl, pent-3-alkynyl, pent-4-alkynyl, hex-1-alkynyl, hex-2-alkynyl, hex-3-alkynyl, hex-4-alkynyl, hex-5-alkynyl, 1-methylprop-2-alkynyl, 2-methylbut-3-alkynyl, 1-methylbut-3-alkynyl, 1-methylbut-2-alkynyl, 3-methylbut-1-alkynyl, 1-ethylprop-2-alkynyl, 3-methylpent-4-alkynyl, 2-methylpent-4-alkynyl, 1-methylpent-4-alkynyl -Alynyl, 2-methylpentan-3-ynyl, 1-methylpentan-3-ynyl, 4-methylpentan-2-ynyl, 1-methylpentan-2-ynyl, 4-methylpentan-1-ynyl, 3-methylpentan-1-ynyl, 2-ethylbutan-3-ynyl, 1-ethylbutan-3-ynyl, 1-ethylbutan-2-ynyl, 1-propylpropan-2-ynyl, 1-isopropylpropan-2-ynyl, 2,2-dimethylbutan-3-ynyl, 1,1-dimethylbutan-3-ynyl, 1,1-dimethylbutan-2-ynyl, or 3,3-dimethylbutan-1-ynyl. In particular, the ynyl group is ethynyl, propan-1-ynyl, or propan-2-ynyl.

[0360] Term "C" 3-10 "Cycloalkyl" should be understood to refer to a saturated monovalent monocyclic, bicyclic (e.g., bridged, spirocyclic) hydrocarbon ring or tricyclic alkane having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The C... 3-10 Cycloalkyl groups can be monocyclic hydrocarbon groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl; or bicyclic hydrocarbon groups, such as borneolyl, indolyl, hexahydroindolyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl, 2,7-diazaspiro[3,5]nonyl, 2,6-diazaspiro[3,4]octyl; or tricyclic hydrocarbon groups, such as adamantyl.

[0361] Unless otherwise defined, the term "3-6 membered heterocyclic group" refers to a saturated or unsaturated non-aromatic ring or ring system, for example, a 4-, 5-, or 6-membered monocyclic ring containing at least one, for example, 1, 2, 3, 4, 5, or more heteroatoms selected from O, S, and N, wherein N and S may optionally be oxidized to various oxidation states to form nitrooxides, -S(O)-, or -S(O)2- states. The heterocyclic group may include fused or bridged rings and spirocyclic rings. In particular, the heterocyclic group may include, but is not limited to: 4-membered rings, such as azirmonobutyl and oxobutyl; 5-membered rings, such as tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoyl, pyrazolyl, or pyrrololinyl; or 6-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazine, or trithiaalkyl.

[0362] The term "alkyloxy (alkoxy)" refers to -O- (alkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, and butoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkyloxy, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, or heterocycloalkyloxy.

[0363] The term "alkylamino" refers to -NH-(alkyl), where alkyl is defined as described above. Non-limiting examples of alkylamino include: methylamino, ethylamino, propylamino, isopropylamino, butylamino, etc.

[0364] The term "(alkyl)2amino" refers to -N-(alkyl)2, where alkyl is defined as described above. Non-limiting examples of (alkyl)2amino include: dimethylamino, methylethylamino, diethylamino, dipropylamino, methylpropylamino, diisopropylamino, dibutylamino, etc.

[0365] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0366] Those skilled in the art will understand that the compounds shown in Formula II can exist in the form of various pharmaceutically acceptable salts. If these compounds have a basic center, they can form acid addition salts; if these compounds have an acidic center, they can form base addition salts; if these compounds contain both an acidic center (e.g., a carboxyl group) and a basic center (e.g., an amino group), they can also form internal salts.

[0367] Depending on their molecular structure, the compounds of the present invention can be chiral, and therefore may exist in various enantiomeric forms. Thus, these compounds can exist in racemic or optically active forms. The compounds of the present invention encompass isomers of each chiral carbon in the R or S configuration, or mixtures thereof, and racemates. The compounds of the present invention or their intermediates can be isolated as enantiomeric compounds by chemical or physical methods known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers are obtained from the mixture by reaction with an optically active resolving agent. Examples of suitable resolving agents are optically active acids, such as tartaric acid in both R and S forms, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids (e.g., N-benzoylproline or N-benzenesulfonylproline), or various optically active camphorsulfonic acids. Chromatographic enantiomeric separation can also be advantageously performed using optically active resolving agents (e.g., dinitrobenzoylphenylglycine immobilized on silica gel, cellulose triacetate or other carbohydrate derivatives, or chiral derivatized isobutylene ester polymers). Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, such as hexane / isopropanol / acetonitrile.

[0368] Wavy lines intersecting chemical bonds Used to indicate the connection position of a group with other atoms or groups in a general formula.

[0369] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen via at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only the complete antibody but also antigen-binding fragments capable of specifically binding to a target antigen.

[0370] A "complete antibody" typically consists of two pairs of polypeptide chains (each pair containing 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 antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 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. Constant domains do not directly participate in antibody-antigen binding, but exhibit various 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 be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The allocation of amino acids in each region or domain can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0371] As used herein, the term "bispecific antibody" or "biantibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). A bispecific antibody comprises two antigen-binding domains that have binding specificity to different antigens (or epitopes), thereby enabling it to bind to two different binding sites and / or target molecules. Each antigen-binding domain in a bispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or its antigen-binding fragments (e.g., Fv fragments, Fab fragments, F(ab')2 fragments, or scFv). In some cases, the individual antigen-binding domains are linked by peptide linkers.

[0372] As used herein, the term “antigen-binding fragment” of a bispecific antibody refers to a polypeptide containing a fragment of a bispecific antibody that retains the ability to specifically bind to the same antigen bound by the bispecific antibody and / or competes with the bispecific antibody for specific binding to the antigen; this is also referred to as the “antigen-binding moiety”.

[0373] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions 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, such as 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 AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al.). The definitions in Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003, are provided. In certain special cases, the precise boundaries of these CDRs can also be defined using a combination of the above numbering systems. For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0374] Unless otherwise stated, all antibodies in this article are defined using the AbM numbering system.

[0375] In certain specific implementations, the antibodies described herein employ a Kabat / AbM hybrid definition. This Kabat / AbM hybrid definition means that, after encoding the antibody variable region sequence according to the Kabat coding system, the Kabat-defined CDR intervals are: heavy chain CDR1 (H31-H35B), heavy chain CDR2 (H50-H65), heavy chain CDR3 (H95-H102), light chain CDR1 (L24-L34), light chain CDR2 (L50-L56), and light chain CDR3 (L89-L97); while the AbM-defined CDR intervals are: heavy chain CDR1 (H26-H35B), heavy chain CDR2 (H50-H58), and heavy chain CDR3 (H95-H102). Light chain CDR1 (L24-L34), light chain CDR2 (L50-L56), light chain CDR3 (L89-L97); the CDR range defined by the Kabat / AbM hybrid system is: heavy chain CDR1 (H26-H35B), heavy chain CDR2 (H50-H65), heavy chain CDR3 (H95-H102), light chain CDR1 (L24-L34), light chain CDR2 (L50-L56), light chain CDR3 (L89-L97). That is, in the CDRs defined by the Kabat / AbM hybrid system, heavy chain CDR2 is defined by the Kabat numbering system, while heavy chain CDR1, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are defined by the AbM numbering system.

[0376] As used herein, the term “frame region” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0377] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies (MAbs), and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0378] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. Full-length antibodies contain two antigen-binding sites formed by VH and VL pairs, which specifically recognize / bind to the same antigen.

[0379] As used herein, the term "Fab fragment" refers to an antibody fragment consisting of a light chain containing VL and CL and a heavy chain containing VH and CH1.

[0380] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. 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 a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)3 can be used, but variants thereof may also be used. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0381] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, referring to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, VHH), typically derived from the variable region of a heavy chain antibody (e.g., a camel antibody or a shark antibody). Single-domain antibodies are also called nanobodies, and the terms are used interchangeably. Typically, a nanobody consists of four framework regions and three complementarity-determining regions, having a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to lack one or both of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0382] As used herein, the terms “Fc” or “Fc domain” or “Fc region” or “Fc domain” have the meanings commonly understood by those skilled in the art and are used interchangeably, referring to a portion of the heavy chain constant region containing CH2 and CH3. The Fc region of an antibody has a variety of different functions but is not involved in antigen binding. “Effective functions” mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phage activity; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation, etc. In some embodiments, the Fc region includes a hinge region, CH2, and CH3. When the Fc region includes a hinge region, the hinge region regulates dimerization between two Fc-containing peptides. The Fc region can be any antibody heavy chain constant region isotype, such as IgG1, IgG2, IgG3, or IgG4.

[0383] The Fc domain can include both native and variant Fc regions. A native Fc region contains an amino acid sequence identical to that of naturally occurring Fc regions, such as the native human IgG1 Fc region (non-A and A allotypes); the native human IgG2 Fc region; the native human IgG3 Fc region; and the native human IgG4 Fc region, as well as their naturally occurring variants. A variant Fc region contains an amino acid sequence that differs from the native Fc region due to at least one amino acid modification. In some embodiments, the variant Fc region may possess altered effector functions compared to the native Fc region (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). In some embodiments, the variant Fc region may possess modifications that promote dimerization. As used herein, a “monomer” of the Fc domain refers to one of the two polypeptides forming the dimer Fc domain, i.e., a polypeptide containing a C-terminal constant region of the immunoglobulin heavy chain capable of stabilizing its own binding.

[0384] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0385] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen binding site / antigen complex. Both the “binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K. D (See Davies et al., Annual Rev Biochem, 1990; 59:439-473). K can be measured by any effective method. D The values ​​of kon and kdis can be measured, for example, using surface plasmon resonance (SPR) in Biacore to measure the dissociation constant, or using bioluminescent interferometry or Kinexa to measure the dissociation constant.

[0386] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length.

[0387] The determination of percentage identity between two sequences can also be achieved using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as an improvement upon that in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are integrated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0388] As used herein, the term "variant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide containing an amino acid sequence altered by the introduction of amino acid residue substitutions, deletions, or additions. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, polypeptides can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, variants have similar, identical, or improved functions to the polypeptide or peptide from which they are derived.

[0389] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues 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 or hydrogen bonds). 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 and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Furthermore, amino acid residues can also be classified into categories defined by optional physical and functional properties. For example, residues containing alcohol groups (S and T), aliphatic residues (I, L, V and M), cycloalkenyl-related residues (F, H, W and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S and T), positively charged residues (H, K and R), small residues (A, C, D, G, N, P, S, T and V), very small residues (A, G and S), residues involved in corner formation (A, C, D, E, G, H, K, N, Q, R, S, P and T), and flexible residues (Q, T, K, S, G, P, D, E and R). Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0390] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this 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.

[0391] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0392] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0393] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Adjuvants include, but are not limited to, inorganic adjuvants (such as aluminum hydroxide and vanadium), biological adjuvants (such as Mycobacterium tuberculosis, BCG, Corynebacterium tumefaciens, Bordetella pertussis, Gram-negative bacillus endotoxins, B subunits of cholera toxin, muramyl dipeptides, and cytokines), synthetic adjuvants (such as double-chain polyadenylates and uridine monophosphate), oil formulations (such as Freund's complete adjuvant and peanut oil emulsions), and nano-adjuvants. Ionic strength enhancers include, but are not limited to, sodium chloride. Reagents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and their analogues. Reagents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, and sorbic acid.

[0394] As used herein, the term "treatment" refers to a method performed to achieve a beneficial or desired clinical outcome. For the purposes of this invention, a beneficial or desired clinical outcome includes (but is not limited to) alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment had been received).

[0395] As used herein, the term "subject" refers to a mammal, such as a mouse, rat, other rodent, rabbit, dog, cat, pig, cattle, sheep, horse, or primate, such as a human. In some embodiments, the subject (e.g., a human) has a tumor (e.g., a B7-H3 and / or PSMA-positive tumor).

[0396] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., tumor) means an amount sufficient to prevent, stop, or delay the onset of said disease; an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from 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 purposes 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 characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0397] Beneficial effects of the invention

[0398] This disclosure provides antibodies that specifically bind to B7-H3, bispecific antibodies against B7-H3 and PSMA, and a B7-H3×PSMA bispecific antibody-drug conjugate (ADC). The bispecific antibodies of this disclosure can efficiently bind to B7-H3 and PSMA and exhibit good endocytic activity. Furthermore, by conjugating cytotoxic drugs to these bispecific antibodies, this disclosure provides a bispecific antibody-toxin conjugate (BsADC). This BsADC molecule has dual-target targeting and killing and bystander killing activities against both target-positive cells, demonstrating excellent tumor suppressor activity in in vivo experiments.

[0399] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Attached Figure Description

[0400] Figures 1-1 to 1-2 Schematic diagram of B7-H3×PSMA dual antibody structure.

[0401] Figures 2-1 to 2-27 Detection of cell binding activity of B7-H3×PSMA bispecific antibody and bispecific antibody ADC.

[0402] Figures 3-1 to 3-30 : B7-H3×PSMA bispecific antibody and bispecific antibody ADC internalization detection.

[0403] Figures 4-1 to 4-20 : Detection of cell killing by dual-antibody ADC.

[0404] Figures 5-1 to 5-7 Dual-resistance ADC side-by-side damage detection.

[0405] Figure 6 The antitumor efficacy of bispecific antibody ADCs in the 22Rv1 xenograft model.

[0406] Figures 7-1 to 7-2The efficacy of bispecific antibody ADCs against LNCAP FGC xenografts in CB17 SCID mice.

[0407] Figures 8-1 to 8-2 The efficacy of bispecific antibody ADCs against 22Rv1 xenograft tumors in nude mice bearing tumors.

[0408] Figures 9-1 to 9-2 The efficacy of bispecific antibody ADCs against PC-3 xenografts in BALB / c nude mice.

[0409] Figure 10 The efficacy of bispecific antibody ADCs against LNCAP FGC xenografts in CB17 SCID mice.

[0410] Figure 11 The efficacy of bispecific antibody ADCs against LNCAP FGC xenografts in CB17 SCID mice.

[0411] Figure 12 The efficacy of bispecific antibody ADCs against 22Rv1 xenograft tumors in nude mice bearing tumors.

[0412] Figures 13-1 to 13-3 : Plasma stability assay for bispecific antibody ADC.

[0413] Figure 14 : Fluorescent staining of LNCaP cells induced by dual anti-ADC DNA damage activity.

[0414] Figure 15 Results of DNA damage activity in LNCaP cells induced by dual anti-ADC therapy.

[0415] Figures 16A to 16D : Bispecific antibody ADC induces apoptosis activity in LNCaP cells, among which, Figure 16A , Figure 16B , Figure 16C , Figure 16D The results of experiments showing the induction of apoptosis in LNCaP cells by C101-LP2, P04-DXD, P16(AC)-MMAF and N001-LP2 are shown respectively.

[0416] Figure 17 Immunohistochemical analysis of co-expression of B7-H3 and PSMA in human prostate cancer tissue microarray.

[0417] Figure 18 The efficacy of bispecific antibody-drug conjugates (ADCs) in the human prostate cancer cell LNCAP-FGC xenograft model.

[0418] Figure 19 The efficacy of bispecific antibody-drug conjugates (ADCs) in a human prostate cancer cell xenograft model of 22Rv1.

[0419] Figure 20The efficacy of bispecific antibody-drug conjugates (ADCs) in a human prostate cancer cell PC-3 xenograft model.

[0420] Figure 21 The efficacy of bispecific antibody ADCs in a human small cell lung cancer cell NCI-H446 xenograft model.

[0421] Figure 22 The efficacy of bispecific antibody ADC in a human non-small cell lung cancer cell NCI-H1975 xenograft model.

[0422] Explanation of sequence information

[0423] Information about the sequence involved in this invention is provided in Table 1.

[0424] Table 1: Sequence Information

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436] Example

[0437] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0438] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0439] Example 1: Preparation of antigen protein and positive control antibody

[0440] 1. Construction of antigen protein and positive control antibody vector

[0441] 1.1 Antigen gene synthesis and expression vector construction

[0442] The amino acid sequences of the extracellular regions of human B7-H3 protein (hB7H3 ECD) and monkey B7-H3 protein (cB7H3 ECD) were obtained from the UniProt database, with UniProt accession numbers Q5ZPR3-1 (amino acids 29–466) and A0A7N9CYV2-1 (amino acids 30–466), respectively. The hB7H3 ECD and cB7H3 ECD sequences were ligated into the background vector pGS003 (ampR) (constructed by Kinsey) for transiently transfected cells, and expression plasmids were obtained through recombination. The amino acid sequences of the His-tagged, mFc (mouse Fc)-tagged, and hFc (human Fc)-tagged proteins corresponding to hB7H3 ECD and cB7H3 ECD are shown in SEQ IDs: 1–6. Human, monkey, and mouse PSMA proteins with His tags, namely hPSMA (PSA-H82Qb), cPSMA (PSA-C5247), and mPSMA (PSA-M5245), were all purchased from ACRO.

[0443] 1.2 Synthesis of control antibody gene and construction of expression vector

[0444] The positive control antibody P04 used the humanized M30-H1L4 antibody targeting B7-H3 disclosed in patent CN103687945A (SEQ ID NO: 85 and 77 in CN103687945A). The M30-H1 and M30-L4 sequences were ligated into the pGS003 vector (ampR) (constructed by Kinsey), and the expression plasmid was obtained through recombination.

[0445] The positive control antibody P13 uses the PSMA-targeting antibody disclosed in patent CN110049999A. The heavy chain (H) and light chain (L) sequences of the P13 antibody are as follows:

[0446] P13 H, as shown in SEQ ID NO: 7.

[0447] P13 L, as shown in SEQ ID NO: 8.

[0448] The positive control antibody P16 uses the humanized antibody targeting PSMA disclosed in patent CN111989138A (SEQ ID NO: 10 and 11 in CN111989138A). The positive control antibody P16(AC) is a mutant antibody of P16, with the first amino acid residue in the constant region of the PSMA antibody heavy chain, namely position 114 (Kabat number), alanine (A), mutated to cysteine ​​(C). The sequences of the antibody heavy chain (H) and light chain (L) are as follows:

[0449] P16(AC)H, as shown in SEQ ID NO: 9.

[0450] P16(AC)L, as shown in SEQ ID NO: 10.

[0451] The construction methods for vectors P13, P16, and P16(AC) are the same as those for P04.

[0452] 2. Transient protein expression

[0453] The relevant proteins were expressed using Expi293F in Expi293F expression medium. 24 hours before transfection, 2.5 × 10⁶ cells were seeded in 500 ml cell culture flasks. 6 100 ml of Expi293F cells / ml were cultured in a 37°C, 8% CO2 incubator using a shaker at 120 rpm. For transfection, 320 μl of ExpiFectamine 293 reagent was added to 5.6 ml of Opti-MEM, mixed thoroughly, and incubated at room temperature for 5 minutes. Simultaneously, 100 μg of expression plasmids containing His-tagged, mFc-tagged, and hFc-tagged antigen proteins hB7H3ECD and cB7H3ECD, and positive antibody molecules P04, P13, P16, and P16(AC) were diluted to 6 ml using Opti-MEM. The diluted transfection reagent and plasmids were thoroughly mixed and incubated at room temperature for 10 minutes. The mixture was then added to the cells, mixed thoroughly, and cultured for 5 days at 37°C, 8% CO2 using a shaker at 120 rpm. The supernatant was collected and filtered through a 0.22 μm filter.

[0454] 3. Protein purification

[0455] His-tagged antigen proteins need to be purified using Ni Excel media (Cytiva). Solution A is 20 mM PB-0.5 M NaCl (pH 7.2), solution B is 20 mM PB-0.5 M NaCl-0.5 M imidazole (pH 7.5), and 0.5 M NaOH is used for washing. The purified protein is obtained by following the sequence of washing-equilibration with solution A-loading-equilibration with solution A-rinsing with 4% solution B-elution with 60% solution B.

[0456] For the antigen of the positive antibody molecule, mFc or hFc tag, collect the expression supernatant, filter it through a 0.22 μM filter membrane, perform chromatography on Protein A (Cytiva), elute with 20 mM citric acid - sodium citrate, pH 3.2, and adjust the pH to neutral with 1 M Tris base.

[0457] Example 2: Screening of anti - B7 - H3 antibody

[0458] 1. Preparation of monoclonal hybridomas

[0459] 1.1 Immunization of mice

[0460] The anti - human B7 - H3 monoclonal antibody is produced by immunizing mice. The experimental Balb / c mice are female, 6 - 8 weeks old (Beijing Speyford Biotechnology Co., Ltd., animal license number: SCXK(Beijing)2019 - 0010). After the mice are purchased, they are raised in the laboratory environment for 1 week, with a 12 / 12 - hour light / dark cycle adjustment, a temperature of 20 - 25 °C, and a humidity of 40 - 60%. The mice that have adapted to the environment are immunized according to the following protocol.

[0461] For the first immunization, Freund's complete adjuvant is used. The immunogen hB7H3 ECD - His (prepared in Example 1) is emulsified with the adjuvant at a volume ratio of 1:1, and is injected subcutaneously. The amount of antigen injected per mouse is 10 μg. The second immunization starts 2 days after the first immunization. The antigen hB7H3 ECD - His is emulsified with Alum (aluminum potassium sulfate, sigma) adjuvant and injected subcutaneously. The amount of antigen injected per mouse is 10 μg.

[0462] Three weeks after the first immunization, the mice are bled from the orbital cavity, and a small amount of blood samples are taken for serum titer detection. After the serum titer reaches 1:200000 or above by indirect ELISA method, the mice are given a booster immunization.

[0463] 1.2 Preparation of myeloma cells

[0464] One week before the fusion experiment, resuscitate the myeloma cells P3X63Ag8.653 (ATCC, #CRL - 1580), and culture them in a complete medium containing 1×8 - Azaguanine (8 - azaguanine). Two days before the fusion, change to DMEM with 10% fetal bovine serum for culture to make them in the logarithmic growth phase. The confluence of P3X63Ag8.653 on the day of fusion is 70% - 80%.

[0465] 1.3 Cell fusion and HA screening

[0466] Obtaining and preparing spleen and lymph node cells: Two mice were used after booster immunization. The peritoneum of the mice was cut open, and the spleen and intra-abdominal lymph nodes were removed. The spleen and lymph nodes were ground with a grinding rod and then filtered through a cell sieve to prepare a single-cell suspension.

[0467] Pretreatment before cell fusion: P3X63Ag8.653 cells were collected from culture flasks, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM for viable cell counting. Spleen cell suspension was centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM for viable cell counting.

