Antibody-drug conjugate targeting PDL1 / b7h3, preparation method therefor and use thereof

Abstract

An antibody-drug conjugate targeting PDL1 and B7H3, a preparation method therefor and the use thereof. The ADC comprises a bispecific antibody conjugated to a cytotoxic drug, and the antibody comprises a domain capable of specifically binding to PDL1 and a domain capable of specifically binding to B7H3. The ADC and the drug thereof can be used for preventing and / or treating diseases (such as tumors) associated with PDL1 and / or B7H3, and / or can be used as an adjunctive drug therapy for said diseases.

Description

Antibody-drug conjugates targeting PDL1 / B7H3, their preparation methods and applications

[0001] Cross-references

[0002] This disclosure is based on and claims priority to Chinese patent applications No. 202510022453.2, filed on January 7, 2025, and No. 202512037769.1, filed on December 30, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to targeted therapy, and more specifically, to antibody-drug conjugates targeting PDL1 and B7H3, methods for their preparation, and uses. Background Technology

[0004] PDL1, also known as CD274, is an immunosuppressive receptor ligand expressed by immune cells and various types of tumor cells. The interaction between this ligand and its receptor PD1 inhibits T cell activation and cytokine secretion. In the tumor microenvironment, this interaction can provide immune escape for tumor cells through cytotoxic T cell inactivation, while inhibition of the PD1 / PDL1 interaction mediates effective antitumor activity in preclinical models. Currently, among the marketed anti-PDL1 monoclonal antibodies, only Avelumab mediates ADCC effects against tumors and shows comparable safety profiles to other PDL1 antibodies. An important reason for this is that ADCC efficacy largely depends on the abundance of antigen expression, and PDL1 cannot be considered a typical tumor antigen, and it exhibits significant heterogeneity within tumor cells.

[0005] B7H3, also known as CD276, is an immune checkpoint molecule belonging to the B7 and CD28 families, involved in the regulation of T-cell immune responses. B7H3 is overexpressed in many types of tumor tissues and is often associated with poor prognosis in human patients. Although the receptor for B7H3 has not yet been characterized, inhibition of the B7H3 signaling pathway can mediate effective anti-tumor activity of immune cells. Recent studies have also shown that B7H3 can influence cancer progression beyond its immunomodulatory role. Given the important role of B7H3 in immune checkpoint regulation, several therapeutic molecules, including monoclonal antibodies, radioimmunotherapy, and antibody-drug conjugates (ADCs), have been developed to modulate B7H3-mediated immune cell regulation for immunotherapy and cancer treatment.

[0006] There is a need in the field for ADCs that combine PDL1 and B7H3 and contain cytotoxic drugs.

[0007] Invention Overview

[0008] This application relates to an antibody-drug conjugate for the prevention and / or treatment and / or as adjunctive therapy for PDL1 and / or B7H3-related diseases. The disclosed ADC comprises a bispecific antibody conjugated to a cytotoxic drug, said antibody comprising a domain specifically binding to PDL1 and a domain specifically binding to B7H3. The ADC and its drug can be used for the prevention and / or treatment and / or as adjunctive therapy for PDL1 and / or B7H3-related diseases (e.g., tumors).

[0009] In a first aspect, this disclosure provides an antibody-drug conjugate containing the structure shown below: Ab-[MLED]x

[0010] in:

[0011] Ab is a bispecific antibody or its antigen-binding fragment containing a first antigen-binding domain that specifically binds to PDL1 and a second antigen-binding domain that specifically binds to B7H3.

[0012] M is the linker site that is linked to the bispecific antibody or its antigen-binding fragment;

[0013] L is a structural segment connecting the joint portions M and E;

[0014] E is a structural segment connecting L and D;

[0015] D is a cytotoxic drug or a fragment thereof; and

[0016] x is any integer from 1 to 10.

[0017] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain includes a first light chain variable region (VL) and a first heavy chain variable region (VH), wherein the first VL and the first VH together form a domain capable of specifically binding PDL1; the second antigen-binding domain includes a second VL and a second VH, wherein the second VL and the second VH together form a domain capable of specifically binding B7H3.

[0018] In some embodiments of the antibody-drug conjugate, the first VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO:61; and / or, the first VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:60.

[0019] The CDRs are defined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system.

[0020] In some embodiments of the antibody-drug conjugate, the first VL comprises:

[0021] (i) LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system;

[0022] (ii) LCDR1 comprising the sequence shown in SEQ ID NO:32, LCDR2 comprising the sequence shown in SEQ ID NO:35, and LCDR3 comprising the sequence shown in SEQ ID NO:38; wherein the CDRs are defined by the Contact numbering system; or,

[0023] (iii) LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the IMGT numbering system;

[0024] And / or,

[0025] The first VH includes:

[0026] (i) HCDR1 containing the sequence shown in SEQ ID NO:18, HCDR2 containing the sequence shown in SEQ ID NO:23, and HCDR3 containing the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system;

[0027] (ii) HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system;

[0028] (iii) HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system;

[0029] (iv) HCDR1 comprising the sequence shown in SEQ ID NO:21, HCDR2 comprising the sequence shown in SEQ ID NO:26, and HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or,

[0030] (v) HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system.