[0468] Cell electrofusion: Spleen cells and P3X63Ag8.653 cells were mixed at a ratio of 2:1, centrifuged at 2000 rpm for 5 min, and the supernatant was removed. The cells were washed twice with electrofusion buffer (BTX, #47-0001), centrifuged at 2000 rpm for 5 min after each wash, and the supernatant was discarded. The cells were resuspended with electrofusion buffer and added to the electrofusion chamber. The electrofusion instrument (BTX, ECM2001) was started, and the fused cells were added to the prepared 1 / 2 HA (Sigma) selection medium.

[0469] Hybridoma cell selection and culture: Mouse hybridoma cells were resuspended in 1 / 2 HA medium, and the cell suspension was added to 96-well cell culture plates at a rate of 200 μl / well and cultured in a cell culture incubator at 37°C. After 1 week of culture, the medium was changed for the first time with 1 / 2 HA medium, and the cells were cultured in a cell culture incubator at 37°C for the second time. After 3 days of culture, the medium was changed for the second time with 1 / 2 HA medium.

[0470] 1.4 Cell Fusion Screening

[0471] Screening of positive cell lines: Two weeks after fusion, cell supernatants were collected for ELISA to detect the binding of cell supernatants to hB7H3 ECD-His protein. After screening cell wells with positive ELISA results, the cell wells were replaced with new medium, and a second ELISA test was performed 2 days later. Cell supernatants with positive retest results were collected for FACS to detect the binding of cell supernatants to B7-H3 protein on the surface of CHO cells.

[0472] The wells that showed positive results were expanded for culture, and then subcloning was performed using the limiting dilution method to select single-clone cell lines.

[0473] 1.5 Cloning detection and scale-up culture

[0474] ELISA and FACS experiments were performed on the supernatant from the wells of monoclonal cells to screen for clones that bind to B7-H3 protein and cell surface B7-H3. Four positive clones were screened and labeled 1.2.3, 10.15.3, 3.17.1, and 10.7.1, respectively. After expanding the clones into larger cultures, the supernatant was collected for affinity purification to obtain purified mouse antibodies, which were then tested for affinity with human B7-H3 protein.

[0475] The kinetic affinity of antigen and antibody was determined using a Biacore 8K analyzer. The specific method is as follows: Mouse antibody molecules were captured using a Protein A chip (Cytiva) at 25°C in HBS-EP+ buffer (pH 7.4, Cytiva) for 30 s at a flow rate of 30 μl / min. hB7H3 ECD-His was serially diluted 2-fold from 100 nmol / L to six concentration points and flowed through the chip at a flow rate of 30 μl / min. The antigen and antibody bound for 120 s, followed by dissociation at 240 s. Regeneration with 1.5% glycine (Cytiva) was performed for 30 s. Kinetic fitting (1:1 model) was performed using Biacore 8K Evaluation software (Cytiva), and the affinity constants are shown in Table 2-1.

[0476] Table 2-1 Affinity determination of murine antibodies with human B7-H3 protein

[0477]

[0478] 2. Monoclonal antibody gene sequencing and preparation of chimeric antibodies

[0479] 2.1 Monoclonal antibody gene sequencing

[0480] After immunization, fusion, and monoclonalization, total RNA was extracted from monoclonal antibody cell lines 1.2.3, 10.15.3, 3.17.1, and 10.7.1 based on affinity assay results. The RNA was then reverse transcribed into cDNA, and the heavy chain variable region and light chain variable region of the antibody were amplified by PCR using the cDNA as a template.

[0481] Sequence analysis of the antibody gene heavy and light chains was performed. Total RNA from the monoclonal antibodies was obtained using data from Invitrogen. Extraction was performed according to the reagent kit (15596-026) and its instructions.

[0482] Next, we used Takara's PrimeScript. TMII. The 1st Strand cDNA Synthesis Kit (6210A) uses total RNA as a template. The random primers in the kit are used for reverse transcription into first-strand cDNA. Then, reverse primers designed with constant regions for the heavy and light strands and adapter primers in the kit are used for PCR amplification.

[0483] The heavy chain and light chain variable regions of four monoclonal antibody cell lines were amplified by PCR and detected by electrophoresis. The PCR fragments were recovered using an agarose gel extraction kit and subjected to TA cloning. Single clones were selected for PCR identification, and samples from correctly identified strains were sequenced. The final protein sequences of the heavy chain variable regions of cells 1.2.3, 10.15.3, 3.17.1, and 10.7.1 were identified as SEQ ID NO: 11, 13, 15, and 17; the protein sequences of the light chain variable regions were identified as SEQ ID NO: 12, 14, 16, and 18. According to the AbM definition, the CDR region sequences of the heavy chain in section 1.2.3 are SEQ ID NO: 19, 108, and 21, and the CDR region sequences of the light chain in section 1.2.3 are SEQ ID NO: 22–24. The CDR region sequences of the heavy chain in section 10.15.3 are SEQ ID NO: 25, 109, and 27, and the CDR region sequences of the light chain in section 10.15.3 are SEQ ID NO: 28–30. The CDR region sequences of the heavy chain in section 3.17.1 are SEQ ID NO: 31, 110, and 33, and the CDR region sequences of the light chain in section 3.17.1 are SEQ ID NO: 34–36. The CDR region sequences of the heavy chain in section 10.7.1 are SEQ ID NO: 37, 111, and 39, and the CDR region sequences of the light chain in section 10.7.1 are SEQ ID NO: 40–42.

[0484] 2.2 Preparation of chimeric antibodies

[0485] The heavy chain and light chain variable regions of the four obtained monoclonal antibody cell lines were cloned into pcDNA3.4(EBV) vectors (ampR) containing the human IgG1 constant region (constructed by Kinsey) and pcDNA3.4(EBV) vectors (ampR) containing the human kappa constant region (constructed by Kinsey), respectively. Expression plasmids were obtained through recombination and then transfected into cells to prepare chimeric antibodies 1.2.3ChAb-hIgG1, 10.15.3ChAb-hIgG1, 3.17.1ChAb-hIgG1, and 10.7.1ChAb-hIgG1. The protein sequence of the human IgG1 constant region is SEQ ID NO:43, and the protein sequence of the human kappa constant region is SEQ ID NO:44.

[0486] 3. Preparation of humanized antibodies

[0487] Humanization of the variable regions of candidate murine antibodies (1.2.3, 10.15.3, 3.17.1, and 10.7.1) was designed using CDR transplantation technology. First, the light and heavy chain variable regions of the candidate murine antibodies were sequence-aligned with human germline genes in the IMGT database. V and J gene sequences from different human germlines with high homology to the murine antibody variable regions were selected as humanization templates. Then, the murine antibody and template sequences were annotated according to the Kabat coding system and AbM CDR definition, and the murine antibody CDR was transplanted into the frame region of the template. Finally, appropriate reversion mutation sites were selected to mutate amino acid residues in the human template frame region back to the corresponding amino acid residues in the murine antibody frame region, resulting in humanized molecules 1.2.3-H6L4, 10.15.3-H2L2, 3.17.1-H2L3, and 10.7.1-H2L2. The humanized heavy chain variable region sequence is SEQ ID NO:45~48, and the humanized light chain variable region sequence is SEQ ID NO:49~52.

[0488] The heavy chain variable region DNA sequence of the humanized antibody sequence was constructed into the human IgG1 constant region pcDNA3.4(EBV) vector (ampR) (constructed by Kinsey), and the expression plasmid was obtained through recombination. The human IgG1 constant region protein sequence is SEQ ID NO:43. The light chain variable region DNA sequence was constructed into the human kappa constant region pcDNA3.4(EBV) vector (ampR) (constructed by Kinsey), and the expression plasmid was obtained through recombination. The human kappa constant region protein sequence is SEQ ID NO:44. Simultaneously, the expression preparation of humanized antibody molecules 1.2.3-H6L4-hIgG1, 10.15.3-H2L2-hIgG1, 3.17.1-H2L3-hIgG1, and 10.7.1-H2L2-hIgG1 was commissioned to Baiying Biotechnology.

[0489] 4. Phage library screening

[0490] 4.1 Construction and Selection of Natural Human-Derived Literature Library

[0491] Take 100 times the volume of the fully human scFv antibody library (1E11-scfv fully human library constructed by Jinsai) and inoculate it into 880 ml of 2YT-AG medium (containing 100 μg / ml ampicillin and 2% glucose). Incubate at 37℃ and 200 rpm until OD600 = 0.5-0.6. Add helper phages at a cell density of 100 times and incubate at 37℃ for 15 min. Then, shake and incubate at 200 rpm for 1 h. Collect the cells by centrifugation and resuspend them in 400 ml of 2YT-AK medium (containing 100 μg / ml ampicillin and 75 μg / ml kanamycin). Incubate overnight at 30℃ and 200 rpm.

[0492] Centrifuge the culture from the previous step at 4100 rpm and 4°C for 30 min, collect the supernatant, add 1 / 4 volume of PEG / NaCl, mix well, and incubate on ice for 1 h; centrifuge at 4100 rpm and 4°C for 30 min, discard the supernatant; resuspend the phage pellet in 2 ml of pre-cooled 1×PBS, centrifuge at 12000 g and 4°C for 10 min; transfer the supernatant to a new 15 ml centrifuge tube to obtain the first round of starting phage. First, take an appropriate amount of PBS-diluted library phage solution and place it in a pre-clear SA immunotherapy tube, incubate at room temperature for 1 h; take out the pre-clear library phage, add 1 mL of PBS, and divide it equally into two blocked 2 ml centrifuge tubes. Add an appropriate amount of antigen to tube R and an equal volume of PBS to tube CK, and incubate at room temperature for 1 h; add 100 μl of M280 magnetic beads blocked with 5% skim milk to tubes R and CK respectively, and bind for 15 min. The phages were separated on a magnetic rack and the supernatant was discarded. Unbound phages were then washed away with PBST, and phages were eluted with 1 ml of Glycine-HCl (pH 2.2). The eluted phages were then re-infected with TG1 cells for amplification of the elution products. The phages were purified by PEG / NaCl precipitation for the next round of screening. A total of four rounds of phage library enrichment and screening were performed, with progressively increasing washing intensities. The titers of the elution products from each round were determined.

[0493] 4.2 Induction of monoclonal expression and ELISA screening

[0494] After the first to fourth rounds of selection, the bacterial culture was limitedly diluted and spread on plates, and cultured overnight. Single colonies were picked and cultured overnight in 96-well deep-well plates containing 0.5 ml / well of 2YT-AG medium. The overnight culture was then transferred at a volume ratio of 1:10 to 96-well deep-well plates containing 0.5 ml / well of 2YT-AG medium and cultured until OD600 = 0.5-0.6. The culture was then induced overnight at 30°C with 2YT-AG medium (containing 100 μg / ml ampicillin and 1 mM IPTG). The next day, the supernatant was transferred to a clean 96-well deep-well plate by centrifugation. 96-well ELISA plates were coated with hB7H3 ECD-mFc and cB7H3 ECD-mFc antigens, respectively. After blocking, 50 μl of monoclonal supernatant was added to each well, and the plates were incubated at 25°C for 1 h. Then, 250 μl of PBST was added to each well, the plates were shaken for 5–10 s, and the solution was discarded. This process was repeated 3 times. Next, 50 μl of anti-His-HRP antibody (Sino Biological) PBS diluted 1:20000 was added to each well, and the plates were incubated at 25°C for 1 h. Then, 250 μl of PBST was added to each well, the plates were shaken for 5–10 s, and the solution was discarded. This process was repeated 3 times. Finally, 50 μl of TMB chromogenic solution was added to each well, and the plates were incubated for 5 min. Then, 50 μl of 2M H2SO4 was added to each well to stop the incubation. The OD450 value was measured using an ELISA reader.

[0495] Single clones specifically binding to hB7H3 ECD-mFc and cB7H3 ECD-mFc were selected for sequencing. Overnight culture of the above-mentioned bacteria in 2YT-AG medium in 96-well deep plates was used for sequencing analysis and ELISA retesting. One scFv monoclonal antibody (P13738) was selected for the preparation of the fully human full-length antibody P13738-hIgG1. The amino acid sequence of its heavy chain variable region is SEQ ID NO:53, and the amino acid sequence of its light chain variable region is SEQ ID NO:54. According to the AbM definition, the heavy chain CDR region sequences are: SEQ ID NO:56, 112, 58, and the light chain CDR region sequences are: SEQ ID NO:59-61. The heavy chain variable region DNA sequence was constructed into the human IgG1 constant region pcDNA3.4(EBV) vector (ampR) (constructed by Kinsey), and the expression plasmid was obtained through recombination. The human IgG1 constant region protein sequence is SEQ ID NO:43. The light chain variable region DNA sequence was constructed into the human kappa constant region pcDNA3.4(EBV) vector (ampR) (Kinsey construction), and the expression plasmid was obtained through recombination. The human kappa constant region protein sequence is SEQ ID NO:44.

[0496] 4.3 Sequence optimization of the fully human monoclonal antibody P13738

[0497] A high isoelectric point (IOP) of an antibody can affect its solubility, stability, tissue distribution, and biological activity. The P13738 molecule was found to have an IOP of 9.1 in previous studies. The IOP was lowered through rational design of the antibody framework region. The study found that the P13738-V1 mutant retained the activity of P13738 while achieving a lower IOP; the IOP of P13738-V1 was 8.54. The amino acid sequence of the heavy chain variable region of P13738-V1 is SEQ ID NO:62, and its amino acid sequence of the light chain variable region is SEQ ID NO:63.

[0498] 5. Detection of anti-B7-H3 antibody binding activity and affinity assay

[0499] 5.1 ELISA Detection of Anti-B7-H3 Antibody Binding Activity

[0500] Coating antigen: The extracellular regions of human and monkey B7-H3, namely hB7H3 ECD-His and cB7H3 ECD-His, were diluted with CBS carbonate to 1 μg / mL and added to 96 wells of an ELISA plate, 100 μL per well, and incubated overnight at 4°C.

[0501] Blocking: After washing the plate three times with 1×PBST, block with 2% Milk / 1×PBS, 200 μL per well, and incubate at room temperature for 2 hours.

[0502] Sample loading: Add fully human antibody P13738-hIgG1 and negative control hIgG1, and incubate at room temperature for 2 hours. After washing the plate 3 times, add secondary antibody: Goat AntiHumanIgG Fc HRP (1:10000), 100 μL per well, and react at room temperature for 1 hour.

[0503] Color development: After washing the plate 6 times, add 100 μL of TMB color development solution to each well and develop the color for 10 minutes at room temperature in the dark.

[0504] Termination: The reaction was terminated by directly adding 100 μL of 1M H2SO4 to each well.

[0505] Detection: Immediately after the reaction is terminated, place the ELISA plate into the ELISA reader and organize the detection data as shown in Table 2-2.

[0506] Table 2-2 Results of the detection of binding of anti-B7-H3 antibody to human and monkey B7-H3.

[0507]

[0508] Note: "-" indicates that they do not combine.

[0509] The results showed that P13738-hIgG1 could bind to human B7-H3 and monkey B7-H3.

[0510] 5.2 Affinity determination of anti-B7-H3 antibody with human and monkey B7-H3 protein

[0511] The kinetic affinity of antigen and antibody was determined using a Biacore 8K analyzer. The specific method is as follows: At 25°C, humanized monoclonal antibody molecules were captured using a Protein A chip (Cytiva) in HBS-EP+ buffer (pH 7.4, Cytiva) for 30 s at a flow rate of 30 μl / min. hB7H3 ECD-His and cB7H3 ECD-His were serially diluted 2-fold from 100 nmol / L to six concentration points, and then flowed through the chip at a flow rate of 30 μl / min. The antigen and antibody bound for 120 s, followed by dissociation for 240 s, and regeneration with 1.5% glycine (Cytiva) for 30 s. Kinetic fitting (1:1 model) was performed using Biacore 8K Evaluation software (Cytiva), and the affinity constants are shown in Tables 2-3.

[0512] Table 2-3 Affinity test results of anti-B7-H3 antibodies against human and monkey B7-H3 proteins

[0513]

[0514] The results showed that the humanized antibodies 1.2.3-H6L4-hIgG1, 10.15.3-H2L2-hIgG1, 3.17.1-H2L3-hIgG1, and 10.7.1-H2L2-hIgG1 had good affinity for human B7-H3 and monkey B7-H3.

[0515] 5.3 Detection of anti-B7-H3 antibody cell binding activity

[0516] This experiment evaluates antibody binding by detecting the fluorescence signal of cell surface antibodies and assessing the intensity of the fluorescence signal. The prepared humanized antibodies 1.2.3-H6L4-hIgG1, 3.7.1-H2L3-hIgG1, 10.15.3-H2L2-hIgG1, 10.7.1-H2L2-hIgG1, negative control hIgG1, and positive control PO4 were used together for in vitro detection.

[0517] The above-mentioned antibodies, negative controls, and positive controls were serially diluted, starting at 200 nM, followed by 5-fold dilutions, resulting in 8 concentration points, and were compared with 2×10⁻⁶ NM. 5B7-H3 positive tumor cells LNCAP (Nanjing Kebai, CBP60346) and NCI-H358 (Nanjing Kebai, CBP60136) were incubated at 4°C for 60 minutes, and excess antibodies were washed away. The cells were then incubated with PE Goat Anti-Human IgG Fcantibody fluorescent secondary antibody at 4°C for 30 minutes. After washing away excess antibodies, the average fluorescence signal on the cell surface was read using BD Canto II. The results are shown in Table 2-4.

[0518] Table 2-4 Results of cell binding assays for anti-B7-H3 antibodies

[0519]

[0520] Note: "-" indicates that they do not combine.

[0521] The results showed that the humanized antibodies 1.2.3-H6L4-hIgG1, 3.7.1-H2L3-hIgG1, 10.15.3-H2L2-hIgG1, and 10.7.1-H2L2-hIgG1 exhibited good binding activity to B7-H3 positive tumor cells LNCAP and NCI-H358. 5.4 Detection of anti-B7-H3 antibody internalization activity

[0522] This experiment evaluated antibody internalization by detecting intracellular fluorescence signals in each cell and assessing the intensity of these signals. The prepared humanized antibodies 1.2.3-H6L4-hIgG1, 3.7.1-H2L3-hIgG1, 10.15.3-H2L2-hIgG1, 10.7.1-H2L2-hIgG1, positive control PO4, and negative control hIgG1 were used together for in vitro detection.

[0523] pHrodo-labeled Goat anti-Human IgG Fc secondary antibody was stored at -80℃ for later use. LNCAP (Nanjing Kebai, CBP60346) cells were digested, stained with nuclear dye (1:5000) at room temperature for 5 min, and then plated into 96-well black-edged translucent plates pre-coated with lysine (1E4 / 100μl / well). PC-3 (Nanjing Kebai, CBP60343) cells were digested, stained with nuclear dye (1:5000) at room temperature for 5 min, and then plated into 96-well black-edged translucent plates (1.5E4 / 100μl / well) and incubated overnight. The above samples were diluted to 66.67 nM and mixed with pHrodo-labeled Goat anti-Human IgG Fc secondary antibody at a 1:1 ratio, then incubated at 37℃ in the dark for 1 h. Cells were added and cultured at 37°C in the dark for 6 h, 16 h, and 24 h, respectively. After washing the cells once with PBS, they were resuspended in 100 μL of PBS. The pHrodo fluorescence value was detected using high-content PBS. The results are shown in Table 2-5.

[0524] Table 2-5 Results of anti-B7-H3 antibody intracellularization experiment

[0525]

[0526] Note: "-" indicates no internalized signal.

[0527] The results showed that the humanized antibodies 1.2.3-H6L4-hIgG1, 3.7.1-H2L3-hIgG1, 10.15.3-H2L2-hIgG1, and 10.7.1-H2L2-hIgG1 had good internalization activity against B7-H3 positive tumor cells LNCAP and PC-3.

[0528] Example 3: Construction and preparation of B7-H3×PSMA bispecific antibody vector

[0529] 1. Construction of bispecific antibody expression vector

[0530] The structures of bispecific antibodies C040, C040-V1, C097, C101, C115, and C116 are as follows: Figure 1-1 As shown in Table 3-1, the Fab1 fragment containing antigen 1 (B7-H3) and the Fab2 fragment containing antigen 2 (PSMA) are included. The heavy chain variable region and light chain variable region of Fab1 are derived from molecules P13738, P13738-V1, 1.2.3-H6L4, 10.15.3-H2L2, 3.17.1-H2L3 and 10.7.1-H2L2, respectively. The heavy chain variable region sequences are SEQ ID NO:53, 62, 45-48, respectively; the light chain variable region sequences are SEQ ID NO:54, 63, 49-52, respectively. The heavy chain variable region and light chain variable region (VH2, VL2) of Fab2 are derived from P13, and the sequences are SEQ ID NO:64 and SEQ ID NO:65, respectively.

[0531] Table 3-1 Structure of Bispecific Antibodies

[0532]

[0533] The structures of C123 and C123(LALA) bispecific antibodies are as follows: Figure 1-2As shown in Table 3-2, scFv-1 contains antigen 2 (B7-H3) binding fragment and Fab fragment containing antigen 1 (PSMA). The heavy chain variable region and light chain variable region of scFv-1 are derived from molecule 3.17.1-H2L3, which are SEQ ID NO:47 and SEQ ID NO:51, respectively. scFv-1 has the structure shown as VH2-flexible peptide 2-VL2. Flexible peptide 2 is shown as SEQ ID NO:82. The heavy chain variable region and light chain variable region (VH1, VL1) of Fab are derived from P13, with sequences of SEQ ID NO:64 and SEQ ID NO:65, respectively.

[0534] Table 3-2 Structure of Bispecific Antibodies

[0535]

[0536] 2. Expression of bispecific antibodies

[0537] Bispecific antibodies were prepared using the ExpiCHO expression system. 24 hours before transfection, 100 ml of ExpiCHO cells (3E+6 cells / ml) were seeded into ExpiCHO Expression Medium and incubated overnight at 37°C with 8% CO2 and shaking at 125 rpm. The cell density on the day of transfection should be 6E+6 cells / ml. 320 μl of ExpiFectamine was added during transfection. TM Add CHO Reagent to 3.68ml of pre-cooled OptiPRO TM In SFM, gently mix and let stand for 2 min. Simultaneously, add 100 μg of the expression plasmid for the bispecific antibody (constructed by GenScript) to a pre-cooled OptiPRO container. TM Dilute SFM to 4 ml. Slowly mix the diluted solution and let stand at room temperature for 2 min. Add the entire mixture to the cells, mix well, and incubate at 37°C, 8% CO2, and 125 rpm for 11 days with shaking. Collect the supernatant and filter through a 0.22 μm filter membrane.