[0031] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:18, HCDR2 comprising the sequence shown in SEQ ID NO:23, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system.

[0032] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system.

[0033] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system.

[0034] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:32, LCDR2 comprising the sequence shown in SEQ ID NO:35, and LCDR3 comprising the sequence shown in SEQ ID NO:38; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:21, HCDR2 comprising the sequence shown in SEQ ID NO:26, and HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system.

[0035] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system.

[0036] In some embodiments of the antibody-drug conjugate, the first VL comprises an amino acid sequence as shown in SEQ ID NO:61, and / or the first VH comprises an amino acid sequence as shown in SEQ ID NO:60.

[0037] In some embodiments of the antibody-drug conjugate, the second VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO:63; and / or, the second VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:62;

[0038] The CDRs are defined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system.

[0039] In some embodiments of the antibody-drug conjugate, the second VL comprises:

[0040] (i) LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system.

[0041] (ii) LCDR1 comprising the sequence shown in SEQ ID NO:53, LCDR2 comprising the sequence shown in SEQ ID NO:56, and LCDR3 comprising the sequence shown in SEQ ID NO:59; wherein the CDRs are defined by the Contact numbering system; or,

[0042] (iii) LCDR1 comprising the sequence shown in SEQ ID NO:54, LCDR2 comprising the sequence shown in SEQ ID NO:57, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the IMGT numbering system;

[0043] And / or,

[0044] The second VH includes:

[0045] (i) HCDR1 containing the sequence shown in SEQ ID NO:39, HCDR2 containing the sequence shown in SEQ ID NO:44, and HCDR3 containing the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system;

[0046] (ii) HCDR1 comprising the sequence shown in SEQ ID NO:40, HCDR2 comprising the sequence shown in SEQ ID NO:45, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system;

[0047] (iii) HCDR1 comprising the sequence shown in SEQ ID NO:41, HCDR2 comprising the sequence shown in SEQ ID NO:46, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system;

[0048] (iv) HCDR1 comprising the sequence shown in SEQ ID NO:42, HCDR2 comprising the sequence shown in SEQ ID NO:47, and HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or,

[0049] (v) HCDR1 comprising the sequence shown in SEQ ID NO:43, HCDR2 comprising the sequence shown in SEQ ID NO:48, and HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0050] In some embodiments of the antibody-drug conjugate, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:41, HCDR2 comprising the sequence shown in SEQ ID NO:46, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system.

[0051] In some embodiments of the antibody-drug conjugate, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:39, HCDR2 comprising the sequence shown in SEQ ID NO:44, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system.

[0052] In some embodiments of the antibody-drug conjugate, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:40, HCDR2 comprising the sequence shown in SEQ ID NO:45, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system.

[0053] In some embodiments of the antibody-drug conjugate, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:53, LCDR2 comprising the sequence shown in SEQ ID NO:56, and LCDR3 comprising the sequence shown in SEQ ID NO:59; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:42, HCDR2 comprising the sequence shown in SEQ ID NO:47, and HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system.

[0054] In some embodiments of the antibody-drug conjugate, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:54, LCDR2 comprising the sequence shown in SEQ ID NO:57, and LCDR3 comprising the sequence shown in SEQ ID NO:58; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:43, HCDR2 comprising the sequence shown in SEQ ID NO:48, and HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0055] In some embodiments of the antibody-drug conjugate, the second VL comprises an amino acid sequence as shown in SEQ ID NO:63, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO:62.

[0056] In some embodiments of the antibody-drug conjugate, (i) the first VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:61, and the first VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:60; and

[0057] (ii) The second VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:63, and the second VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:62.

[0058] The CDRs are defined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system.

[0059] In some embodiments of the antibody-drug conjugate, (a) the first VL comprises:

[0060] (i) LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system;

[0061] (ii) LCDR1 comprising the sequence shown in SEQ ID NO:32, LCDR2 comprising the sequence shown in SEQ ID NO:35, and LCDR3 comprising the sequence shown in SEQ ID NO:38; wherein the CDRs are defined by the Contact numbering system; or,

[0062] (iii) LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the IMGT numbering system;

[0063] and,

[0064] (b) The first VH includes:

[0065] (i) HCDR1 containing the sequence shown in SEQ ID NO:18, HCDR2 containing the sequence shown in SEQ ID NO:23, and HCDR3 containing the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system;

[0066] (ii) HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system;

[0067] (iii) HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system;

[0068] (iv) HCDR1 comprising the sequence shown in SEQ ID NO:21, HCDR2 comprising the sequence shown in SEQ ID NO:26, and HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or,

[0069] (v) HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system;

[0070] and

[0071] (c) The second VL includes:

[0072] (i) LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system.