[0538] 3. Affinity purification of B7-H3×PSMA bispecific antibody

[0539] The supernatant from cells expressing the bispecific antibody was filtered through a 0.22 μm filter and then purified using Protein A medium (GE) to obtain the purified bispecific antibody. The protein purification steps are as follows:

[0540] Cleaning: Clean 3 column volumes with 1M NaOH;

[0541] Equilibration: 20mM PB-0.15M NaCl (pH 7.4) equilibration system pH is consistent with the equilibrium solution;

[0542] Sample loading: Load the cell supernatant filtrate and retain it for 1 min;

[0543] Equilibration: The UV and pH values ​​of the 20mM PB-0.15M NaCl (pH 7.4) equilibration system are consistent with those of the equilibration solution;

[0544] Eluting: Elute for 2 column volumes with 20 mM PB-1 M NaCl (pH 7.4);

[0545] Equilibrium: The conductivity of the 20mM PB-0.15M NaCl (pH 7.4) equilibration system is consistent with that of the equilibration solution;

[0546] Elution: Elute with 20 mM citrate buffer (pH 3.0). Start collecting the sample when the UV absorbance reaches 280 to 20 mAu, and stop collecting when it drops to 20 mAu. Adjust the sample pH to 6–8 with 1 M Tris.

[0547] 4. Two-step purification of B7-H3×PSMA bispecific antibody

[0548] The proteins captured in the first-step affinity purification were subjected to a two-step purification process using ion exchange chromatography. A HiTrap QHP (Cytiva) column was used. Solution A was 20 mM Tris-HCl, pH 9.0, and solution B was 20 mM Tris-HCl-1 M NaOH, pH 9.0. The sample was diluted with solution A to a conductivity < 5 mS / cm and pH adjusted to 9.0. Gradient elution was then performed for elution and collection. Fractions with good purity were combined and analyzed by SDS-PAGE and HPLC-SEC. The results are shown in Table 3-3.

[0549] Table 3-3 Purity Characterization of Bispecific Antibiotic Molecules

[0550]

[0551]

[0552] Example 4: Preparation of small molecule compounds

[0553] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0554] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ ohms. -6The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0555] MS measurements were performed using an Agilent 1200 / 1290DAD-6110 / 6120 Quadrupole MS LC-MS system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS), a waters ACQuity UPLC-QD / SQD system (manufacturer: waters, MS model: waters ACQuity Qda Detector / waters SQ Detector), and a THERMO Ultimate3000-Q Exactive system (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0556] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260II HPLC and a Waters Acquity UPLC H-Class HPLC system.

[0557] Chiral HPLC analysis was performed using a Waters Acquity UPCC high-performance liquid chromatograph.

[0558] High-performance liquid chromatography was performed using Waters MS-triggered Prep-LC with SQD2 detector, Waters MS triggered Prep-LC with Acquity QDA detector, Waters MS-triggered Prep-LC with QDA detector, and GILSON Prep LC with UV detector.

[0559] The CombiFlash rapid preparation system uses a CombiFlash Rf200 (TELEDYNE ISCO).

[0560] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0561] Silica gel column chromatography generally uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0562] Mean inhibition rate of kinases and IC 50 The values ​​were determined using a NovoStar microplate reader (BMG GmbH, Germany).

[0563] The starting materials known in this invention can be synthesized using or according to methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0564] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0565] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0566] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

[0567] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0568] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0569] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0570] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0571] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0572] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system. The volume ratio of the solvent was adjusted according to the polarity of the compounds. Small amounts of basic or acidic reagents such as triethylamine and acetic acid were optionally added for adjustment.

[0573] ADC preparation and analysis instruments: Eppendoff benchtop centrifuges, models 5810R, 5430R, and 5418R.

[0574] The pH meter used was a METTLER TOLEDO Seven Ecellence; the electronic scale used was a METTLER TOLEDO ME1002E; the isothermal mixer used was an Eppendorf 5382KN644547; the inverted mixer used was an IKA LoopSTER; the Nanodrop spectrophotometer used was a Thermo Scientific 2000-C; the microplate reader used was a BioTek EPOCH2; the LC-MS used were a 6224TOF and a 6530LC / Q-TOF; and the HPLC used was an Agilent Technologies 1260 Infinity II.

[0575] Synthesis of intermediates

[0576] 1,2,5-Dioxopyrrolidone-1-yl-6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate Int-1

[0577]

[0578] Step 1: 5-Bromo-2-chloro-4-methoxypyrimidine Int-1b

[0579] 5-Bromo-2,4-dichloropyrimidine Int-1a (5.0 g, 22 mmol) was dissolved in methanol (60 mL), and a methanol solution of sodium methoxide (30%, 4.0 g, 22.0 mol) was added to the solution. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was concentrated, extracted with ethyl acetate, and the organic phase was dried and concentrated to give compound Int-1b (5.0 g, yield: 100%).

[0580] MSm / z(ESI): 223.1(M+1) + .

[0581] Step 2: 5-Bromo-4-methoxy-2-(methylthio)pyrimidine Int-1c

[0582] Sodium methanethiol (1.52 g, 22 mmol) was added to N,N-dimethylformamide (25 mL) to react with compound Int-1b (5.0 g, 22.0 mmol). The reaction mixture was stirred at 40 °C for 1 hour. After the reaction was completed, the reaction mixture was poured into water and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, dried, and concentrated. The crude product was purified by silica gel column chromatography system B to give compound Int-1c (3.0 g, yield: 60%).

[0583] MSm / z(ESI): 235.0(M+1) + .

[0584] Step 3: 6-(4-methoxy-2-(methylthio)pyrimidin-5-yl)hexyl-5-alkynic acid Int-1d

[0585] Compound Int-1c (3.0 g, 12.8 mmol) was dissolved in isopropanol (20 mL), and 5-hexyneic acid (1.43 g, 12.8 mmol), cuprous iodide (243 mg, 1.29 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) chloride (300 mg, 0.41 mmol), and sodium carbonate solution (5 M, 8 mL) were added. The reaction mixture was stirred at 80 °C for 16 hours under a nitrogen atmosphere. After the reaction was completed, the reaction mixture was filtered, and the filtrate was extracted with ethyl acetate (50 mL × 3). The combined organic phases were dried and concentrated. The crude product was purified by silica gel column chromatography system B to give compound Int-1d (3.0 g, yield: 87%).

[0586] MSm / z(ESI): 267.1(M+1) + .

[0587] Step 4: 6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-alkynyl acid Int-1e

[0588] Compound Int-1d (3.0 g, 11.2 mmol) was dissolved in a mixed solvent of methanol and water (40 mL, V / V = 1:1), and potassium peroxymonosulfate (11.6 g, 33.6 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction mixture was poured into water and extracted with ethyl acetate (50 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate to obtain the crude product. The crude product was purified by silica gel column chromatography system B to obtain compound Int-1e (2.5 g, yield: 75%).

[0589] MSm / z(ESI): 299.1(M+H) + .

[0590] Step 5: 2,5-Dioxopyrrolidone-1-yl-6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate Int-1

[0591] N,N-diisopropylcarbodiimide (434 mg, 3.45 mmol) was added to a tetrahydrofuran (10 mL) solution of compound Int-1e (700 mg, 2.3 mmol) and N-hydroxysuccinimide (396 mg, 3.45 mmol), and the reaction was stirred at room temperature for 3 hours. After the reaction was completed, the crude product obtained by direct concentration of the reaction solution was purified by silica gel column chromatography system B to obtain compound Int-1 (600 mg, yield: 60%).

[0592] MSm / z(ESI): 395.5(M+1) + .

[0593] 2,2,5-Dioxopyrrolidone-1-yl-6-(5-cyano-6-(methanesulfonyl)pyridin-3-yl)hexyl-5-acetylacetate Int-2

[0594]

[0595] Step 1: 5-Bromo-2-(methylthio)nicotinonitrile Int-2b

[0596] 5-Bromo-2-chloronicotinonitrile Int-2a (2 g, 9.2 mmol) was dissolved in ethylene glycol dimethyl ether (20 mL), and sodium methanethiol (640 mg, 9.2 mmol) was added under ice bath conditions. The reaction was stirred at room temperature for 4 hours. After the reaction was completed, the reaction was quenched with ammonium chloride aqueous solution, and the system was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and the organic phases were combined and concentrated. The concentrate was purified by silica gel column chromatography system B to give compound Int-2b (1.5 g, yield: 71%).

[0597] MSm / z(ESI): 228.9, 230.9(M+1) + .

[0598] Step 2: 6-(5-cyano-6-(methylthio)pyridin-3-yl)hexyl-5-acynic acid Int-2c

[0599] Compound Int-2b (1.5 g, 6.6 mmol) was dissolved in a mixed solvent of tetrahydrofuran (5 mL) and triethylamine (5 mL), and 5-hexyneic acid (1.5 g, 13.2 mmol), bis(triphenylphosphine)palladium dichloride (913 mg, 1.3 mmol), and cuprous iodide (133 mg, 0.7 mmol) were added. The reaction was stirred at 70 °C for 2 hours. After the reaction was complete, the reaction solution was filtered, and the mother liquor was directly mixed with the solution. The crude product was purified by silica gel column chromatography system B to give compound Int-2c (0.7 g, yield: 41%). MS m / z (ESI): 261.1 (M+1) + .

[0600] Step 3: 6-(5-cyano-6-(methanesulfonyl)pyridin-3-yl)hexyl-5-alkynic acid Int-2d

[0601] Compound Int-2c (0.7 g, 2.7 mmol) was dissolved in methanol (10 mL) and water (10 mL), and potassium peroxide monosulfonate (9.3 g, 27 mmol) was added. The reaction was stirred at room temperature for two hours. After the reaction was complete, the mixture was filtered, the mother liquor was poured into water, extracted three times with dichloromethane, dried over anhydrous sodium sulfate, and the combined organic phases were concentrated. The filtrate was collected to give crude compound Int-2d (815 mg), which was used directly in the next reaction without purification.

[0602] MSm / z(ESI): 293.0(M+1) + .

[0603] Step 4: 2,5-Dioxopyrrolidone-1-yl-6-(5-cyano-6-(methanesulfonyl)pyridin-3-yl)hexyl-5-acetylacetate Int-2

[0604] The crude compound Int-2d (815 mg) was dissolved in dichloromethane (10 mL), and N-hydroxysuccinimide (345 mg, 3.0 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.04 g, 5.4 mmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was poured into water and extracted three times with dichloromethane. The extract was dried over anhydrous sodium sulfate, and the organic phases were combined and concentrated. The concentrate was purified by silica gel column chromatography system B to give compound Int-2 (400 mg, yield: 37%).

[0605] MSm / z(ESI): 390.0(M+1) + .

[0606] 3,2,5-Dioxopyrrolidone-1-yl-6-(4-(methoxymethyl)-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate Int-3

[0607]

[0608] Step 1: 5-Bromo-2-chloro-4-(methoxymethyl)pyrimidine Int-3b

[0609] 5-Bromo-2-chloropyrimidine Int-3a (10 g, 0.05 mol), silver nitrate (36 g, 0.2 mol), ammonium persulfate (57 g, 0.25 mol), and 2-methoxyacetic acid (5.4 g, 0.06 mol) were dissolved in acetonitrile (300 mL) and water (300 mL) and stirred at 60 °C for 2 hours. The reaction mixture was poured into water and extracted three times with ethyl acetate, washed once with saturated brine, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography system B to give compound Int-3b (1.2 g, yield: 13%).

[0610] MSm / z(ESI): 236.9(M+1) + .

[0611] Step 2: 6-(2-chloro-4-(methoxymethyl)pyrimidin-5-yl)hexyl-5-ynylynic acid Int-3c

[0612] Compound Int-3b (1.2 g, 4.5 mmol) was dissolved in tetrahydrofuran (10 mL), and 5-hexyneic acid (0.76 g, 6.8 mmol), cuprous iodide (86 mg, 0.45 mmol), bis(triphenylphosphine)palladium dichloride (632 mg, 0.9 mmol), and triethylamine (1.4 g, 13.5 mmol) were added. The reaction mixture was stirred at 60 °C for 3 hours under a nitrogen atmosphere. After the reaction was completed, the reaction mixture was filtered, and the filtrate was separated into layers with ethyl acetate and water. The aqueous phase was extracted with ethyl acetate (20 mL × 3), and the combined organic phases were dried and concentrated. The crude product was purified by silica gel column chromatography system A to give compound Int-3c (600 mg, yield: 50%). MS m / z (ESI): 269.1 (M+1) + .

[0613] Step 3: 6-(4-(methoxymethyl)-2-(methylthio)pyrimidin-5-yl)hexyl-5-alkynic acid Int-3d

[0614] Compound Int-3c (600 mg, 2.2 mmol) was dissolved in dimethyl sulfoxide (6 mL), and sodium methanethiol (154 mg, 2.2 mmol) and anhydrous magnesium sulfate (528 mg, 4.4 mmol) were added. The reaction mixture was stirred at 50 °C for 1 hour. After the reaction was completed, the reaction mixture was poured into water and extracted with ethyl acetate (50 mL × 3). The combined organic phases were dried and concentrated to obtain crude compound Int-3d (600 mg), which was directly used for the next reaction without purification.

[0615] MSm / z(ESI): 281.1(M+1) + .

[0616] Step 4: 6-(4-(methoxymethyl)-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-alkynic acid Int-3e

[0617] The compound Int-3d (600 mg) obtained in the previous step was dissolved in a mixed solvent of acetone and water (20 mL, V / V = 1:1), and potassium peroxide monosulfonate (7.6 g, 22 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was poured into water and extracted three times with ethyl acetate, washed once with saturated brine, concentrated under reduced pressure, and the crude product obtained was purified by silica gel column chromatography system A to give compound Int-3e (200 mg, yield: 30%).

[0618] MSm / z(ESI): 313.0(M+1) + .

[0619] Step 5: 2,5-Dioxopyrrolidone-1-yl-6-(4-(methoxymethyl)-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate Int-3

[0620] Compound Int-3e (200 mg, 0.64 mmol) and N-hydroxysuccinimide (110 mg, 0.96 mmol) were dissolved in dichloromethane (10 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (246 mg, 1.28 mmol) was added. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the crude product obtained by direct concentration of the reaction solution was purified by silica gel column chromatography system B to give compound Int-3 (100 mg, yield: 38%).

[0621] MSm / z(ESI): 410.1(M+1) + .

[0622] Preparation Example 1

[0623] (R)-N-((1S,9S)-9-ethyl-5-fluoro-4,9-dihydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4',6,7] indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0624] (R)-N-((1R,9S)-9-ethyl-5-fluoro-4,9-dihydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4',6,7] indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0625]

[0626]

[0627] Step 1 N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide 1b

[0628] 20 g (90.8 mmol) of 3-bromo-5-fluoro-4-methoxyaniline 1a was dissolved in dichloromethane (40 mL). After cooling to 0 °C, acetyl chloride (14.3 g, 181.6 mmol) and triethylamine (27.6 mg, 272.4 mmol) were slowly added. The reaction mixture was stirred at 0 °C for 0.5 hours. After the reaction was complete, water (30 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The residue was purified by silica gel column chromatography using eluent system B to give title compound 1b (20 g, yield: 84%).

[0629] MSm / z(ESI): 262.1(M+H) + .

[0630] Step 2 (E)-4-(5-acetamido-3-fluoro-2-methoxyphenyl)but-3-enoic acid 1c

[0631] Compound 1b (20 g, 76.3 mmol) was dissolved in dioxane (30 mL) and water (10 mL), and butyric acid (7.23 g, 83.9 mmol), palladium acetate (1.71 g, 7.6 mmol), tris(o-methylphenyl)phosphine (4.64 g, 15.2 mmol) and N,N-diisopropylethylamine (30 mg, 229 mmol) were added. The reaction mixture was stirred at 100 °C for 16 hours. After the reaction was complete, the reaction mixture was filtered, and the residue was purified by silica gel column chromatography using eluent system B to give the title compound 1c (20 g, yield: 87%).

[0632] MSm / z(ESI): 268.1(M+H) + .

[0633] Step 3: 4-(5-acetamido-3-fluoro-2-methoxyphenyl)butyric acid 1d

[0634] Compound 1c (20 g, 74.8 mmol) was dissolved in tetrahydrofuran (50 mL), and 10% Pd / C (0.8 g, 7.4 mmol) was added. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. After the reaction was complete, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The residue was purified by silica gel column chromatography using eluent system A to give the title compound 1d (20 g, yield: 99%).

[0635] MSm / z(ESI): 270.1(M+1) + .

[0636] Step 4: N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide 1e

[0637] Compound 1d (20 g, 74.4 mmol) was dissolved in trifluoroacetic acid (60 mL), cooled to 0 °C, and then trifluoroacetic anhydride (31.24 g, 148.8 mmol) was slowly added. The reaction mixture was stirred at room temperature for 7 hours. After the reaction was complete, the reaction mixture was slowly poured into water (50 mL), extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with saturated sodium bicarbonate aqueous solution until neutral, then washed with saturated brine, dried, and concentrated. The residue was purified by silica gel column chromatography using eluent system B to give title compound 1e (9.2 g, yield: 46%).

[0638] MSm / z(ESI): 252.1(M+1) + .

[0639] Step 5 (Z)-N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide 1f

[0640] Potassium tert-butoxide (9.83 g, 87.5 mmol) was dissolved in tetrahydrofuran (40 mL) and tert-butanol (10 mL), and the mixture was cooled to 0 °C. Then, N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide 1e (10 g, 39.8 mmol) was dissolved in tetrahydrofuran (10 mL) and slowly added to the reaction mixture. After ten minutes, isoamyl nitrite (7.46 g, 63.6 mmol) was added. The reaction mixture was stirred at 0 °C for 1 hour. After the reaction was complete, the reaction mixture was quenched with saturated ammonium chloride solution, extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with saturated brine, dried, and concentrated. The residue was purified by silica gel column chromatography using eluent system B to give the title compound 1f (6 g, yield: 51%).

[0641] MSm / z(ESI): 281.1(M+1) + .

[0642] Step 6: 1g of N-(7-amino-3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0643] (Z)-N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide 1f (6 g, 21.4 mmol) was dissolved in dioxane (60 mL) and 2N hydrochloric acid solution (20 mL), and 10% Pd / C (1.13 g, 10.7 mmol) was added. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 5 hours. After the reaction was completed, the reaction solution was filtered and concentrated to obtain 1 g (5 g) of crude product, which was used directly in the next step without purification.

[0644] MSm / z(ESI): 267.1(M+1) + .

[0645] Step 7: (9H-fluorene-9-yl)methyl (8-acetamido-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate, 1 hour

[0646] 1 g (5 g, 18.8 mmol) of the compound obtained in the previous step was dissolved in dioxane (50 mL), and the pH was adjusted to 7-8 with sodium bicarbonate. Then, 6.36 g (18.8 mmol) of 9-fluorenylmethyl-N-succinimide carbonate was slowly added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was slowly poured into water (40 mL), extracted with ethyl acetate (40 mL × 3), the organic phases were combined, washed with saturated brine, dried, and concentrated.

[0647] The residue was purified by silica gel column chromatography with eluent system B to give the title compound 1h (5.3 g, yield: 54%).

[0648] MSm / z(ESI): 489.2(M+1) + .

[0649] Step 8 (9H-fluorene-9-yl)methyl (8-amino-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate 1i

[0650] Compound 1h (5 g, 10.25 mmol) was dissolved in dioxane (50 mL) and 12N hydrochloric acid (10 mL). The reaction mixture was stirred at 60 °C for 2 hours. After the reaction was complete, the reaction mixture was slowly poured into water (50 mL), extracted with ethyl acetate (40 mL × 3), the organic phases were combined, washed with saturated brine, dried, and concentrated. The residue was purified by silica gel column chromatography with eluent system B to give the title compound 1i (3.75 g, yield: 78%).

[0651] MSm / z(ESI): 447.2(M+1) + .

[0652] Step 9 (9H-fluorene-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4',6,7]indo[1,2-b]quinoline-1-yl)carbamate 1k

[0653] Compound 1i (3 g, 6.6 mmol) was dissolved in toluene (30 mL), and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyranindolazine-3,6,10(4H)-trione 1j (2.61 g, 9.9 mmol) and p-toluenesulfonic acid (2.52 g, 13.2 mmol) were added. The reaction mixture was stirred at 110 °C for 5 hours. After the reaction was complete, the mixture was extracted with ethyl acetate (30 mL × 3), the organic phases were combined, washed with saturated brine, dried, and concentrated. The residue was purified by silica gel column chromatography with eluent system B to give the title compound 1k (1.8 g, yield: 36%).

[0654] MSm / z(ESI): 674.2(M+1) + .

[0655] Step 10 (9H-fluorene-9-yl)methyl ((9S)-9-ethyl-5-fluoro-4,9-dihydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4',6,7]indo[1,2-b]quinoline-1-yl)carbamate 1l

[0656] Compound 1k (1.8 g, 2.7 mmol) was dissolved in 40% hydrobromic acid (40 mL). The reaction mixture was stirred at 100 °C for 2 hours. After the reaction was complete, the reaction mixture was extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with saturated brine, dried, and concentrated. The residue was purified by silica gel column chromatography with eluent system B to give title compound 1l (1.08 g, yield: 60%).

[0657] MSm / z(ESI): 660.2(M+1) + .

[0658] Step 11 (9S)-1-amino-9-ethyl-5-fluoro-4,9-dihydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4',6,7]indo[1,2-b]quinoline-10,13-dione 1m

[0659] Compound 1L (500 mg, 0.7 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (166 mg, 2.3 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the diethylamine in the reaction mixture was evaporated to dryness using an oil pump. The crude product 1M obtained by evaporation was directly added to the next step of the reaction after being slurried with ethyl acetate.

[0660] MSm / z(ESI): 438.1(M+1) + .

[0661] Step 12 (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((R)-3-hydroxybutyrylamino)-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4',6,7] indo[1,2-b]quinoline-4-yl-(R)-3-hydroxybutyrate 1n

[0662] Compound 1m (100 mg, 0.2 mmol) was dissolved in N,N-dimethylformamide (3 mL), and (R)-3-hydroxybutyric acid (36 mg, 0.34 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (174 mg, 0.46 mmol), and N,N-diisopropylethylamine (88 mg, 0.68 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with saturated brine, dried, and concentrated. The residue was purified by silica gel column chromatography using eluent system B to give the title compound 1n (90 mg, yield: 64%).

[0663] MSm / z(ESI): 610.2(M+1) + .

[0664] Step Thirteen

[0665] (R)-N-((1S,9S)-9-ethyl-5-fluoro-4,9-dihydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4',6,7] indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0666] (R)-N-((1R,9S)-9-ethyl-5-fluoro-4,9-dihydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4',6,7] indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0667] Compound 1n (90 mg, 0.15 mmol) was dissolved in methanol (5 mL), and 1 M lithium hydroxide aqueous solution (2 mL) was added at room temperature. The reaction was stirred at room temperature for 15 minutes. After the reaction was completed, the methanol was evaporated, and the remaining aqueous phase was lyophilized to obtain the crude product. The crude product was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge C18 150×19 mm, 5 μm; mobile phase 1: water (containing 0.1% TFA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 23%-33%, flow rate: 20 mL / min) to obtain compound 1-1 (35 mg, yield: 30%) and compound 1-2 (28 mg, yield: 24%).

[0668] Single-configuration compound 1-1 (shorter retention time):

[0669] MSm / z(ESI): 524.2(M+1) + .

[0670] 1 H NMR (400MHz, DMSO-d6) δ10.42(s,1H),8.39(d,J=8.8Hz,1H),7.84(d,J=11.6H z,1H),7.26(s,1H),6.50(s,1H),5.57–5.49(m,1H),5.41(s,2H),5.27–5.11( m,2H),4.04(dd,J=13.2,6.0Hz,1H),3.18–3.04(m,2H),2.36–2.17(m,2H),2. 15–1.96(m,2H),1.92–1.77(m,2H),1.08(d,J=6.4Hz,3H),0.93–0.81(m,3H).

[0671] Single-configuration compounds 1-2 (longer retention times):

[0672] MSm / z(ESI): 524.2(M+1) + .

[0673] 1H NMR (400MHz, DMSO-d6) δ10.42(s,1H),8.42(d,J=8.8Hz,1H),7.84(d,J=11.6Hz,1H),7.26(s ,1H),5.59–5.51(m,1H),5.42(s,2H),5.29–5.14(m,2H),4.04(dd,J=13.2,6.4Hz,1H),3.17 (dd,J=12.0,4.8Hz,1H),3.10–2.92(m,1H),2.28(dd,J=13.6,7.2Hz,1H),2.18(dd,J=13.6, 5.6Hz,1H),2.13–1.95(m,2H),1.93–1.78(m,2H),1.08(d,J=6.0Hz,3H),0.91–0.82(m,3H).

[0674] Preparation Example 2

[0675] (R)-N-((1S,9S)-4-bromo-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4',6,7]indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0676] (R)-N-((1R,9S)-4-bromo-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4',6,7]indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0677]

[0678] Step 1 4-Bromo-3-fluoro-5-iodoaniline 3b

[0679] 3-Fluoro-5-iodoaniline 3a (50 g, 210.95 mmol) was dissolved in DMF (250 mL), and NBS (41.30 g, 232.04 mmol) was slowly added under ice bath conditions. The reaction was stirred at room temperature for 16 hours. After the reaction was complete, water was added, and the system was extracted with dichloromethane. The organic phase was washed with saturated brine, collected, dried over anhydrous sodium sulfate, and the solvent was evaporated to give crude product 3b (66 g).

[0680] MSm / z (ESI): 315.9, 317.9 (M+1) + .

[0681] 1H NMR (400MHz, DMSO-d6) δ7.01(d,1H),6.47(dd,1H),5.74(s,2H).

[0682] Step 2: N-(4-bromo-3-fluoro-5-iodophenyl)acetamide 3c

[0683] Compound 3b (66 g, 208.92 mmol) was dissolved in dichloromethane (660 mL), and triethylamine (42.28 g, 417.84 mmol) was added. The system was cooled to 0 °C, and acetyl chloride (19.68 g, 250.70 mmol) was slowly added dropwise. After the addition was complete, the system was stirred at room temperature for 4 hours. After the reaction was complete, the solvent was evaporated to obtain the crude product. The crude product was redissolved in ethyl acetate, and the pH of the system was adjusted to 2–3 using dilute hydrochloric acid. The system was then extracted with ethyl acetate, washed with saturated brine, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product, which was then slurried using a dichloromethane / methanol (10:1) mixture to give the title compound 3c (60 g, yield: 80%).

[0684] MSm / z (ESI): 357.9, 359.9 (M+1) + .

[0685] 1 H NMR (400MHz, DMSO-d6) δ10.27(s,1H),7.96(s,1H),7.66(dd,1H),2.06(s,3H).

[0686] Step 3 (E)-4-(5-acetamido-2-bromo-3-fluorophenyl)but-3-enoic acid 3d

[0687] Compound 3c (20 g, 55.87 mmol) was dissolved in a mixed solvent of dioxane (200 mL) and water (40 mL), and but-3-enoic acid (4.81 g, 55.87 mmol), DIPEA (14.45 g, 111.74 mmol), palladium acetate (630 mg, 2.79 mmol), and tris(o-methylphenyl)phosphine (1.7 g, 5.59 mmol) were added. The reaction was stirred at 100 °C for 16 hours under nitrogen protection. After the reaction was complete, water and dichloromethane were added, and the system was washed 3–5 times with saturated sodium bicarbonate solution. The aqueous phase was collected. The pH of the aqueous phase was adjusted to 2–3 with hydrochloric acid and extracted 5–7 times with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was evaporated to give the crude product compound 3d (17 g).

[0688] MSm / z(ESI): 316.0, 317.9(M+1) + .

[0689] Step 4: 4-(5-acetamido-2-bromo-3-fluorophenyl)butyric acid 3e

[0690] Compound 3d (8 g, 25.3 mmol) was dissolved in methanol (80 mL), and a platinum-carbon catalyst (800 mg) was added. The reaction was stirred at room temperature for two hours under hydrogen atmosphere. After the reaction was complete, the mixture was filtered, the filtrate was collected, and the solvent was evaporated to dryness to obtain crude product 3e (8 g). The crude product was used directly in the next reaction without purification.

[0691] MSm / z(ESI): 318.0, 320.0(M+1) + .

[0692] 1 H NMR (400MHz, DMSO-d6) δ12.14(s,1H),10.24(s,1H),7.63(dd,1H),7.25(s,1H),2.75-2.67(m,2H),2.29(t,2H),2.05(s,3H),1.79(dd,2H).

[0693] Step 5: N-(4-bromo-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide 3f

[0694] Compound 3e (8 g, 25.1 mmol) was dissolved in trifluoroacetic acid (80 mL), and trifluoroacetic anhydride (15.82 g, 75.3 mmol) was slowly added under ice bath conditions. The reaction was stirred at 0 °C for 4 hours. After the reaction was complete, water was added under ice bath conditions, and the pH of the system was adjusted to 9–10 using 15% sodium hydroxide solution. The mixture was then extracted with dichloromethane, and the organic phase was washed with saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was evaporated to give compound 3f (4.8 g, yield: 53%).

[0695] MSm / z(ESI): 329.0, 331.0(M+1) + .

[0696] Step 6: (Z)-N-(4-bromo-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide 3g

[0697] Potassium tert-butoxide (1.62 g, 14.4 mmol) was dissolved in a mixed solvent of tetrahydrofuran (80 mL) and tert-butanol (20 mL). A tetrahydrofuran solution (20 mL) of compound 3f (2.15 g, 7.2 mmol) was slowly added under ice bath conditions. After stirring at 0 °C for 10 min, isoamyl nitrite (1.27 g, 10.8 mmol) was added, and the reaction was continued at 0 °C for 50 min. After the reaction was complete, dilute hydrochloric acid was added to adjust the pH of the system to 4–5. The mixture was extracted with ethyl acetate, and the organic phase was washed with saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. The crude product was then slurried with methyl tert-butyl ether to give the title compound 3 g (1.1 g, yield: 46%).

[0698] MSm / z(ESI): 329.0, 331.0(M+1) + .

[0699] 1 H NMR (400MHz, CD3OD) δ8.52(d,1H),3.21(dd,2H),3.07(dd,2H),2.24(s,3H).

[0700] Step 7: N-(7-amino-4-bromo-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (3 hours)

[0701] 3 g (500 mg, 1.52 mmol) of the compound was dissolved in dioxane (10 mL), and 1 mL of 1 M hydrochloric acid and 100 mg of platinum-carbon catalyst were added. The reaction was stirred at room temperature for 4 hours under hydrogen atmosphere. After the reaction was completed, the mixture was filtered, the filtrate was collected, concentrated, and 3 h (500 mg) of crude product was obtained and used directly in the next reaction.

[0702] MSm / z(ESI): 315.1, 317.1(M+1) + .

[0703] Step 8 (9H-fluorene-9-yl)methyl (8-acetamido-5-bromo-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate 3i

[0704] Compound 3h (500 mg) was dissolved in dioxane (10 mL). The pH of the filtrate from step 7 was adjusted to 8–9 using saturated sodium carbonate solution, followed by the addition of fluorenemethyloxycarbonyl chloride (432 mg, 1.67 mmol). The reaction was stirred at room temperature for 1 hour. After the reaction was complete, the system was extracted with ethyl acetate, and the organic phase was washed with saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. The residue was purified by silica gel column chromatography using eluent system B to give the title compound 3i (300 mg, yield: 37%).

[0705] MSm / z(ESI): 537.0, 539.0 (M+1) + .

[0706] Step 9 (9H-fluorene-9-yl)methyl (8-amino-5-bromo-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate 3j

[0707] Compound 3i (700 mg, 1.30 mmol) was dissolved in methanol (10 mL), and concentrated hydrochloric acid (12 mol / L, 2 mL) was added. The reaction was stirred at 60 °C for 1 hour. After the reaction was completed, the residue was purified by silica gel column chromatography with eluent system B to give title compound 3j (500 mg, yield: 77%).

[0708] Step 10 ((9H-fluorene-9-yl)methyl((9S)-4-bromo-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]indo[1,2-b]quinoline-1-yl)carbamate 3k

[0709] Compound 3j (200 mg, 0.40 mmol) was dissolved in toluene (5 mL), and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]-indene-3,6,10(4H)-trione 1j (117 mg, 0.44 mmol) and p-toluenesulfonic acid monohydrate (77 mg, 0.40 mmol) were added. The reaction was stirred at 120 °C for 2 hours. After the reaction was completed, the solvent was evaporated to give the crude product, and the residue was purified by silica gel column chromatography with eluent system B to give the title compound 3k (250 mg, yield: 86%).

[0710] MSm / z(ESI): 722.0, 724.0(M+1) + .

[0711] 1H NMR (400MHz, CDCl3) δ8.19–7.31(m,9H),5.68(dd,1H),5.33–5.18(m,2H),4.66(s,2H),4.34(d,1H),4.21 –4.04(m,2H),3.26(s,1H),3.00–2.94(m,1H),2.04(s,2H),1.81(s,2H),1.70–1.50(m,2H),1.27(dd,3H).

[0712] Step 11 (9S)-1-amino-4-bromo-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4',6,7]indo[1,2-b]quinoline-10,13-dione 3l

[0713] Compound 3k (200 mg, 0.28 mmol) was dissolved in DMF (1 mL), and diethylamine (0.1 mL) was added. The reaction was stirred at room temperature for 30 minutes. After the reaction was complete, the residue was purified by silica gel column chromatography with eluent system A to give title compound 3l (100 mg, yield: 72%).

[0714] MSm / z(ESI): 500.0, 502.1(M+1) + .

[0715] Step 12 (R)-N-((1S,9S)-4-bromo-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4',6,7]indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0716] (R)-N-((1R,9S)-4-bromo-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4',6,7]indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0717] Compound 3l (100 mg, 0.20 mmol) was dissolved in DMF (2 mL), and (R)-3-hydroxybutyric acid (25 mg, 0.24 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (114 mg, 0.30 mmol) and N,N-diisopropylethylamine (52 mg, 0.40 mmol) were added. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: WELCH Xtimate C18 21.2×250 mm, 10 μm; mobile phase 1: water (0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 38%-48%, flow rate: 25 mL / min) to obtain compound 3-1 (1.62 mg, yield: 6%) and compound 3-2 (1.32 mg, yield: 7.4%).

[0718] Compound 3-1 with a single configuration (shorter retention time):

[0719] MSm / z(ESI): 586.0, 588.0(M+1) + .

[0720] 1 H NMR (400MHz, CD3OD) δ8.10(d,J=10.0Hz,1H),7.90(s,1H),5.96–5.88(m,1H),5.83(d,J=16.4Hz,1H),5.71–5.48(m,4H), 4.52–4.46(m,1H),2.71–2.65(m,2H),2.59–2.52(m,2H),2.23–2.18(m,3H),1.49(d,J=6.4Hz,3H),1.25(t,J=7.2Hz,3H).

[0721] Compound 3-2 with a single configuration (longer retention time):

[0722] MSm / z(ESI): 586.0, 588.0(M+1) + .

[0723] 1H NMR (400MHz, CD3OD) δ7.79(d,J=9.6Hz,1H),7.59(s,1H),5.67–5.59(m,1H),5.52(d,J=16.4Hz,1H),5.41–5.33(m,2H),5.32–5.23( m,2H),4.24–4.18(m,1H),2.34(d,J=6.8Hz,2H),2.28–2.21(m,2H),1.93–1.86(m,3H),1.17(d,J=6.4Hz,3H),0.94(t,J=7.2Hz,3H).

[0724] Preparation Example 3

[0725] (R)-N-((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-1,2,3,4,10,11,14,16-octahydro-13H-cycloheptane[3',4',6,7] indene[1,2-b]quinolin-1-yl)-3-hydroxybutyramide

[0726] (R)-N-((1R,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-1,2,3,4,10,11,14,16-octahydro-13H-cycloheptane[3',4',6,7] indene[1,2-b]quinolin-1-yl)-3-hydroxybutyramide

[0727]

[0728]

[0729] Step 1: 1-Bromo-3-fluoro-2-methyl-5-nitrobenzene 6b

[0730] 2-Fluoro-1-methyl-4-nitrobenzene 6a (20.0 g, 0.13 mol) was dissolved in n-heptane (50 mL), and concentrated sulfuric acid (50 mL) was added. The mixture was heated to 60 °C, and N-bromosuccinimide (35.6 g, 0.20 mol) was added in portions at this temperature. The reaction mixture was maintained at 60 °C for 2 hours. The reaction mixture, cooled to room temperature, was added dropwise to ice water, extracted with toluene, and the combined organic phases were washed successively with sodium sulfite solution, water, and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound 6b (30.0 g). The product was used directly in the next reaction without purification.

[0731] MSm / z(ESI): 233.9(M+1) + .

[0732] 1H NMR (400MHz, CDCl3) δ8.29-8.23(m,1H),7.88(dd,1H),2.44(d,3H).

[0733] Step 2: 3-Bromo-5-fluoro-4-methylaniline 6c

[0734] Compound 6b (30.0 g, 0.13 mol) was dissolved in methanol (200 mL), and platinum carbon (3.0 g, 5%) was added. After purging with hydrogen, the mixture was reacted at room temperature for 16 hours under a hydrogen atmosphere. After the reaction was complete, the reaction solution was filtered, and the filtrate was concentrated to give the title compound 6c (20.0 g, yellow oil). The product was used directly in the next reaction without purification.

[0735] MSm / z (ESI): 204.0 (M+1) + .

[0736] Step 3: N-(3-bromo-5-fluoro-4-methylphenyl)acetamide 6d

[0737] 3-Bromo-5-fluoro-4-methylaniline 6c (20.0 g, 0.10 mol) was dissolved in dichloromethane (100 mL) under ice bath conditions, followed by the addition of triethylamine (20.2 g, 0.20 mol) and acetyl chloride (11.8 g, 0.15 mol). The reaction was maintained under ice bath conditions for 3 hours. After the reaction was complete, the reaction solution was quenched with water, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was then slurried in a mixture of dichloromethane and petroleum ether (V / V = 10:1) to obtain the title compound 6d (10.0 g, yield: 32%).

[0738] MSm / z (ESI): 246.0 (M+1) + .

[0739] Step 4 (E)-5-(5-acetamido-3-fluoro-2-methylphenyl)pent-4-enoic acid 6e

[0740] N-(3-bromo-5-fluoro-4-methylphenyl)acetamide 6d (10.0 g, 40.8 mmol) was dissolved in dioxane (40 mL) and water (10 mL). Pentyl-4-enoic acid (6.1 g, 61.20 mmol), palladium acetate (0.7 g, 4.10 mmol), tris(o-methylphenyl)phosphine (2.5 g, 8.20 mmol), and N,N-diisopropylethylamine (15.9 g, 122.01 mmol) were added. The reaction mixture was stirred at 100 °C for 16 hours. After the reaction was complete, water and dichloromethane were added, and the system was washed three times with saturated sodium bicarbonate solution. The aqueous phase was collected. The pH of the aqueous phase was adjusted to 2–3 with hydrochloric acid and extracted three times with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was evaporated to give the title compound 6e (10 g). The product was used directly in the next reaction without purification.

[0741] MSm / z(ESI): 266.1(M+1) + .

[0742] The subsequent synthetic route followed that of Preparation Example 2, except that intermediate 3d was replaced with intermediate 6e (10 g, 0.04 mol). The final products were purified by high-performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge C18 150×19 mm, 5 μm; mobile phase 1: water (0.1% TFA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 37%-47%, flow rate: 20 mL / min) to obtain title products 6-1 (12.6 mg, yield: 14%) and 6-2 (17.2 mg, yield: 19%).

[0743] Single-configuration compound 6-1 (shorter retention time):

[0744] MSm / z(ESI): 536(M+1) + .

[0745] 1H NMR(400MHz,DMSO-d6)δ8.60-8.55(m,1H),7.73-7.72(m,1H),7.28(s,1H), 6.51(s,1H),5.54-5.53(m,1H),5.42(s,2H),5.32-5.26(m,1H),5.24-5.22( m,1H),4.65(s,1H),4.01(s,1H),3.22-3.18(m,2H),2.42-2.40(m,3H),2.2 8-2.24(m,4H),2.10-1.67(m,6H),1.06(d,J=6.0Hz,3H),0.85-0.82(m,3H).

[0746] Compound 6-2 with a single configuration (longer retention time):

[0747] MSm / z(ESI): 536(M+1) + .

[0748] 1 H NMR(400MHz,DMSO-d6)δ8.67-8.66(m,1H),7.73-7.71(m,1H),7.28(s,1H ),6.49(s,1H),5.53-5.52(m,1H),5.41-5.40(m,3H),5.35-5.33(m,1H), 4.67-4.65(m,1H),4.03-3.96(m,1H),3.22-3.18(m,2H),2.42(s,4H),2. 40-2.15(m,4H),2.11-1.52(m,6H),1.08-1.07(m,3H),0.87-0.85(m,3H).

[0749] Preparation Example 4

[0750]

[0751] Compound 6m (700 mg) was prepared by high performance liquid chromatography (GILSON Prep LC with UV detector, column: Xbridge 5 μm C18 150×30 mm; mobile phase 1: water (0.1% TFA); mobile phase 2: acetonitrile; 15 min gradient, gradient ratio: acetonitrile phase 22%-32%, flow rate: 50 mL / min) to obtain compounds 6m-1 (290 mg, shorter retention time) and 6m-2 (300 mg, longer retention time).

[0752] MSm / z(ESI): 450.2(M+H) + .

[0753] Preparation Example 5

[0754] N-((7S,15R)-7-benzyl-17-(((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-1,2,3,4,10,11,14,16-octahydro-13H-cyclohepta[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-15-methyl-2,5,8,11,17-pentoxo-14-oxo-3,6,9,12-tetraazaheptadecyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide 13

[0755]

[0756] Step 1 (R)-3-hydroxybutyrate benzyl ester 13b

[0757] (R)-3-hydroxybutyric acid 13a (50 g, 0.48 mol) was dissolved in N,N-dimethylformamide (100 mL), and benzyl bromide (90 g, 0.52 mol) and cesium carbonate (312.8 g, 0.96 mol) were added. The reaction mixture was placed at room temperature for 12 hours. After the reaction was completed, water (1000 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography system B to give the title compound 13b (40 g, yield: 40%).

[0758] MSm / z (ESI): 217.1 (M+Na) + .

[0759] Step 2: (R)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid benzyl ester 13d

[0760] Methyl acetate 13c (15 g, 40.6 mmol) was dissolved in dichloromethane (150 mL), and compound 13b (39 g, 203.2 mmol) and pyridine 4-methylbenzenesulfonic acid (2 g, 8.1 mmol) were added. The reaction mixture was placed at 40 °C for 12 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography system B to give the title compound 13d (10 g, yield: 50%).

[0761] MSm / z (ESI): 525.2 (M+Na) + .

[0762] Step 3: (R)-3-((2-aminoacetamido)methoxy)benzyl butyrate 13e

[0763] Compound 13d (10 g, 19.9 mmol) was dissolved in N,N-dimethylformamide (50 mL), and diethylamine (3 g, 39.8 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solution was concentrated to obtain crude product 13e (5 g), which was used directly in the next reaction without purification.

[0764] MSm / z(ESI): 281.1(M+H) + .

[0765] Step 4: benzyl(5S,13R)-5-benzyl-1-(9H-fluorene-9-yl)-13-methyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazapentadecan-15-carboxylic acid 13f

[0766] Compound 13e (5 g, 17.8 mmol) was dissolved in N,N-dimethylformamide (20 mL), and (S)-2,5-dioxopyrrolidone-1-yl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-phenylpropionate (7 g, 19.5 mmol) and N,N-diisopropylethylamine (4.5 g, 35.6 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solution was concentrated to give crude product 13f (10 g), which was used directly in the next reaction without purification.

[0767] MSm / z (ESI): 672.3 (M+Na) + .

[0768] Step 5: 13g of (R)-3-((2-((S)-2-amino-3-phenylpropamido)acetamido)methoxy)benzyl butyrate

[0769] Compound 13f (10 g, 15.4 mmol) was dissolved in N,N-dimethylformamide (50 mL), and diethylamine (2 g, 30.8 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the system was diluted with water (100 mL), extracted with ethyl acetate (100 mL x 3), the organic phases were combined and washed with saturated brine, dried, and concentrated. The concentrate was purified by silica gel column chromatography system A to give product 13 g (4 g, yield: 61%).

[0770] MSm / z(ESI): 428.1(M+H) + .

[0771] Step 6: (11S,19R)-11-benzyl-1-(9H-fluorene-9-yl)-19-methyl-3,6,9,12,15-pentoxo-2,18-dioxo-4,7,10,13,16-pentazabenzofuran-21-acid benzyl ester 13h

[0772] Compound 13 g (4 g, 9.3 mmol) was dissolved in N,N-dimethylformamide (30 mL), and (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (3.6 g, 10.2 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (5.3 g, 13.9 mmol) and N,N-diisopropylethylamine (2.3 g, 18.6 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was diluted with water (100 mL), extracted with ethyl acetate (50 mL x 3), the organic phases were combined and washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography system A to give the title compound 13h (3 g, yield: 42%).

[0773] MSm / z(ESI): 786.3(M+H) + .

[0774] Step 7 (11S,19R)-11-benzyl-1-(9H-fluorene-9-yl)-19-methyl-3,6,9,12,15-pentoxo-2,18-dioxo-4,7,10,13,16-pentazabenzofuran-21-carboxylic acid 13i

[0775] Compound 13h (3 g, 3.9 mmol) was dissolved in ethanol (20 mL) and ethyl acetate (10 mL), and palladium / carbon (2 g, 19.5 mmol) was added to displace hydrogen gas. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. After the reaction was completed, the mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrates were combined and concentrated under reduced pressure to obtain crude compound 13i (2 g). The product was used directly in the next reaction without purification.

[0776] MSm / z(ESI): 696.3(M+H) + .

[0777] Step 8 (7S,15R)-1-amino-7-benzyl-15-methyl-2,5,8,11-tetraoxo-14-oxo-3,6,9,12-tetraazaheptadecane-17-carboxylic acid 13j

[0778] Compound 13i (2 g, 3 mmol) was dissolved in N,N-dimethylformamide (20 mL), and diethylamine (0.44 g, 6 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, tetrahydrofuran (20 mL) was added to the reaction mixture, and the precipitated solid was filtered to collect the filter cake, yielding the title compound 13j (1 g, yield: 76%).

[0779] MSm / z(ESI): 452.2(M+H) + .

[0780] Step 9 (3R,11S)-11-benzyl-3-methyl-24-(2-(methanesulfonyl)pyrimidin-5-yl)-7,10,13,16,19-pentaoxo-4-oxo-6,9,12,15,18-pentazatetracarbonyl-23-enoic acid 13l

[0781] Compound 13j (300 mg, 0.66 mmol) was dissolved in N,N-dimethylformamide (10 mL), and 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynate (267 mg, 0.72 mmol) and N,N-diisopropylethylamine (171 mg, 1.32 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was diluted with water (50 mL), extracted with ethyl acetate (30 mL x 3), the organic phases were combined and washed with saturated brine, dried and concentrated, and the crude product was purified by high performance liquid chromatography (GILSON Prep LC with UV detector, column: Ultimate 10 μm C18 250 x 30 mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15 min gradient, gradient ratio: acetonitrile phase 25%-95%, flow rate: 50 mL / min) to obtain the title compound 13l (100 mg, 21%).

[0782] MSm / z (ESI): 724.3 (M+Na) + .

[0783] Step 10: N-((7S,15R)-7-benzyl-17-(((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-1,2,3,4,10,11,14,16-octahydro-13H-cyclohepta[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-15-methyl-2,5,8,11,17-pentoxo-14-oxo-3,6,9,12-tetraazaheptadecyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide 13

[0784] Compound 13l (100 mg, 0.14 mmol) was dissolved in N,N-dimethylformamide (6 mL), and compound 6m-2 (64 mg, 0.14 mmol), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (84 mg, 0.28 mmol) and triethylamine (29 mg, 0.28 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was diluted with water (20 mL), extracted with ethyl acetate (20 mL x 3), the organic phases were combined and washed with saturated brine, dried and concentrated, and the crude product was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5 μm C18 150 x 19 mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15 min gradient, gradient ratio: acetonitrile phase 40%-100%, flow rate: 20 mL / min) to obtain title compound 13 (10 mg, yield: 6%).

[0785] MSm / z(ESI): 1133.3(M+H) + .

[0786] 1 H NMR(400MHz,CD3OD)δ8.92(s,1H),7.60(d,2H),7.16(dd,6H),5.57(d,5H),5.36(s,2H),4.70(d,2H),4.57(d,2H),4.45(d,2H),3.80-3 .72(m,4H),3.48(s,2H),3.13(s,2H),2.60-2.51(m,4H),2.47-2.40(m,6H),1.93(d,6H),1.30(d,2H),1.23(d,3H),1.01-0.96(m,3H).

[0787] Preparation Example 6

[0788] (R)-N-((1R,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyran[3',4':6,7]indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0789] (R)-N-((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyran[3',4':6,7]indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide

[0790] Step 1 N-(3-fluoro-4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)acetamide 24b

[0791] N-(3-fluoro-5-iodo-4-methylphenyl)acetamide 24a (7 g, 23.9 mmol) was dissolved in 1,4-dioxane (70 mL), and pinacol diboronate (12.14 g, 47.8 mmol), Pd(dppf)Cl2 (3.47 g, 4.78 mmol), and potassium acetate (7.04 g, 71.7 mmol) were added. The reaction was carried out under nitrogen protection at 110 °C for 16 hours. After the reaction was complete, the mixture was filtered, the filtrate was collected, and the solvent was evaporated to obtain the crude product. The crude product was purified by column chromatography system B to give the title compound 24b (4.1 g, yield: 59%).

[0792] MSm / z(ESI): 294.1(M+1) + .

[0793] Step 2: N-(3-fluoro-5-hydroxy-4-methylphenyl)acetamide 24c

[0794] Compound 24b (4.1 g, 14 mmol) was dissolved in a mixed solvent of tetrahydrofuran and water (40 mL, V / V = 1:1), and sodium chlorite (1.52 g, 16.8 mmol) was added. The reaction was stirred at room temperature for 30 minutes. After the reaction was complete, ethyl acetate was added, and the organic phase was washed three times with an aqueous sodium hydroxide solution (15% wt). The aqueous phase was collected, and the pH was adjusted to 3–5 with hydrochloric acid, followed by extraction with dichloromethane. The organic phase was collected, and the solvent was evaporated to give the crude product. The crude product was purified by silica gel column chromatography system B to give the title compound 24c (1.9 g, yield: 74%).

[0795] MSm / z(ESI): 184.0(M+1) + .

[0796] Step 3: Methyl 4-(5-acetamido-3-fluoro-2-methylphenoxy)butyrate 24d

[0797] Compound 24c (1.9 g, 10.4 mmol) was dissolved in N,N-dimethylformamide (20 mL), and methyl 4-bromobutyrate (2.82 g, 15.6 mmol), potassium carbonate (2.87 g, 20.8 mmol), and potassium iodide (2.59 g, 15.6 mmol) were added. The reaction was stirred at 85 °C for 16 hours. After the reaction was complete, ethyl acetate was added, and the system was washed three times with saturated brine. The organic phase was collected. The solvent was evaporated to dryness to give the crude product, which was purified by silica gel column chromatography system B to give the title compound 24d (2 g, yield: 68%).

[0798] MSm / z(ESI): 284.0(M+1) + .

[0799] Step 4: 4-(5-acetamido-3-fluoro-2-methylphenoxy)butyric acid 24e

[0800] Compound 24d (2 g, 7.1 mmol) was dissolved in a mixed solvent of tetrahydrofuran and water (20 mL, V / V = 1:1), and lithium hydroxide (340 mg, 14.2 mmol) was added to the above system. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the pH of the system was adjusted to 3–5 with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate. The organic phase was collected and dried over anhydrous sodium sulfate. The solvent was evaporated to give crude product 24e (1.4 g, yield: 73%), which was used directly in the next reaction without purification.

[0801] MSm / z(ESI): 270.0(M+1) + .

[0802] Step 5: N-(8-fluoro-9-methyl-5-oxo-2,3,4,5-tetrahydrobenzo[b]oxacyclohexane-6-yl)acetamide 24f

[0803] Compound 24e (1.4 g, 5.2 mmol) was dissolved in trifluoroacetic acid (20 mL), and trifluoroacetic anhydride (3.28 g, 15.6 mmol) was added. The reaction was stirred at 40 °C for 1 hour. After the reaction was complete, water (10 mL) was slowly added, and stirring was continued at 40 °C for another hour. After the reaction was complete, the system was extracted with ethyl acetate, and the organic phase was collected. The solvent was evaporated to obtain the crude product, which was purified by silica gel column chromatography system B to obtain compound 24f (900 mg, yield: 69%).

[0804] MSm / z(ESI): 252.1(M+1) + .

[0805] 1H NMR (400MHz, CDCl3) δ11.24(s,1H),8.10(d,1H),4.15(t,2H),2.84–2.77(m,2H),2.17–2.10(m,5H),2.07(d,3H).

[0806] Step 6: (Z)-N-(8-fluoro-4-(hydroxyimino)-9-methyl-5-oxo-2,3,4,5-tetrahydrobenzo[b]oxacyclohexane-6-yl)acetamide 24g

[0807] Tetrahydrofuran (20 mL) and tert-butanol (5 mL) were added to a flask, and the mixture was cooled to 0 °C. Potassium tert-butoxide (806 mg, 6.43 mmol) was added. Then, compound 24f (900 mg, 3.59 mmol) was slowly added, and the reaction mixture was stirred at 0 °C for 10 minutes. Isoamyl nitrite (631 mg, 5.39 mmol) was then added, and the reaction was stirred at 0 °C for 1 hour. After the reaction was complete, hydrochloric acid was added to adjust the pH to 4–5, and the mixture was extracted with ethyl acetate. The organic phase was washed with brine and dried over anhydrous sodium sulfate, concentrated, and the solvent was evaporated to obtain 24 g (900 mg) of crude product. The crude product was used directly in the next step without purification. MS m / z (ESI): 281.0 (M+1) + .

[0808] Step 7 (9H-fluorene-9-yl)methyl (6-acetamido-8-fluoro-9-methyl-5-oxo-2,3,4,5-tetrahydrobenzo[b]oxetane-4-yl)carbamate 24h

[0809] 24 g (900 mg, 3.21 mmol) of compound was dissolved in dioxane (10 mL), and palladium on carbon catalyst (200 mg) and 1 M dilute hydrochloric acid (2 mL) were added. The reaction was stirred at room temperature for 2 hours under hydrogen atmosphere. After the reaction was complete, the mixture was filtered, and the filtrate was collected. The pH was adjusted to 10 with saturated sodium bicarbonate aqueous solution, followed by the addition of 929 mg (3.59 mmol) of fluorenyl chloroformate. The reaction was stirred at room temperature for 1 hour. After the reaction was complete, the system was extracted with dichloromethane, and the organic phase was collected. The solvent was evaporated to obtain the crude product, which was purified by silica gel column chromatography system B to give the title compound 24h (500 mg, yield: 29%).

[0810] MSm / z(ESI): 489.1(M+1) + .

[0811] Step 8 (9H-fluorene-9-yl)methyl (6-acetamido-8-fluoro-9-methyl-5-oxo-2,3,4,5-tetrahydrobenzo[b]oxetane-4-yl)carbamate 24i

[0812] Compound 24h (500 mg, 1.02 mmol) was dissolved in dichloromethane (5 mL), and a 4 M hydrogen chloride methanol solution (5 mL) was added. The reaction was stirred at 60 °C for 2 hours. After the reaction was complete, the solvent was evaporated to obtain the crude product, which was purified by silica gel column chromatography to obtain the title compound 24i (350 mg, yield: 77%).

[0813] MSm / z(ESI): 447.1(M+1) + .

[0814] Step 9 (9H-fluoro-9-yl)methyl ((10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate 24j

[0815] Compound 24i (350 mg, 0.78 mmol) was dissolved in toluene (5 mL), and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]-indene-3,6,10(4H)-trione (248 mg, 0.94 mmol) and p-toluenesulfonic acid monohydrate (223 mg, 1.18 mmol) were added. The reaction was stirred at 120 °C for 2 hours. After the reaction was completed, the solvent was evaporated to obtain the crude product, which was purified by silica gel column chromatography system B to obtain the title compound 24j (450 mg, yield: 85%).

[0816] MSm / z(ESI): 674.2(M+1) + .

[0817] Step 10 (10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-1,2,3,10,13,16-hexahydro-11H,14H-oxohepeno[4,3,2-de]pyrano[3',4':6,7]indo[1,2-b]quinoline-11,14-one 24k

[0818] Compound 24j (100 mg, 0.15 mmol) was dissolved in N,N-dimethylformamide, and diethylamine (0.6 mL) was added. The reaction was stirred at room temperature for 30 minutes. After the reaction was complete, the solvent was evaporated to give crude 24k (65 mg, yield: 97%). MS m / z (ESI): 452.1 (M+1) + .

[0819] Step 11

[0820] (R)-N-((1R,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepyn[4,3,2-de]pyran[3',4':6,7]indene[1,2-b]quinoline-1-yl)-3-hydroxybutyramide 24

[0821] Compound 24k (65 mg, 0.14 mmol) was dissolved in N,N-dimethylformamide (3 mL), and (R)-3-hydroxybutyric acid (18 mg, 0.17 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (82 mg, 0.22 mmol), and N,N-diisopropylethylamine (37 mg, 0.29 mmol) were added. The reaction was stirred at room temperature for 15 minutes. After the reaction was completed, the crude product was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2, column: Xbridge C18150 x 19 mm, 10 μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 10-minute gradient, gradient ratio: acetonitrile phase 37%-47%, flow rate: 20 mL / min) to obtain title compounds 24-1 (7.37 mg, yield: 10%) and 24-2 (4.70 mg, yield: 6%).

[0822] Single-configuration compound 24-1 (shorter retention time)

[0823] MSm / z(ESI): 538.2(M+1) + .

[0824] 1H NMR(400MHz,DMSO-d6)δ8.48(d,1H),7.68(d,1H),7.47(d,1H),7.29(s,1H),7.11(d,1H),5.61(dd,1H),5.51–5.39(m,4H),4.61–4.50(m,1H), 4.30–4.16(m,1H),3.92–3.80(m,1H),2.35(d,3H),2.29(s,1H),2.20(d d,2H),2.05–1.94(m,1H),1.92–1.77(m,2H),0.87(t,3H),0.82(d,3H).

[0825] Compound 24-2 with a single configuration (longer retention time)

[0826] MSm / z(ESI): 538.2(M+1) + .

[0827] 1 H NMR(400MHz,DMSO-d6)δ8.48(d,1H),7.68(d,1H),7.47(d,1H),7.29(s,1H ),7.11(d,1H),5.68(dd,1H),5.48(d,2H),5.43(s,2H),4.54–4.42(m,1H) ,4.33–4.24(m,1H),3.92–3.81(m,1H),2.35(d,3H),2.29(s,1H),2.26–2. 16(m,2H),2.03–1.93(m,1H),1.92–1.78(m,2H),1.00(d,3H),0.87(t,3H).

[0828] Preparation Example 7

[0829] N-((7S,15R)-7-benzyl-17-(((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-1,2,3,4,10,14,16-octahydro-13H-cyclohepta[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-15-methyl-2,5,8,11,17-pentoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide

[0830]

[0831] Step 1 (3R,11S)-11-benzyl-24-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)-3-methyl-7,10,13,16,19-pentaoxo-4-oxa-6,9,12,15,18-pentazatetracarbonyl-23-enoic acid 28g

[0832] Compound Int-1 (600 mg, 1.5 mmol) was dissolved in N,N-dimethylformamide (6 mL). N,N-diisopropylethylamine (580 mg, 4.5 mmol) and compound 13j (677 mg, 1.5 mmol) were added sequentially to the solution, and the reaction mixture was stirred at room temperature for 3 hours. The crude product was concentrated and purified by high-performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: Xbridge C18 150 × 19 mm, 5 μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 29%-100%, flow rate: 25 mL / min) to obtain 28 g (350 mg, yield: 35%) of the title compound.

[0833] MSm / z (ESI): 754.1 (M+Na) + .

[0834] Step 2: N-((7S,15R)-7-benzyl-17-(((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-1,2,3,4,10,14,16-octahydro-13H-cyclohepta[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-15-methyl-2,5,8,11,17-pentoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide 28

[0835] 28 g (25 mg, 0.034 mmol) of compound was dissolved in N,N-dimethylformamide (3 mL), and compound 6m-2 (15 mg, 0.033 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (25 mg, 0.065 mmol), and N,N-diisopropylethylamine (171 mg, 1.32 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was completed, the crude product was concentrated and purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: WELCH Xtimate C18 21.2×250mm 10μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 40%-70%, flow rate: 30mL / min) to obtain title compound 28 (16mg, yield: 42%).

[0836] MSm / z(ESI): 1163.2(M+1) + .

[0837] 1 H NMR(400MHz, CDCl3)δ8.56(s,1H),8.43(s,1H),7.52–7.48(m,2H),7.40–7.29( m,3H),7.24–7.04(m,6H),6.94(s,1H),5.60–5.55(m,2H),5.35–5.32(m,1H),5. 16(d,2H),5.04–4.56(m,4H),4.25–3.73(m,9H),3.30(s,3H),3.26–2.93(m,4H ),2.60–2.42(m,7H),2.26(s,3H),2.17–1.74(m,8H),1.31(d,3H),0.97(t,3H).

[0838] Preparation Example 8

[0839] (R)-2-Cyclopropyl-N-((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[0840] (S)-2-Cyclopropyl-N-((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[0841]

[0842] first step

[0843] (9H-fluoro-9-yl)methyl((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyran[3',4':6,7]indene[1,2-b]quinoline-1-yl)carbamate 35a

[0844] Compound 24j (500 mg, 0.74 mmol) was purified to obtain compound 35a (230 mg, yield: 46%) by separating the chiral isomers using a Gilson preparative instrument and a Daicel chiral column (column: CHIRALPAK IE 3.0 cm ID×25 cm, 10 μm; mobile phase 1: MeOH; mobile phase 2: DCM; gradient ratio: MeOH / DCM=80 / 20, flow rate: 25 mL / min).

[0845] MSm / z(ESI): 674.1(M+1) + .

[0846] Step 2

[0847] (1S,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-1,2,3,10,13,16-hexahydro-11H,14H-oxohepeno[4,3,2-de]pyran[3',4':6,7] indene[1,2-b]quinoline-11,14-one 35b

[0848] Compound 35a (50 mg, 0.07 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (0.5 mL) was added. The reaction was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was evaporated under vacuum to give the title compound 35b (34 mg), which was used directly in the next reaction without purification.

[0849] MSm / z(ESI): 452.1(M+1) + .

[0850] Step 3

[0851] (R)-2-Cyclopropyl-N-((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[0852] (S)-2-Cyclopropyl-N-((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[0853] Compound 35b (35 mg, 0.08 mmol) was dissolved in N,N-dimethylformamide (5 mL), and 2-cyclopropyl-2-hydroxyacetic acid (11 mg, 0.09 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (46 mg, 0.12 mmol), and N,N-diisopropylethylamine (21 mg, 0.16 mmol) were added. The reaction was stirred at room temperature for 30 minutes. After the reaction, 2-cyclopropyl-N-((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)-2-hydroxyacetamide (15 mg) was purified by Waters MS-triggered Prep-LC with SQD2 detector. The chromatographic column was an Xbridge 5 μm C18 150 x 19 mm column. The mobile phase 1 was water (containing 0.1% FA) and the mobile phase 2 was acetonitrile. The gradient was 40%-60% acetonitrile phase and the flow rate was 20 mL / min. The sample was chirally separated (SFC 80 column: Daicel CHIRALCEL OD, 250 mm x 30 mm ID, 10 μm; mobile phase: CO2 / MeOH [0.2% NH3 (7M Solution in MeOH)] = 70 / 30, flow rate: 70 g / min) to give compounds 35-1 (5 mg, yield: 12%) and 35-2 (5 mg, yield: 12%).

[0854] 35-1 (A compound with a single configuration and a short retention time)

[0855] MSm / z(ESI): 550.1(M+1) + .

[0856] 1 H NMR(400MHz,DMSO-d6)δ8.14(d,1H),7.72(d,1H),7.30(s,1H),6.53(s,1H),5.68-5.61(m,1H),5.55-5.52(m,2H),5.44(s,2H),4.67 (dd,1H),4.23(dd,1H),3.46(t,1H),2.36(s,3H),2.02-1.97(m,2H),1.92-1.80(m,2H),0.87(t,3H),0.84(d,1H),0.36-0.25(m,4H).

[0857] 35-2 (A compound with a single configuration and a long retention time)

[0858] MSm / z(ESI): 550.1(M+1) + .

[0859] 1 H NMR(400MHz,DMSO-d6)δ8.17(d,1H),7.71(d,1H),7.30(s,1H),6.53(s,1H),5.62(d,1H),5.49(t,2H),5.44(s,2H),4.62-4.53(m,1H),4.29 (dd,1H),3.61(t,1H),2.36(d,3H),2.06-1.94(m,2H),1.85(dd,2H),0.87(t,3H),0.85-0.83(m,1H),0.25-0.19(m,2H),0.08–0.02(m,2H).

[0860] Preparation Example 9

[0861]

[0862] Preparation of step 1, 2-cyclopropyl-2-hydroxyacetic acid (40b)

[0863] (S)-2-amino-2-cyclopropylacetic acid (23 g, 200 mmol) 40a was dissolved in 2M sulfuric acid (100 mL), and 4M sodium nitrite aqueous solution (450 mL) was added under ice-water bath. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was diluted with water, washed with ethyl acetate, and the resulting filtrate was directly concentrated to obtain crude product 40b (11.5 g).

[0864] MSm / z(ESI): 117.1(M+H) + .

[0865] Step 2: Preparation of 2-cyclopropyl-2-hydroxyacetic acid benzyl ester (40c)

[0866] Compound 40b (11.5 g, 0.099 mol) was dissolved in acetonitrile (50 mL), followed by the addition of potassium carbonate (54.65 g, 0.396 mol), benzyl bromide (17.03 g, 0.099 mol), and tetrabutylammonium iodide (3.66 g, 0.0099 mol). The reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was filtered through diatomaceous earth, the filter cake was washed with ethyl acetate, and the filtrate was concentrated and purified by silica gel column chromatography system B to give the title compound 40c (5.8 g, yield: 28%).

[0867] MSm / z(ESI): 207.1(M+H) + .

[0868] Preparation of benzyl(R)-2-cyclopropyl-2-hydroxyacetic acid ester (40d) in step 3

[0869] Compound 40c (12.1 g, 58.4 mmol) was purified using an SFC 150 preparative instrument and a chiral column to separate the chiral isomers (column: Daicel CHIRALCEL AD, 250 mm x 30 mm ID, 10 μm; mobile phase CO2 / MeOH [0.2% NH3 (7MS solution in MeOH)] = 90 / 10, flow rate: 120 g / min) to give compound 40d (5.8 g, yield: 48%). MS m / z (ESI): 207.1 (M+H) + .

[0870] Step 4: Preparation of (R)-10-cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxo-4,7-diazaundecane-11-acid benzyl ester (40e)

[0871] Compound 40d (5.8 g, 28 mmol) was dissolved in dichloromethane (20 mL), and methyl (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)acetate (5 g, 14 mmol) and pyridine 4-methylbenzenesulfonic acid (1.4 g, 5.6 mmol) were added. The reaction mixture was placed at 40 °C for 12 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography system B to give the title compound 40e (1.2 g, yield: 30%).

[0872] MSm / z (ESI): 537.2 (M+Na) + .

[0873] Step 5: Preparation of (R)-2-((2-aminoacetamido)methoxy)-2-cyclopropylacetate benzyl ester (40f)

[0874] Compound 40e (1.2 g, 2.33 mmol) was dissolved in N,N-dimethylformamide (10 mL), and diethylamine (0.34 g, 4.66 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the product was directly used for the next reaction without purification.

[0875] MSm / z(ESI): 293.1(M+H) + .

[0876] Step 6: Preparation of (5S,13R)-5-benzyl-13-cyclopropyl-1-(9H-fluorene-9-yl)-3,6,9-trioxy-2,12-dioxy-4,7,10-triazatetradecane-14-acid benzyl ester (40g)

[0877] Compound 40f (0.6 g, 2.05 mmol) was dissolved in N,N-dimethylformamide (5 mL), and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-phenylalanine glycine (1.09 g, 2.46 mmol) and N,N-diisopropylethylamine (0.53 g, 4.1 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the product was directly used for the next reaction without purification.

[0878] MSm / z (ESI): 684.3 (M+Na) + .

[0879] Step 7: Preparation of (R)-2-((2-((S)-2-amino-3-phenylpropamido)acetamido)methoxy)-2-cyclopropylbenzyl acetate (40 h)

[0880] 40 g (1.2 g, 1.8 mmol) of compound was dissolved in N,N-dimethylformamide (10 mL), and diethylamine (0.27 g, 3.6 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, water (20 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The concentrate was purified by silica gel column chromatography system B to give the title compound 40h (0.6 g, yield: 70%).

[0881] MSm / z(ESI): 440.2(M+H) + .

[0882] Step 8: Preparation of (11S,19R)-11-benzyl-19-cyclopropyl-1-(9H-fluoro-9-yl)-3,6,9,12,15-pentoxo-2,18-dioxo-4,7,10,13,16-pentazaeicosuccinate benzyl ester (40i)

[0883] Compound 40h (0.6 g, 1.36 mmol) was dissolved in N,N-dimethylformamide (10 mL), and (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycine (0.58 g, 1.63 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.78 g, 2.04 mmol) and N,N-diisopropylethylamine (0.38 g, 2.72 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, water (10 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The concentrate was purified by silica gel column chromatography system B to give the title compound 40i (0.8 g, yield: 76%).

[0884] MSm / z(ESI): 798.2(M+H) + .

[0885] Step 9: Preparation of (11S,19R)-11-benzyl-19-cyclopropyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo-2,18-dioxo-4,7,10,13,16-pentazaeicosuccinate benzyl ester (40j)

[0886] Compound 40i (0.8 g, 1.03 mmol) was dissolved in a mixed solvent of ethanol and ethyl acetate (10 mL, V / V = 1:1), and Pd / C (0.2 g, 1.03 mmol) was added. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. After the reaction was completed, the mixture was filtered through diatomaceous earth, the filter cake was washed with ethyl acetate, and the combined filtrates were concentrated under reduced pressure to give the title compound 40j (0.55 g, yield: 71%).

[0887] MSm / z (ESI): 708.1 (M+Na) + .

[0888] Step 10: Preparation of (2R,10S)-16-amino-10-benzyl-2-cyclopropyl-6,9,12,15-tetraoxo-3-oxo-5,8,11,14-tetraazahexadecanoic acid (40kJ)

[0889] Compound 40j (0.55 g, 0.8 mmol) was dissolved in N,N-dimethylformamide (10 mL), and diethylamine (117 mg, 1.6 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, tetrahydrofuran (20 mL) was added to the reaction mixture, and the precipitated solid was filtered to collect the filter cake. The title compound 40k (0.2 g, yield: 54%) was given.

[0890] MSm / z(ESI): 464.2(M+H) + .

[0891] Step 11: Preparation of (2R,10S)-10-benzyl-2-cyclopropyl-23-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxo-3-oxo-5,8,11,14,17-pentazatricarbon-22-alkynical acid (40l)

[0892] Compound 40k (200 mg, 0.43 mmol) was dissolved in N,N-dimethylformamide (10 mL), and 2,5-dioxopyrrolidone-1-yl-6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate Int-1 (152 mg, 0.39 mmol) and N,N-diisopropylethylamine (110 mg, 0.78 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (15 mL x 3). The organic phases were combined and washed with saturated brine, dried, and concentrated to obtain the crude product. The crude product was purified by high performance liquid chromatography (GILSON Prep LC with UV detector, column: Ultimate 10u C18 250 x 30 mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 25%-95%, flow rate: 50 mL / min) to obtain the title compound 40 l (100 mg, yield: 35%).

[0893] MSm / z (ESI): 766.2 (M+Na) + .

[0894] Step 12 Preparation of N-((2R,10S)-10-benzyl-2-cyclopropyl-1-((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-1,2,3,4,10,14,16-octahydro-13H-cycloheptyl[yl]pyrano[3',4':6,7] indene[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-amide (40)

[0895] Compound 40L (60 mg, 0.08 mmol) was dissolved in N,N-dimethylformamide (6 mL), and compound 6m-2 (45 mg, 0.08 mmol), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (61 mg, 0.16 mmol), and triethylamine (26 mg, 0.16 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine, dried, concentrated, and the crude product was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5um C18150 x 19 mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 40%-100%, flow rate: 20 mL / min) to give the title compound 40 (10 mg, yield: 6%).

[0896] MSm / z(ESI): 1175.3(M+H) + .

[0897] 1 H NMR(400MHz,DMSO-d6)δ8.79(s,1H),8.65(d,2H),8.44(s,1H),8.31(s,1H),8.17(d,2H),8.06(s,1H),7.72( d,1H),7.30(s,1H),7.18(dt,5H),6.52(s,1H),5.60(s,1H),5.44(t,3H),5.30(d,1H),4.82(dd,1H),4.59(dd ,1H),4.48(dd,1H),4.07(s,3H),3.76–3.66(m,5H),3.60–3.53(m,2H),3.39(s,3H),3.23(s,3H),3.00(dd,1 H),2.78(d,1H),2.40(d,3H),2.34–2.22(m,3H),1.86(qdd,9H),1.12–1.03(m,1H),0.86(t,3H),0.45(d,4H).

[0898] Preparation Example 10

[0899] N-((2R,10S)-10-benzyl-2-cyclopropyl-1-((1S,10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-oxohepeno[4,3,2-de]pyrano[3',4':6,7]indene[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide

[0900]

[0901] Compound 35b (20 mg, 0.04 mmol) was dissolved in N,N-dimethylformamide (3 mL), and compound 40l (33 mg, 0.04 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (30 mg, 0.08 mmol), and N,N-dimethylethylenediamine (14 mg, 0.11 mmol) were added. The reaction was stirred at room temperature for 30 minutes. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: Gemini5u C18 100 x 21.2 mm; mobile phase 1: water (containing 0.1% NH4); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 45%-75%, flow rate: 30 mL / min) to give title compound 41 (18 mg, yield: 38%). MSm / z(ESI): 1177(M+1) + .

[0902] 1H NMR(400MHz,CD3OD)δ8.61(s,1H),7.68–7.52(m,2H),7.31–7.08(m,5H),5.83–5.69(m,2H),5.55 (dd,2H),5.40(d,1H),4.68(d,2H),4.60(s,2H),4.44–4.35(m,2H),4.28(d,1H),4.12(s,3H),3. 80–3.71(m,4H),3.48(d,2H),3.17–3.06(m,3H),2.97–2.85(m,2H),2.73(s,1H),2.58(dd,4H),2 .46–2.41(m,4H),2.01–1.86(m,4H),1.03(dd,3H),0.94(d,1H),0.49–0.40(m,2H),0.34(s,2H).

[0903] Preparation Example 11

[0904] N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropyl-2-yl)amino-3-methyl-1-oxobut-2-yl)-1-(6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-ynamide)-3,6,9,12,15,18-hexaoxane-21-amide(001)

[0905]

[0906] Step 1: (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7] indo[1,2-b]quinoline-4-yl)amino)-1-oxopropyl-2-yl(amino)-3-methyl-1-oxobutyl-2-yl)carbamate (001b)

[0907] N,N,N',N'-Tetramethylchloroformamidine hexafluorophosphate (333.8 mg, 1.19 mmol) was added to a solution of compound 001a (400.0 mg, 0.99 mmol), (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanine (407.0 mg, 0.99 mmol), and N-methylimidazole (170.9 mg, 2.08 mmol) in N,N-dimethylformamide (18.4 mL). The mixture was stirred at 23 °C for 3 hours. After the reaction was complete, the reaction mixture was added dropwise to saturated brine (50 mL), and a yellow solid precipitated. The solid was filtered, and the filter cake was dissolved in methanol and dichloromethane, washed twice with water, dried, filtered, and concentrated under reduced pressure to obtain compound 001b (560.0 mg, 71%).

[0908] MSm / z(ESI): 796.2(M+1).

[0909] Step 2: (S)-2-amino-N-((S)-1-(((S,9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,2H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (001c)

[0910] Diethylamine (22.1 mg, 0.30 mmol) was added to a solution of compound 001b (80.0 mg, 0.10 mmol) in 1 mL of N,N-dimethylformamide at 0 °C. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to give compound 001c (70.0 mg). The product was used for the next reaction without purification.

[0911] MSm / z(ESI): 574.2(M+1).

[0912] Step 3: tert-butyl-28-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)-23-oxo-4,7,10,13,16,19-hexaoxa-22-azaoctacarbon-27-alkynyl ester (001e)

[0913] N,N-diisopropylethylamine (98.1 mg, 0.76 mmol) was added to a solution of compound Int-1 (100.0 mg, 0.25 mmol) and compound 001d (103.6 mg, 0.25 mmol) in N,N-dimethylformamide (5 mL), and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, ethyl acetate (50 mL) was added, and the mixture was washed twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 001e (170.0 mg, 97%).

[0914] MSm / z(ESI): 634.2(M-56+1).

[0915] Step 4: 28-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)-23-oxo-4,7,10,13,16,19-hexaoxa-22-azaoctacarbon-27-alkynyl acid (001f)

[0916] Trifluoroacetic acid (132.2 mg, 1.16 mmol) was added to a 5 mL solution of compound 001e (80.0 mg, 0.12 mmol) in dichloromethane at 0 °C, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solution was concentrated under reduced pressure to obtain compound 001f (80.0 mg). The product was used directly in the next reaction without purification.

[0917] MSm / z(ESI): 634.2(M+1).

[0918] Step 5: N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropyl-2-yl)amino-3-methyl-1-oxobut-2-yl)-1-(6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylamido)-3,6,9,12,15,18-hexaoxane-21-amide(001)

[0919] Compound 001c (57.7 mg, 0.10 mmol) was added to a solution of compound 001f (70.1 mg, 0.11 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (57.3 mg, 0.15 mmol), and N,N-diisopropylethylamine (39.0 mg, 0.30 mmol) in N,N-dimethylformamide (3 mL), and stirred at room temperature for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: Ultimate C18, 21.2×250 mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to obtain compound 001 (17.3 mg, 14%).

[0920] MSm / z(ESI): 595.5(M / 2+1).

[0921] 1 H NMR(400MHz,DMSO-d6)δ9.75(s,1H),8.80(s,1H),8.27(d,1H),7.97–7.89(m,3H),7.82(d,1H),7. 30(s,1H),6.51(s,1H),5.43(s,2H),5.26(s,2H),4.53(t,1H),4.25(dd,1H),4.07(s,3H),3.59(t, 2H),3.49–3.47(m,21H),3.39(s,4H),3.22–3.14(m,6H),2.99–2.96(m,2H),2.42–2.32(m,2H),2.2 4(t,2H),2.05–1.96(m,3H),1.90–1.83(m,2H),1.80–1.75(m,2H),1.39(d,3H),0.90–0.84(m,9H).

[0922] Preparation Example 12

[0923] N-((S)-1-((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-1-(6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylamido)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide(002)

[0924]

[0925] Step 1: 34-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetraaza-33-alkynyl tert-butyl ester (002b)

[0926] Compound 002a (100 mg, 0.201 mmol) and compound Int-1 (87 mg, 0.221 mmol) were dissolved in N,N-dimethylformamide (1 mL). N,N-diisopropylethylamine (78 mg, 0.603 mol) was added dropwise to the reaction system at 0 °C. The reaction mixture was heated to 25 °C and stirred for 2 hours. After the reaction was complete, the solvent was removed by rotary evaporation. The mixture was dissolved in water (20 mL) and extracted three times with dichloromethane (30 mL). The organic phase was washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate. The product was then evaporated to dryness to give compound 002b (142 mg, yield: 91%), which was used directly in the next reaction without purification.

[0927] MSm / z(ESI):722.3(M+1-56).

[0928] Step 2: 34-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetraaza-33-alkynyl acid (002c).

[0929] Compound 002b (60 mg, 0.077 mmol) was dissolved in dichloromethane (1 mL), and then trifluoroacetic acid (0.2 mL, 298 mg, 2.612 mmol) was added at 0 °C. The mixture was then heated to 25 °C and stirred for 1 hour. After the reaction was complete, the reaction solution was evaporated to dryness to obtain compound 002c (50 mg). The product was used directly in the next reaction without purification.

[0930] MSm / z(ESI):722.2(M+1).

[0931] Step 3: N-((S)-1-((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-1-(6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylamido)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide (002)

[0932] Compound 002c (50 mg, 0.069 mmol), HATU (40 mg, 0.104 mmol), and N,N-diisopropylethylamine (27 mg, 0.208 mmol) were dissolved in N,N-dimethylformamide (1 mL). Compound 001c (40 mg, 0.070 mmol) was added at 0 °C, and the reaction was stirred at 25 °C for 2 hours. After the reaction was completed, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was prepared by high performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20APPump, Shimadzu SPD-20AP UV Detector; column: Ultimate C18, 21.2*250mm, 5μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20mL / min) to obtain compound 002 (22.6mg, yield: 26%).

[0933] MSm / z(ESI): 639.4(M / 2+1).

[0934] 1 H NMR(400MHz,DMSO-d6)δ9.75(s,1H),8.80(s,1H),8.27(d,1H),7.97–7.89(m,3H),7.82(d,1H),7.31(s, 1H),6.50(s,1H),5.43(s,2H),5.26(s,2H),4.57–4.50(m,1H),4.27–4.24(m,1H),4.08(s,3H),3.60(t,2 H),3.50–3.48(m,30H),3.42–3.39(m,5H),3.22–3.14(m,4H),2.99–2.96(m,2H),2.47–2.35(m,2H),2.2 6–2.22(m,2H),2.06–1.95(m,3H),1.93–1.84(m,2H),1.82–1.74(m,2H),1.40(d,3H),0.90–0.84(m,9H).

[0935] Preparation Example 13

[0936] N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropyl-2-yl)amino-3-methyl-1-oxobut-2-yl)-1-(6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylamido)-3,6,9,12,15,18,21,24,27,30-decaoxane-33-amide(003)

[0937]

[0938] Step 1: 40-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)-35-oxo-4,7,10,13,16,19,22,25,28,31-decaoxa-34-azatetraone-39-alkynyl tert-butyl ester (003b)

[0939] Compound 003a (100 mg, 0.17 mmol) and compound Int-1 (67.5 mg, 0.17 mmol) were dissolved in N,N-dimethylformamide (5 mL) solution, and N,N-diisopropylethylamine (66.1 mg, 0.51 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, water and ethyl acetate were added for extraction, and the organic phase was washed twice with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography system A to obtain compound 003b (110 mg, 74.4%).

[0940] MSm / z(ESI): 886.4(M+1).

[0941] Step 2: 40-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)-35-oxo-4,7,10,13,16,19,22,25,28,31-decaoxa-34-azatetraone-39-alkynic acid (003c).

[0942] Compound 003b (100 mg, 0.17 mmol) was dissolved in dichloromethane (2 mL), cooled to 0 °C, and trifluoroacetic acid (0.4 mL) was slowly added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain crude compound 003c (90 mg). The crude product was used directly in the next reaction without purification.

[0943] MSm / z(ESI): 810.4(M+1).

[0944] Step 3

[0945] N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropyl-2-yl)amino-3-methyl-1-oxobut-2-yl)-1-(6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylamido)-3,6,9,12,15,18,21,24,27,30-decaoxane-33-amide(003)

[0946] Compound 003c (80 mg, 0.10 mmol) and compound 001c (56.7 mg, 0.10 mmol) were dissolved in N,N-dimethylformamide (2 mL) solution, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (56.3 mg, 0.15 mmol) and N,N-diisopropylethylamine (38.2 mg, 0.30 mmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, and the crude product was purified by high performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector; column: Ultimate 5μm C18 250×21.2mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 21-minute gradient, gradient ratio: acetonitrile phase 20%-48%, flow rate: 20mL / min) to obtain compound 003 (24.1mg, 17.9%).

[0947] MSm / z(ESI): 683.4(M / 2+1).

[0948] 1H NMR(400MHz,DMSO-d6)δ9.78(s,1H),8.82(s,1H),8.29(d,1H),8.02–7.81(m,4H),7.33(s,1H),6. 53(s,1H),5.45(s,2H),5.28(s,2H),4.55(t,1H),4.31–4.22(m,1H),4.09(s,3H),3.61(t,2H),3. 51–3.50(m,34H),3.43–3.41(m,5H),3.24–3.18(m,5H),3.01–2.98(m,2H),2.70–2.68(m,1H),2.2 8–2.24(m,2H),2.09–1.96(m,4H),1.93–1.76(m,5H),1.41(d,3H),1.25(s,2H),0.92–0.86(m,9H).

[0949] Preparation Example 14

[0950] 1-(6-(5-cyano-6-(methanesulfonyl)pyridin-3-yl)hexyl-5-acetylamido)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-3,6,9,12,15,18-hexaoxane-21-amide (004)

[0951]

[0952] Following the synthetic route of Preparation Example 11, the starting material in the first step was replaced with Int-2. The crude product was purified by high performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector; column: Ultimate C18, 21.2*250mm, 5μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20mL / min). The purified product was lyophilized to obtain compound 004 (10.7mg, 17%).

[0953] MSm / z(ESI): 592.4(M+2 / 2).

[0954] 1H NMR(400MHz,DMSO-d6)δ9.82(s,1H),9.04(d,1H),8.78(d,1H),8.33(d,1H),8.04–7.94(m,3H),7.88(d,1H),7.37 (s,1H),6.56(s,1H),5.49(s,2H),5.32(s,2H),4.62–4.56(m,1H),4.33–4.29(m,1H),3.67–3.64(m,2H),3.57–3. 53(m,20H),3.51(s,3H),3.49–3.45(m,4H),3.28–3.26(m,2H),3.24–3.21(m,2H),3.05–3.02(m,2H),2.48–2.39( m,2H),2.34–2.31(m,2H),2.08-2.03(m,2H)1.99-1.91(m,2H)1.86–1.82(m,2H),1.46(d,3H),0.97–0.89(m,9H).

[0955] Preparation Example 15

[0956] 1-(6-(5-cyano-6-(methanesulfonyl)pyridin-3-yl)hexyl-5-ynamide)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide (005)

[0957]

[0958] Following the synthetic route of Preparation Example 12, the starting material in the first step was replaced with Int-2. The crude product was purified by high performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector; column: Ultimate C18, 21.2*250mm, 5μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-46%, flow rate: 20mL / min). The purified product was lyophilized to obtain compound 005 (10.7mg, 13.7%).

[0959] MSm / z(ESI):1271.4(M+1).

[0960] 1 H NMR(400MHz,DMSO-d6)δ9.76(s,1H),8.98(d,1H),8.72(d,1H),8.27(d,1H),7.99–7.90(m,3H),7.83(d,1H),7. 31(s,1H),6.51(s,1H),5.44(s,2H),5.27(s,2H),4.55–4.52(m,1H),4.27–4.24(m,1H),3.61–3.58(m,2H),3.50 (s,28H),3.45(s,3H),3.42–3.39(m,4H),3.23–3.21(m,2H),3.18-3.16(m,2H)2.99–2.97(m,2H),2.42-2.33(m, 2H),2.29–2.25(m,2H),2.02–1.97(m,3H),1.91–1.84(m,2H),1.82-1.76(m,2H)1.40(d,3H),0.91–0.83(m,9H).

[0961] Preparation Example 16

[0962] 1-(6-(5-cyano-6-(methanesulfonyl)pyridin-3-yl)hexyl-5-ynamide)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-3,6,9,12,15,18,21,24,27,30-decaoxane-33-amide (006)

[0963]

[0964] Following the synthetic route of Preparation Example 13, the starting material in the first step was replaced with Int-2. After the reaction, the reaction solution was directly purified by high performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector; column: Ultimate C18, 21.2*250mm, 5μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 21%-46%, flow rate: 20mL / min) to obtain compound 006 (11.2mg, yield: 18%).

[0965] MSm / z(ESI):1360.5(M+1).

[0966] 1 H NMR (400MHz, CD3OD) δ8.85(d,1H),8.47(d,1H),7.97(d,1H),7.82(d,J=9.2Hz,1H),7. 66(s,1H),5.62(d,1H),5.42(d,1H),5.26(s,2H),4.72–4.58(m,2H),4.27(d,1H),3.77 -3.74(m,2H),3.63 -3.61(m,38H)3.56-3.53(m,2H),3.39-3.33(m,4H),3.23-3.20(m,2H),3.08-3.04(m,2H),2.62–2.55( m,4H),2.43-2.39(m,2H),2.21–2.12(m,3H),2.03–1.92(m,4H),1.58-1.56(m,3H),1.05-1.00(m,9H).

[0967] Preparation Example 17

[0968] N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropyl-2-yl)amino-3-methyl-1-oxobut-2-yl)-1-(6-(4-(methoxymethyl)-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-ynamide)-3,6,9,12,15,18-hexaoxane-amide(007)

[0969]

[0970] Following the synthetic route of Preparation Example 11, the starting material in the first step was replaced with Int-3. The crude product was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: Ultimate C18, 21.2×250 mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to obtain compound 007 (32.0 mg, 26%).

[0971] MSm / z(ESI): 602.5(M / 2+1).

[0972] 1 H NMR(400MHz,DMSO-d6)δ9.75(s,1H),9.04(s,1H),8.27(d,1H),7.97–7.90(m,3H),7.82(d,1H),7 .30(s,1H),6.50(s,1H),5.43(s,2H),5.26(s,2H),4.70(s,2H),4.53(t,1H),4.25(dd,1H),3.59( t,2H),3.49–3.47(m,20H),3.41–3.38(m,8H),3.22–3.14(m,4H),2.97(t,2H),2.57(t,2H),2.46 –2.35(m,2H),2.27(t,2H),2.06–1.96(m,3H),1.91–1.78(m,4H),1.39(d,3H),0.90–0.84(m,9H).

[0973] Preparation Example 18

[0974] N-((S)-1-((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-1-(6-(4-(methoxymethyl)-2-(methanesulfonyl)pyrimidin-5-yl)hexadecyl)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide(008)

[0975]

[0976] Following the synthetic route of Preparation Example 12, the starting material in the first step was replaced with Int-3. After the reaction was completed, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector; column: Ultimate C18, 21.2*250mm, 5μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20mL / min) to obtain compound 008 (18.1mg, yield: 21%).

[0977] MSm / z(ESI): 646.4(M / 2+1).

[0978] 1 H NMR (400MHz, DMSO) δ9.76(s,1H),9.05(s,1H),8.28(d,1H),7.97–7.91(m,3H),7.83(d,1H),7.31(s ,1H),6.51(s,1H),5.43(s,2H),5.26(s,2H),4.71(s,2H),4.57–4.50(m,1H),4.28–4.24(m,1H),3. 60(t,2H),3.50–3.48(m,28H),3.42–3.39(m,8H),3.23–3.14(m,4H),2.98(t,2H),2.58(t,2H),2.4 9–2.36(m,2H),2.28(t,2H),2.08–1.96(m,3H),1.93–1.79(m,4H),1.40(d,3H),0.91–0.85(m,9H).

[0979] Preparation Example 19

[0980] N-(((S)-1-(((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropyl-2-yl)amino-3-methyl-1-oxobut-2-yl)-1-(6-(4-(methoxymethyl)-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-ynamide)-3,6,9,12,15,18,21,24,27,30-decaoxane-33-amide(009)

[0981]

[0982] Following the synthetic route of Preparation Example 13, the starting material in the first step was replaced with Int-3. The crude product was purified by high performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector; column: Ultimate 5μm C18 250×21.2mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-48%, flow rate: 20mL / min) to obtain compound 009 (23.2mg, 19.8%).

[0983] MSm / z(ESI): 690.4(M / 2+1).

[0984] 1 H NMR(400MHz,DMSO-d6)δ9.78(s,1H),9.07(s,1H),8.29(d,1H),7.99–7.83(m,4H),7.33(s,1H),6.53(s,1 H),5.45(s,2H),5.29(s,2H),4.72(s,2H),4.57–4.53(m,1H),4.29–4.25(m,1H),3.61(t,2H),3.52–3.50( m,34H),3.43–3.42(m,7H),3.21–3.18(m,4H),3.01–2.98(m,2H),2.70–2.68(s,1H),2.62–2.56(m,3H),2. 36–2.27(m,3H),2.08–1.98(m,3H),1.91–1.82(m,4H),1.41(d,3H),1.27–1.24(m,2H),0.92–0.86(m,9H).

[0985] Compound 28 obtained in the above preparation examples is also referred to as PL-066 or PL066, compound 41 is also referred to as PL-096 or PL096, compound 001 is also referred to as LP-1 or LP1, compound 002 is also referred to as LP-2 or LP2, compound 003 is also referred to as LP-3 or LP3, compound 004 is also referred to as LP-4 or LP4, compound 005 is also referred to as LP-5 or LP5, and compound 006 is also referred to as LP-6 or LP6.

[0986] Example 5: B7-H3×PSMA dual antibody ADC conjugation

[0987] 1. Preparation method of antibody-drug conjugates

[0988] 1.1 Preparation method of antibody-drug conjugate PO4-DXD

[0989] P04-DXD was obtained by conjugating the B7-H3 monoclonal antibody P04 with Deruxtecan, and its molecular structure is as follows:

[0990]

[0991] Transfer 35 mg of P04 antibody to a new 50 mL centrifuge tube. Add 30 mM histidine / acetic acid buffer (30 mM HIS / HAc) pH 5.5 to a final antibody concentration of 5 mg / mL. Add 0.1 M EDTA solution to a final EDTA concentration of 5 mM. Add 2.8 molar amounts of tris(2-carboxyethyl)phosphine (TCEP). Incubate at 37 °C for 2.5 h, continuously mixing. Under ice bath conditions, add Deruxtecan (the linker-payload portion of DS-8201) solution prepared with N,N-dimethylacetamide (DMA) at a final drug-to-antibody molar ratio of 10:1. Add DMA at 10% of the total reaction volume. Shake to mix and incubate at 4 °C for 1 h, continuously mixing. The conjugate was desalted and purified using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with dextran-coated charcoal (Sigma, C6241) for 1 h, the dextran-coated charcoal was removed by filtration. The solution was concentrated using an Amicon ultrafiltration centrifuge tube (50 kDa, 15 mL) to a storage solution of 30 mM His / HAc pH 5.5, yielding the antibody-drug conjugate PO4-DXD. The concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[0992] 1.2 Preparation method of antibody-drug conjugate P13-DXD

[0993] Transfer 50 mg of P13 antibody to a new 50 mL centrifuge tube, add 30 mM His / HAc pH 5.5 to achieve an antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 37 °C for 2 h, and continuously mix. Under ice bath conditions, add Deruxtecan DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 1 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 KMWCO). After co-incubating the conjugate with Dextran coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran coated charcoal. The antibody-drug conjugate P13-DXD was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc solution at pH 5.5. The concentration, drug / antibody ratio, purity, and free small molecule drug content were measured and are shown in Table 4-1. 1.3 Preparation method of antibody-drug conjugate CO40-DXD

[0994] Transfer 45 mg of C040 antibody to a new 50 mL centrifuge tube, add 50 mM phosphate-buffered saline (PBS) pH 7.4 to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 12 times the molar amount of TCEP, incubate at 37 °C for 2 h, and continuously mix. Under ice bath conditions, add DMA-prepared Deruxtecan solution at a final drug-to-antibody molar ratio of 10:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 1 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C040-DXD was obtained by concentrating the drug using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM histidine / acetic acid buffer (30 mM His / HAc pH 5.5). The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[0995] 1.4 Preparation method of antibody-drug conjugate C097-DXD

[0996] Transfer 40 mg of C097 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 18 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add Deruxtecan DMA solution at a final drug-to-antibody molar ratio of 20:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 1 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 KMWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C097-DXD was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc solution at pH 5.5. The concentration, drug / antibody ratio, purity, and free small molecule drug content were measured and are shown in Table 4-1. 1.5 Preparation method of antibody-drug conjugate C101-DXD

[0997] Transfer 40 mg of C101 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 20 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add Deruxtecan DMA solution at a final drug-to-antibody molar ratio of 23:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 1 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 KMWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C101-DXD was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the medium with a 30 mM His / HAc solution at pH 5.5. The concentration, drug / antibody ratio, purity, and free small molecule drug content were measured and are shown in Table 4-1. 1.6 Preparation method of antibody-drug conjugate C116-DXD

[0998] Transfer 15 mg of C116 antibody to a new 50 mL centrifuge tube, add 30 mM His / HAc solution (pH 5.5) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add Deruxtecan DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran coated charcoal. The antibody-drug conjugate C116-DXD was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc solution at pH 5.5. The concentration, drug / antibody ratio, purity, and free small molecule drug content were measured and are shown in Table 4-1. 1.7 Preparation method of antibody-drug conjugate C040-PL066

[0999] Transfer 15 mg of C040 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.97) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-066DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, incubate at 22 °C for 3 h, then at 4 °C for 17 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C040-PL066 was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1000] 1.8 Preparation method of antibody-drug conjugate C040-PL096

[1001] Transfer 15 mg of C040 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.97) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-096DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, incubate at 22 °C for 3 h, then at 4 °C for 17 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C040-PL096 was obtained by concentrating the solution in Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1002] 1.9 Preparation method of antibody-drug conjugate C101-PL066

[1003] Transfer 20 mg of C101 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 20 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-066DMA solution at a final drug-to-antibody molar ratio of 20:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C101-PL066 was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1004] 1.10 Preparation method of antibody-drug conjugate C101-PL096

[1005] Transfer 10 mg of C101 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 20 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-096DMA solution at a final drug-to-antibody molar ratio of 20:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C101-PL096 was obtained by concentrating the solution in Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1006] 1.11 Preparation method of antibody-drug conjugate C097-PL066

[1007] Transfer 20 mg of C097 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-066DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C097-PL066 was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1008] 1.12 Preparation method of antibody-drug conjugate C097-PL096

[1009] Transfer 10 mg of C097 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-096DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C097-PL096 was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc solution at pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1010] 1.13 Preparation method of antibody-drug conjugate C115-PL066

[1011] Transfer 40 mg of C115 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-066DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C115-PL066 was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1012] 1.14 Preparation method of antibody-drug conjugate C115-PL096

[1013] Transfer 40 mg of C115 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-096DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C115-PL096 was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1014] 1.15 Preparation method of antibody-drug conjugate C116-PL066

[1015] Transfer 40 mg of C116 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-066DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C116-PL066 was obtained by concentrating the solution in Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1016] 1.16 Preparation method of antibody-drug conjugate C116-PL096

[1017] Transfer 40 mg of C116 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add PL-096DMA solution at a final drug-to-antibody molar ratio of 15:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C116-PL096 was obtained by concentrating the solution using Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1018] 1.17 Preparation method of antibody-drug conjugate C123-LP-2

[1019] Transfer 30 mg of C123 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 18 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add LP-2DMA solution at a final drug-to-antibody molar ratio of 23:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 K MWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C123-LP-2 was obtained by concentrating the solution in Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1020] 1.18 Preparation method of antibody-drug conjugate C123(LALA)-LP-2

[1021] Transfer 30 mg of C123 (LALA) antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to a final antibody concentration of 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 18 times the molar amount of TCEP, incubate at 22 °C for 3 h, and continuously mix. Under ice bath conditions, add LP-2DMA solution at a final drug-to-antibody molar ratio of 23:1, supplement with DMA at 10% of the total reaction volume, vortex to mix, and incubate at 4 °C for 20 h, continuously mixing. Desalt and purify the conjugate using a 10 mL desalting column (40 KMWCO). After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C123(LALA)-LP-2 was obtained by concentrating the solution in Amicon ultrafiltration centrifuge tubes (50 kDa, 15 mL) and replacing the solution with a 30 mM His / HAc pH 5.5 solution. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-1.

[1022] 1.19 Preparation method of antibody-drug conjugate C101-LP-1

[1023] Transfer 16 mg of C101 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 20 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-1DMA solution at a final drug-to-antibody molar ratio of 20:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C101-LP-1 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-2.

[1024] 1.20 Preparation method of antibody-drug conjugate C101-LP-2

[1025] Transfer 16 mg of C101 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 20 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-2DMA solution at a final drug-to-antibody molar ratio of 20:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C101-LP-2 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-2.

[1026] 1.21 Preparation method of antibody-drug conjugate C101-LP-3

[1027] Transfer 16 mg of C101 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 20 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-3DMA solution at a final drug-to-antibody molar ratio of 20:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C101-LP-3 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-2.

[1028] 1.22 Preparation method of antibody-drug conjugate C101-LP-4

[1029] Transfer 16 mg of C101 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 20 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-4DMA solution at a final drug-to-antibody molar ratio of 20:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C101-LP-4 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-2.

[1030] 1.23 Preparation method of antibody-drug conjugate C101-LP-5

[1031] Transfer 16 mg of C101 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 20 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-5DMA solution at a final drug-to-antibody molar ratio of 20:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C101-LP-5 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-2.

[1032] 1.24 Preparation method of antibody-drug conjugate C101-LP-6

[1033] Transfer 16 mg of C101 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 20 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-6DMA solution at a final drug-to-antibody molar ratio of 20:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C101-LP-6 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-2.

[1034] 1.25 Preparation method of antibody-drug conjugate C116-LP-1

[1035] Transfer 16 mg of C116 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-1DMA solution at a final drug-to-antibody molar ratio of 14:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C116-LP-1 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-2.

[1036] 1.26 Preparation method of antibody-drug conjugate C116-LP-2

[1037] Transfer 16 mg of C116 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-2DMA solution at a final drug-to-antibody molar ratio of 14:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C116-LP-2 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-2.

[1038] 1.27 Preparation method of antibody-drug conjugate C116-LP-3

[1039] Transfer 16 mg of C116 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-3DMA solution at a final drug-to-antibody molar ratio of 14:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C116-LP-3 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. The results of its concentration, drug / antibody ratio, purity, and free small molecule drug content are shown in Table 4-2.

[1040] 1.28 Preparation method of antibody-drug conjugate C116-LP-4

[1041] Transfer 16 mg of C116 antibody to a new 50 mL centrifuge tube, add 50 mM PBS (pH 7.4) to adjust the antibody concentration to 5 mg / mL, add 0.1 M EDTA solution to a final EDTA concentration of 5 mM, add 15 times the molar amount of TCEP, incubate at 22 °C for 3 h, and mix continuously. Under ice bath conditions, add LP-4DMA solution at a final drug-to-antibody molar ratio of 14:1, supplement with DMA at 10% of the total reaction volume, and then add 100 mM Tris to adjust the pH to 8.0. Shake to mix, incubate at 4 °C for 18 h, and mix continuously. After co-incubating the conjugate with Dextran-coated charcoal (Sigma, C6241) for 1 h, filter to remove the Dextran-coated charcoal. The antibody-drug conjugate C116-LP-4 was obtained by concentrating the solution in Amicon (50 kDa, 15 mL) ultrafiltration centrifuge tubes and replacing the solution with 20 mL M Histidine-HCl, 8% Sucrose, pH 5.5. Th...

Claims

1. Antibody-drug conjugates comprising: a bispecific antibody that specifically binds to B7-H3 and PSMA or an antigen-binding fragment thereof, and a bioactive molecule linked thereto.

2. The antibody-drug conjugate of claim 1, having the structure shown in Formula I, Ab-[LD] β (type I) in, Ab is a bispecific antibody that specifically binds to B7-H3 and PSMA or its antigen-binding fragment; D is a structural fragment of a bioactive molecule; L is a linker connecting Ab and D; and β is an integer or decimal selected from 1 to 10.

3. The antibody-drug conjugate of claim 2, wherein, D is selected from the structural fragment of a compound represented by Formula II or Formula III, its racemate, stereoisomer, isotopic label, or pharmaceutically acceptable salt after dehydrogenation, wherein: (1) The structure of the compound represented by formula II is shown below: Among them, R1 and R2 may be the same or different, and are independently selected from H, OH, CN, halogens, and C. 1-10 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Alkoxy, halogenated C 1-10 Alkyl, Halogenated C 1-10 Alkoxy, cyano C 1-10 Alkyl, cyano C 1-10 Alkoxy, C 3-10 cycloalkyl; Y1 is selected from O or CH2; R 51 R 52 They are either the same or different, and are independently selected from H and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkyl-NH-, (C 1-6 Alkyl)2N-, C 1-6 Alkyl-NH-C 1-6 Alkyl, (C 1-6 Alkyl)2N-C 1-6 Alkyl, C 1-6 Alkoxyalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-14 Aryl and 5-14 heteroaryl groups; n is selected from 0, 1, or 2; (2) The structure of the compound represented by Formula III is shown below:

4. The antibody-drug conjugate of claim 3, wherein, R1 is selected from H, OH, CN, halogen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl or halogenated C 1-6 Alkoxy; Preferably, R1 is selected from H, OH, Br, methyl, difluoromethoxy, 2,2,2-trifluoroethoxy, vinyl, cyclopropyl or ethynyl; Preferably, R1 is selected from methyl or cyclopropyl; Preferably, R2 is selected from H, halogen, CN, or C. 1-6 alkyl; Preferably, R2 is selected from H or F; Preferably, R 51 Selected from H, methyl, ethyl, isopropyl, or cyclopropyl; Preferably, R 52 Selected from H or methyl.

5. The antibody-drug conjugate according to claim 3 or 4, wherein, The structure of the compound represented by Formula II is shown below: Among them, R1, R2, R 51 R 52 Y1 and n have the definitions described in claim 3 or 4.

6. The antibody-drug conjugate according to any one of claims 3-5, wherein, The structure of the compound of formula II is shown below:

7. The antibody-drug conjugate according to any one of claims 2-6, wherein, The structure of D is shown below:

8. The antibody-drug conjugate according to any one of claims 2-7, wherein, L is selected from the connector shown in Formula IV or Formula V: Where Z is N or CR 22 R 21 R 22 and R 23 Each is independently selected from H, halogen, cyano, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, 3-8 membered heterocyclic groups, C 1-6 Alkyl-OC 1-6 Alkylene; condition is that when Z is N, R 21 R 23 Not both H; Preferably, n1 is selected from an integer from 1 to 36; more preferably, n1 is selected from an integer from 3 to 12, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; most preferably, n1 is selected from an integer from 4 to 10. Preferably, Z is N or C-CN; Preferably, R 21 Selected from H, halogen, cyano, C 1-6 Alkoxy or C 1-6 Alkyl-OC 1-6 Alkylene; Preferably, Z is N, R 21 Selected from halogen, cyano, C 1-3 Alkoxy or C 1-3 Alkyl-OC 1-3 Alkylene; Preferably, Z is N, R 21 Selected from methoxy or CH3-O-CH2-; Preferably, Z is C-CN, R 21 Selected from H; Preferably, R 23 Selected from H; Preferably, bit 1 is connected to Ab, and bit 2 is connected to D.

9. The antibody-drug conjugate according to any one of claims 2-8, wherein, L is selected from: Among them, bit 1 is connected to Ab, and bit 2 is connected to D.

10. The antibody-drug conjugate according to any one of claims 2-9, wherein, LD is selected from:

11. The antibody-drug conjugate according to any one of claims 1-10, wherein, The bispecific antibody or its antigen-binding fragment that specifically binds to B7-H3 and PSMA comprises a first antigen-binding domain that specifically binds to one of B7-H3 and PSMA and a second antigen-binding domain that specifically binds to the other of B7-H3 and PSMA.

12. The antibody-drug conjugate of claim 11, wherein, The antigen-binding domains that specifically bind to B7-H3 include: (a) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:45 or 11; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:49 or 12; (b) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46 or 13; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:50 or 14; (c) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:47 or 15; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:51 or 16; (d) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof, and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:48 or 17; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof, and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:52 or 18; or, (e) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62 or 53; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:63 or 54; The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions); preferably, the substitutions are conservative substitutions. Preferably, the antigen-binding domain that specifically binds to B7-H3 includes: (a) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:45 or 11; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:49 or 12. (b) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46 or 13; and / or, three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:50 or 14; or, (c) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:47 or 15; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:51 or 16. (d) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:48 or 17; and / or, three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:52 or 18; or, (e) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62 or 53; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:63 or 54; Preferably, the antigen-binding domain that specifically binds to B7-H3 includes: (1) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:19, the heavy chain CDR2 shown in SEQ ID NO:108, and the heavy chain CDR3 shown in SEQ ID NO:21, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:22, the light chain CDR2 shown in SEQ ID NO:23, and the light chain CDR3 shown in SEQ ID NO:24; (2) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:25, the heavy chain CDR2 shown in SEQ ID NO:109, and the heavy chain CDR3 shown in SEQ ID NO:27, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:28, the light chain CDR2 shown in SEQ ID NO:29, and the light chain CDR3 shown in SEQ ID NO:30; (3) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:31, the heavy chain CDR2 shown in SEQ ID NO:110, and the heavy chain CDR3 shown in SEQ ID NO:33, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:34, the light chain CDR2 shown in SEQ ID NO:35, and the light chain CDR3 shown in SEQ ID NO:36; (4) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:37, the heavy chain CDR2 shown in SEQ ID NO:111, and the heavy chain CDR3 shown in SEQ ID NO:39, and / or, a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:40, the light chain CDR2 shown in SEQ ID NO:41, and the light chain CDR3 shown in SEQ ID NO:42; or (5) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:56, the heavy chain CDR2 shown in SEQ ID NO:112, and the heavy chain CDR3 shown in SEQ ID NO:58, and / or a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:59, the light chain CDR2 shown in SEQ ID NO:60, and the light chain CDR3 shown in SEQ ID NO:61; Preferably, the antigen-binding domain that specifically binds to B7-H3 includes: (1a) VH containing the sequence shown in SEQ ID NO:45 or a variant thereof and VL containing the sequence shown in SEQ ID NO:49 or a variant thereof; (1b) VH containing the sequence shown in SEQ ID NO:11 or a variant thereof and VL containing the sequence shown in SEQ ID NO:12 or a variant thereof; (2a) VH containing the sequence shown in SEQ ID NO:46 or a variant thereof and VL containing the sequence shown in SEQ ID NO:50 or a variant thereof; (2b) VH containing the sequence shown in SEQ ID NO:13 or a variant thereof and VL containing the sequence shown in SEQ ID NO:14 or a variant thereof; (3a) VH containing the sequence shown in SEQ ID NO:47 or a variant thereof and VL containing the sequence shown in SEQ ID NO:51 or a variant thereof; (3b) VH containing the sequence shown in SEQ ID NO:15 or a variant thereof and VL containing the sequence shown in SEQ ID NO:16 or a variant thereof; (4a) VH containing the sequence shown in SEQ ID NO:48 or a variant thereof and VL containing the sequence shown in SEQ ID NO:52 or a variant thereof; (4b) VH containing the sequence shown in SEQ ID NO:17 or a variant thereof and VL containing the sequence shown in SEQ ID NO:18 or a variant thereof; (5a) A VH comprising the sequence shown in SEQ ID NO:62 or a variant thereof and a VL comprising the sequence shown in SEQ ID NO:63 or a variant thereof; or (5b) VH containing the sequence shown in SEQ ID NO:53 or a variant thereof and VL containing the sequence shown in SEQ ID NO:54 or a variant thereof; The variant has 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 with the sequence from which it originates, or 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 original sequence; preferably, the substitution is a conservative substitution.

13. The antibody-drug conjugate of claim 11 or 12, wherein, The antigen-binding domains that specifically bind to PSMA include: heavy chain CDR1 or variants thereof, heavy chain CDR2 or variants thereof, and heavy chain CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:64; and / or, light chain CDR1 or variants thereof, light chain CDR2 or variants thereof, and light chain CDR3 or variants thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:65; The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions); preferably, the substitutions are conservative substitutions. Preferably, the antigen-binding domain that specifically binds to PSMA includes: three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:64; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:65; Preferably, the antigen-binding domain that specifically binds to PSMA includes: a heavy chain variable region (VH) comprising heavy chain CDR1 shown in SEQ ID NO:98, heavy chain CDR2 shown in SEQ ID NO:113, and heavy chain CDR3 shown in SEQ ID NO:100, and / or a light chain variable region (VL) comprising light chain CDR1 shown in SEQ ID NO:101, light chain CDR2 shown in SEQ ID NO:102, and light chain CDR3 shown in SEQ ID NO:103; Preferably, the antigen-binding domain that specifically binds to PSMA comprises: a VH containing the sequence shown in SEQ ID NO:64 or a variant thereof, and a VL containing the sequence shown in SEQ ID NO:65 or a variant thereof; wherein the variant has 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 with the sequence from which it originates, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions); preferably, the substitution is a conserved substitution.

14. The antibody-drug conjugate according to any one of claims 11-13, wherein, One of the first antigen-binding domain and the second antigen-binding domain is Fab or sdAb / VHH, and the other is scFv or Fab or sdAb / VHH. Preferably, the bispecific antibody or its antigen-binding fragment further comprises an Fc domain, the Fc domain comprising a first Fc domain monomer and a second Fc domain monomer, wherein the first antigen-binding domain and the second antigen-binding domain are optionally linked to the N-terminus of the first Fc domain monomer and the second Fc domain monomer via a first peptide linker.

15. The antibody-drug conjugate of claim 14, wherein, The first antigen-binding domain is Fab, and the second antigen-binding domain is Fab containing crossMab-type domain exchanges. Preferably, the CrossMab-style domain swapping is selected from: (a) CrossMab Fab: In the Fab, VL and VH are interchanged, and CH1 and CL are interchanged; (b) CrossMab VH-VL: In the Fab, VL and VH are interchanged; or (c)CrossMab CH1-CL: In the Fab, CH1 and CL are interchanged.

16. The antibody-drug conjugate of claim 15, wherein, The bispecific antibody or its antigen-binding fragment comprises: (i) Peptide chain IA, which includes the VL of the first antigen-binding domain and the light chain constant region (CL); (ii) A peptide chain IB, comprising the VH of the first antigen-binding domain, the CH1 region of the heavy chain, and a monomer of the first Fc domain. (iii) A peptide chain IC comprising the VH of the second antigen-binding domain, a light chain constant region (CL), and a second Fc domain monomer. (iv) Peptide chain ID, which includes the VL region of the second antigen-binding domain and the CH1 region of the heavy chain; Preferably, the first Fc domain monomer and the second Fc domain monomer form a dimer.

17. The antibody-drug conjugate of claim 15 or 16, wherein, The first antigen-binding domain is selected from the antigen-binding domain that specifically binds to B7-H3 as defined in claim 12, and the second antigen-binding domain is selected from the antigen-binding domain that specifically binds to PSMA as defined in claim 13. Preferably, the first antigen-binding domain comprises: (1) VH containing the sequence shown in SEQ ID NO:45 and VL containing the sequence shown in SEQ ID NO:49; (2) VH containing the sequence shown in SEQ ID NO:46 and VL containing the sequence shown in SEQ ID NO:50; (3) VH containing the sequence shown in SEQ ID NO:47 and VL containing the sequence shown in SEQ ID NO:51; (4) VH containing the sequence shown in SEQ ID NO:48 and VL containing the sequence shown in SEQ ID NO:52; (5) VH containing the sequence shown in SEQ ID NO:62 and VL containing the sequence shown in SEQ ID NO:63; or (6) VH containing the sequence shown in SEQ ID NO:53 and VL containing the sequence shown in SEQ ID NO:54; Preferably, the second antigen-binding domain comprises: a VH containing the sequence shown in SEQ ID NO:64 and a VL containing the sequence shown in SEQ ID NO:

65.

18. The antibody-drug conjugate according to any one of claims 15-17, wherein, The first and second Fc domain monomers are wild-type IgG Fc domains or each independently contains one or more amino acids, and the modifications can promote the dimerization of the first and second Fc domain monomers. Preferably, the first and second Fc domain monomers may or may not contain disulfide bonds; Preferably, the dimerization-promoting modification includes a "knob" modification in one of the two Fc domain monomers and a "hole" modification in the other of the two Fc domain monomers to form a "knobs-into-holes" modification. Preferably, the "knob" modification is selected from the following mutations in the EU number: T366W, T366W / S354C or T366W / S354C / K409A, for example T366W or T366W / S354C; Preferably, the "hole" modification is selected from the following mutations in the EU number: T366S / L368A / Y407V, T366S / L368A / Y407V / Y349C or T366S / L368A / Y407V / Y349C / F405K, for example T366S / L368A / Y407V or T366S / L368A / Y407V / Y349C.

19. The antibody-drug conjugate of claim 18, wherein, The first and second Fc domain monomers respectively comprise sequences selected from the following sequences: SEQ ID NO:97 and 97, SEQ ID NO:94 and 78, SEQ ID NO:78 and 94.

20. The antibody-drug conjugate according to any one of claims 15-19, wherein, The peptide chain ID further includes a flexible amino acid sequence between the VL region of the second antigen-binding domain and the CH1 region of the heavy chain. Preferably, the flexible amino acid sequence consists of one or more glycine (G) and / or serine (S); Preferably, the flexible amino acid sequence is SS.

21. The antibody-drug conjugate according to any one of claims 15-20, wherein, The CL of the peptide chain IC contains the following mutation with EU number: R108A / T109S.

22. The antibody-drug conjugate according to any one of claims 15-21, wherein, The bispecific antibody or its antigen-binding fragment comprises: (1) Peptide chain IA containing the sequence shown in SEQ ID NO:55, peptide chain IB containing the sequence shown in SEQ ID NO:104, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:

81. (2) Peptide chain IA containing the sequence shown in SEQ ID NO:66, peptide chain IB containing the sequence shown in SEQ ID NO:72, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:

81. (3) Peptide chain IA containing the sequence shown in SEQ ID NO:67, peptide chain IB containing the sequence shown in SEQ ID NO:73, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:

81. (4) Peptide chain IA containing the sequence shown in SEQ ID NO:68, peptide chain IB containing the sequence shown in SEQ ID NO:74, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:

81. (5) Peptide chain IA containing the sequence shown in SEQ ID NO:69, peptide chain IB containing the sequence shown in SEQ ID NO:75, peptide chain IC containing the sequence shown in SEQ ID NO:79, peptide chain ID containing the sequence shown in SEQ ID NO:81; or, (6) Peptide chain IA containing the sequence shown in SEQ ID NO:70, peptide chain IB containing the sequence shown in SEQ ID NO:76, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:

81.

23. The antibody-drug conjugate of claim 14, wherein, The first antigen-binding domain is Fab, the second antigen-binding domain is scFv, and the bispecific antibody or its antigen-binding fragment comprises: (i) Peptide chain II-A, which includes the VL of the first antigen-binding domain and the light chain constant region (CL); (ii) Peptide chain II-B, which includes the VH of the first antigen-binding domain, the CH1 region of the heavy chain, and a monomer of the first Fc domain. and (iii) Peptide chain II-C, which includes the second antigen-binding domain and the second Fc domain monomer; Preferably, the second Fc domain monomer forms a dimer with the first Fc domain monomer; Preferably, the scFv has a structure shown as [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a second peptide linker; Preferably, the first peptide linker and the second peptide linker are each independently the same or different peptide linkers; Preferably, the first peptide linker and the second peptide linker are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), such as flexible peptides containing (G4S)n, where n is an integer not less than 0, such as 1, 2, 3 or 4. Preferably, the VH and VL of the scFv may or may not contain disulfide bonds.

24. The antibody-drug conjugate of claim 23, wherein, The first antigen-binding domain is selected from the antigen-binding domain that specifically binds PSMA as defined in claim 13, and the second antigen-binding domain is selected from the antigen-binding domain that specifically binds B7-H3 as defined in claim 12. Preferably, the first antigen-binding domain comprises: a VH containing the sequence shown in SEQ ID NO:64 and a VL containing the sequence shown in SEQ ID NO:65; Preferably, the second antigen-binding domain comprises: a VH containing the sequence shown in SEQ ID NO:47 and a VL containing the sequence shown in SEQ ID NO:

51.

25. The antibody-drug conjugate of claim 23 or 24, wherein, The first and second Fc domain monomers are wild-type IgG Fc domains or each independently contains one or more amino acids, and the modifications can promote the dimerization of the first and second Fc domain monomers. Preferably, the first and second Fc domain monomers may or may not contain disulfide bonds; Preferably, the dimerization-promoting modification includes a "knob" modification in one of the two Fc domain monomers and a "hole" modification in the other of the two Fc domain monomers to form a "knobs-into-holes" modification. Preferably, the "knob" modification is selected from the following mutations in the EU number: T366W, T366W / S354C or T366W / S354C / K409A, for example T366W or T366W / S354C; Preferably, the "hole" modification is selected from the following mutations in the EU number: T366S / L368A / Y407V, T366S / L368A / Y407V / Y349C or T366S / L368A / Y407V / Y349C / F405K, for example T366S / L368A / Y407V or T366S / L368A / Y407V / Y349C.

26. The antibody-drug conjugate according to any one of claims 23-25, wherein, The second Fc domain monomer contains the following mutation with EU number: C220S.

27. The antibody-drug conjugate according to any one of claims 23-26, wherein, The first and second Fc domain monomers each independently contain modifications that can reduce or eliminate effector functions (e.g., ADCC activity); Preferably, the modification is selected from the following mutations in the EU number: L234A and / or L235A.

28. The antibody-drug conjugate according to any one of claims 23-27, wherein, The first and second Fc domain monomers respectively comprise sequences selected from the following sequences: SEQ ID NO:97 and 97, SEQ ID NO:95 and 88, SEQ ID NO:88 and 95, SEQ ID NO:96 and 92, SEQ ID NO:92 and 96.

29. The antibody-drug conjugate according to any one of claims 23-28, wherein, The bispecific antibody or its antigen-binding fragment comprises: (1) Peptide chain II-A containing the sequence shown in SEQ ID NO:84, peptide chain II-B containing the sequence shown in SEQ ID NO:86, and peptide chain II-C containing the sequence shown in SEQ ID NO:89; or (2) Peptide chain II-A containing the sequence shown in SEQ ID NO:84, peptide chain II-B containing the sequence shown in SEQ ID NO:91, and peptide chain II-C containing the sequence shown in SEQ ID NO:

93.

30. Antibody-drug conjugates, selected from: in: C040 is a bispecific antibody comprising the following peptide chains: peptide chain IA comprising the sequence shown in SEQ ID NO:55, peptide chain IB comprising the sequence shown in SEQ ID NO:104, peptide chain IC comprising the sequence shown in SEQ ID NO:79, and peptide chain ID comprising the sequence shown in SEQ ID NO:

81. C097 is a bispecific antibody containing the following peptide chains: peptide chain IA containing the sequence shown in SEQ ID NO:67, peptide chain IB containing the sequence shown in SEQ ID NO:73, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:

81. C101 is a bispecific antibody containing the following peptide chains: peptide chain IA containing the sequence shown in SEQ ID NO:68, peptide chain IB containing the sequence shown in SEQ ID NO:74, peptide chain IC containing the sequence shown in SEQ ID NO:79, and peptide chain ID containing the sequence shown in SEQ ID NO:

81. C115 is a bispecific antibody comprising the following peptide chains: peptide chain IA comprising the sequence shown in SEQ ID NO:69, peptide chain IB comprising the sequence shown in SEQ ID NO:75, peptide chain IC comprising the sequence shown in SEQ ID NO:79, and peptide chain ID comprising the sequence shown in SEQ ID NO:

81. C116 is a bispecific antibody comprising the following peptide chains: peptide chain IA comprising the sequence shown in SEQ ID NO:70, peptide chain IB comprising the sequence shown in SEQ ID NO:76, peptide chain IC comprising the sequence shown in SEQ ID NO:79, and peptide chain ID comprising the sequence shown in SEQ ID NO:

81. C123 is a bispecific antibody containing the following peptide chains: peptide chain II-A containing the sequence shown in SEQ ID NO:84, peptide chain II-B containing the sequence shown in SEQ ID NO:86, and peptide chain II-C containing the sequence shown in SEQ ID NO:

89. C123(LALA) is a bispecific antibody containing the following peptide chains: peptide chain II-A containing the sequence shown in SEQ ID NO:84, peptide chain II-B containing the sequence shown in SEQ ID NO:91, and peptide chain II-C containing the sequence shown in SEQ ID NO:

93.

31. A composition comprising one or more antibody-drug conjugates as described in any one of claims 1-30; Preferably, the DAR value (drug-antibody conjugate ratio) of the composition is 1-10; Preferably, the composition has a DAR value of 4-9, for example 5-9. Preferably, the composition has a DAR value of 5.5-8.5, for example 7.0-8.5 or 5.5-6.

0.

32. A bispecific antibody or antigen-binding fragment thereof that specifically binds to B7-H3 and PSMA, comprising a first antigen-binding domain that specifically binds to one of B7-H3 and PSMA and a second antigen-binding domain that specifically binds to the other of B7-H3 and PSMA. Preferably, the bispecific antibody or its antigen-binding fragment is defined as in any one of claims 11-29.

33. An antibody or antigen-binding fragment thereof that specifically binds to B7-H3, comprising: (a) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:45 or 11; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:49 or 12; (b) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46 or 13; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:50 or 14; (c) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:47 or 15; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:51 or 16; (d) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof, and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:48 or 17; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof, and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:52 or 18; or, (e) Heavy chain CDR1 or a variant thereof, heavy chain CDR2 or a variant thereof and heavy chain CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62 or 53; and / or, light chain CDR1 or a variant thereof, light chain CDR2 or a variant thereof and light chain CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:63 or 54; in, The variant has one or more amino acid substitutions, deletions, or additions compared to its source sequence (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, such as conservative substitutions); preferably, the substitutions are conservative substitutions. Preferably, the antibody or its antigen-binding fragment comprises: (a) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:45 or 11; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:49 or 12. (b) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46 or 13; and / or, three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:50 or 14; or, (c) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:47 or 15; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:51 or 16. (d) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:48 or 17; and / or, three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:52 or 18; or, (e) Three heavy chain CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62 or 53; and / or three light chain CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:63 or 54.

34. The antibody or antigen-binding fragment thereof according to claim 33, comprising: (1) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:19, the heavy chain CDR2 shown in SEQ ID NO:108, and the heavy chain CDR3 shown in SEQ ID NO:21, and a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:22, the light chain CDR2 shown in SEQ ID NO:23, and the light chain CDR3 shown in SEQ ID NO:24; (2) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:25, the heavy chain CDR2 shown in SEQ ID NO:109, and the heavy chain CDR3 shown in SEQ ID NO:27, and a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:28, the light chain CDR2 shown in SEQ ID NO:29, and the light chain CDR3 shown in SEQ ID NO:30; (3) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:31, the heavy chain CDR2 shown in SEQ ID NO:110, and the heavy chain CDR3 shown in SEQ ID NO:33, and a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:34, the light chain CDR2 shown in SEQ ID NO:35, and the light chain CDR3 shown in SEQ ID NO:36; (4) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:37, the heavy chain CDR2 shown in SEQ ID NO:111, and the heavy chain CDR3 shown in SEQ ID NO:39, and a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:40, the light chain CDR2 shown in SEQ ID NO:41, and the light chain CDR3 shown in SEQ ID NO:42; or (5) A heavy chain variable region (VH) comprising the heavy chain CDR1 shown in SEQ ID NO:56, the heavy chain CDR2 shown in SEQ ID NO:112, and the heavy chain CDR3 shown in SEQ ID NO:58, and a light chain variable region (VL) comprising the light chain CDR1 shown in SEQ ID NO:59, the light chain CDR2 shown in SEQ ID NO:60, and the light chain CDR3 shown in SEQ ID NO:

61.

35. The antibody or antigen-binding fragment thereof according to claim 34, comprising: (1a) VH containing the sequence shown in SEQ ID NO:45 or a variant thereof and VL containing the sequence shown in SEQ ID NO:49 or a variant thereof; (1b) VH containing the sequence shown in SEQ ID NO:11 or a variant thereof and VL containing the sequence shown in SEQ ID NO:12 or a variant thereof; (2a) VH containing the sequence shown in SEQ ID NO:46 or a variant thereof and VL containing the sequence shown in SEQ ID NO:50 or a variant thereof; (2b) VH containing the sequence shown in SEQ ID NO:13 or a variant thereof and VL containing the sequence shown in SEQ ID NO:14 or a variant thereof; (3a) VH containing the sequence shown in SEQ ID NO:47 or a variant thereof and VL containing the sequence shown in SEQ ID NO:51 or a variant thereof; (3b) VH containing the sequence shown in SEQ ID NO:15 or a variant thereof and VL containing the sequence shown in SEQ ID NO:16 or a variant thereof; (4a) VH containing the sequence shown in SEQ ID NO:48 or a variant thereof and VL containing the sequence shown in SEQ ID NO:52 or a variant thereof; (4b) VH containing the sequence shown in SEQ ID NO:17 or a variant thereof and VL containing the sequence shown in SEQ ID NO:18 or a variant thereof; (5a) A VH comprising the sequence shown in SEQ ID NO:62 or a variant thereof and a VL comprising the sequence shown in SEQ ID NO:63 or a variant thereof; or (5b) VH containing the sequence shown in SEQ ID NO:53 or a variant thereof and VL containing the sequence shown in SEQ ID NO:54 or a variant thereof; in, The variant has 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 with the sequence from which it originates, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions); preferably, the substitutions are conservative substitutions.

36. The antibody or antigen-binding fragment thereof according to any one of claims 33-35, further comprising a constant region derived from mammalian (e.g., human) immunoglobulin; Preferably, the heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human immunoglobulins (e.g., IgG, such as IgG1, IgG2, IgG3, or IgG4), and / or, the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from human immunoglobulins (e.g., κ or λ). Preferably, the heavy chain of the antibody or its antigen-binding fragment comprises the human IgG1 constant region sequence shown in SEQ ID NO:43, and / or, the light chain of the antibody or its antigen-binding fragment comprises the human kappa constant region sequence shown in SEQ ID NO:

44.

37. The antibody or antigen-binding fragment thereof according to any one of claims 33-36, wherein, The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, and diabody; and / or the antibody is a murine antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or a multispecific antibody.

38. A multispecific antibody comprising the antibody or antigen-binding fragment thereof as described in any one of claims 33-37; Preferably, the multispecific antibody specifically binds to B7-H3 and additionally specifically binds to one or more other targets; Preferably, the multispecific antibody further comprises at least one second antibody or antigen-binding fragment thereof having a second binding specificity against a second target; Preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody.

39. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof as described in any one of claims 33-37 or the multispecific antibody as described in claim 38 and a bioactive molecule thereto.

40. An isolated nucleic acid molecule encoding: (i) the bispecific antibody of claim 32 or its antigen-binding fragment or at least one peptide chain thereof; (ii) the antibody of any one of claims 33-37 or its antigen-binding fragment, or its heavy chain variable region and / or light chain variable region, or its heavy chain and / or light chain; or (iii) the multispecific antibody of claim 38 or at least one peptide chain thereof.

41. A vector comprising the isolated nucleic acid molecule of claim 40; preferably, the vector is a cloning vector or an expression vector.

42. A host cell comprising the isolated nucleic acid molecule of claim 40 or the vector of claim 41.

43. A method for preparing the bispecific antibody or antigen-binding fragment thereof as claimed in claim 32, the antibody or antigen-binding fragment thereof as claimed in any one of claims 33-37, or the multispecific antibody as claimed in claim 38, comprising culturing the host cell of claim 42 under conditions that allow protein expression, and recovering the bispecific antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof, or the multispecific antibody from the cultured host cell culture.

44. A pharmaceutical composition comprising: an antibody-drug conjugate according to any one of claims 1-30, or a composition according to claim 31, or a bispecific antibody or antigen-binding fragment thereof according to claim 32, or an antibody or antigen-binding fragment thereof according to any one of claims 33-37, or a multispecific antibody according to claim 38, or an antibody-drug conjugate according to claim 39, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises additional pharmaceutically active agents; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, antiangiogenic agent, cytokine, specific tumor cell-targeting antibody, or immune checkpoint inhibitor.

45. Use of the antibody-drug conjugate of any one of claims 1-30, or the composition of claim 31, or the bispecific antibody or antigen-binding fragment thereof of claim 32, or the antibody or antigen-binding fragment thereof of any one of claims 33-37, or the multispecific antibody of claim 38, or the antibody-drug conjugate of claim 39, or the pharmaceutical composition of claim 44 in the preparation of a medicament for treating tumors; Preferably, the tumor is B7-H3 and / or PSMA positive; Preferably, the tumor is selected from prostate cancer, lung cancer, breast cancer, stomach cancer, liver cancer, and cervical cancer; Preferably, the tumor is prostate cancer, such as metastatic castration-resistant prostate cancer (mCRPC); Preferably, the tumor is lung cancer, such as small cell lung cancer and non-small cell lung cancer; Preferably, the tumor is breast cancer, such as triple-negative breast cancer; Preferably, the tumor is head and neck cancer, such as squamous cell carcinoma of the head and neck; Preferably, the antibody-drug conjugate, composition, bispecific antibody or its antigen-binding fragment, antibody or its antigen-binding fragment, multispecific antibody or pharmaceutical composition is administered in combination with another pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, antiangiogenic agent, cytokine, specific tumor cell-targeting antibody, or immune checkpoint inhibitor.

46. ​​A method for treating a tumor in a subject, comprising administering to a subject in need an effective amount of the antibody-drug conjugate of any one of claims 1-30, or the composition of claim 31, or the bispecific antibody or antigen-binding fragment thereof of claim 32, or the antibody or antigen-binding fragment thereof of any one of claims 33-37, or the multispecific antibody of claim 38, or the antibody-drug conjugate of claim 39, or the pharmaceutical composition of claim 44; Preferably, the tumor is B7-H3 and / or PSMA positive; Preferably, the tumor is selected from prostate cancer, lung cancer, breast cancer, stomach cancer, liver cancer, and cervical cancer; Preferably, the tumor is prostate cancer, such as metastatic castration-resistant prostate cancer (mCRPC); Preferably, the tumor is lung cancer, such as small cell lung cancer and non-small cell lung cancer; Preferably, the tumor is breast cancer, such as triple-negative breast cancer; Preferably, the tumor is head and neck cancer, such as squamous cell carcinoma of the head and neck; Preferably, the antibody-drug conjugate, composition, bispecific antibody or its antigen-binding fragment, antibody or its antigen-binding fragment, multispecific antibody or pharmaceutical composition is administered in combination with another pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, antiangiogenic agent, cytokine, specific tumor cell-targeting antibody or immune checkpoint inhibitor. Preferably, the method further includes administering additional antitumor therapy to the subject, such as surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

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