[0073] (ii) LCDR1 comprising the sequence shown in SEQ ID NO:53, LCDR2 comprising the sequence shown in SEQ ID NO:56, and LCDR3 comprising the sequence shown in SEQ ID NO:59; wherein the CDRs are defined by the Contact numbering system; or,

[0074] (iii) LCDR1 comprising the sequence shown in SEQ ID NO:54, LCDR2 comprising the sequence shown in SEQ ID NO:57, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the IMGT numbering system;

[0075] and,

[0076] (d) The second VH includes:

[0077] (i) HCDR1 containing the sequence shown in SEQ ID NO:39, HCDR2 containing the sequence shown in SEQ ID NO:44, and HCDR3 containing the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system;

[0078] (ii) HCDR1 comprising the sequence shown in SEQ ID NO:40, HCDR2 comprising the sequence shown in SEQ ID NO:45, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system;

[0079] (iii) HCDR1 comprising the sequence shown in SEQ ID NO:41, HCDR2 comprising the sequence shown in SEQ ID NO:46, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system;

[0080] (iv) HCDR1 comprising the sequence shown in SEQ ID NO:42, HCDR2 comprising the sequence shown in SEQ ID NO:47, and HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or,

[0081] (v) HCDR1 comprising the sequence shown in SEQ ID NO:43, HCDR2 comprising the sequence shown in SEQ ID NO:48, and HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0082] In some embodiments of the antibody-drug conjugate, the bispecific antibody comprises:

[0083] (1) A first antigen-binding domain that specifically binds to PDL1, selected from any of the following:

[0084] (i) A first VL, comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 comprising the sequence shown in SEQ ID NO:18, HCDR2 comprising the sequence shown in SEQ ID NO:23, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system;

[0085] (ii) A first VL, comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system;

[0086] (iii) A first VL, comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system;

[0087] (iv) A first VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:32, an LCDR2 comprising the sequence shown in SEQ ID NO:35, and an LCDR3 comprising the sequence shown in SEQ ID NO:38; and a first VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:21, an HCDR2 comprising the sequence shown in SEQ ID NO:26, and an HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or

[0088] (v) A first VL, comprising: LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system;

[0089] and,

[0090] (2) A second antigen-binding domain that specifically binds to B7H3, selected from any of the following:

[0091] (i) a second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:52, an LCDR2 comprising the sequence shown in SEQ ID NO:55, and an LCDR3 comprising the sequence shown in SEQ ID NO:58; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:41, an HCDR2 comprising the sequence shown in SEQ ID NO:46, and an HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system;

[0092] (ii) A second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:52, an LCDR2 comprising the sequence shown in SEQ ID NO:55, and an LCDR3 comprising the sequence shown in SEQ ID NO:58; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:39, an HCDR2 comprising the sequence shown in SEQ ID NO:44, and an HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system;

[0093] (iii) A second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:52, an LCDR2 comprising the sequence shown in SEQ ID NO:55, and an LCDR3 comprising the sequence shown in SEQ ID NO:58; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:40, an HCDR2 comprising the sequence shown in SEQ ID NO:45, and an HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system;

[0094] (iv) A second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:53, an LCDR2 comprising the sequence shown in SEQ ID NO:56, and an LCDR3 comprising the sequence shown in SEQ ID NO:59; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:42, an HCDR2 comprising the sequence shown in SEQ ID NO:47, and an HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or,

[0095] (v) A second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:54, an LCDR2 comprising the sequence shown in SEQ ID NO:57, and an LCDR3 comprising the sequence shown in SEQ ID NO:58; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:43, an HCDR2 comprising the sequence shown in SEQ ID NO:48, and an HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0096] In some embodiments of the antibody-drug conjugate, the bispecific antibody comprises:

[0097] (1) A first antigen-binding domain that specifically binds to PDL1, selected from any of the following:

[0098] (i) a first VL, the first VL comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, the first VH comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:18, HCDR2 with the amino acid sequence shown in SEQ ID NO:23, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28;

[0099] (ii) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:19, HCDR2 with the amino acid sequence shown in SEQ ID NO:24, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28;

[0100] (iii) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:20, HCDR2 with the amino acid sequence shown in SEQ ID NO:25, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28;

[0101] (iv) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:32, LCDR2 with the amino acid sequence shown in SEQ ID NO:35, and LCDR3 with the amino acid sequence shown in SEQ ID NO:38; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:21, HCDR2 with the amino acid sequence shown in SEQ ID NO:26, and HCDR3 with the amino acid sequence shown in SEQ ID NO:29; or

[0102] (v) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:33, LCDR2 with the amino acid sequence shown in SEQ ID NO:36, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:22, HCDR2 with the amino acid sequence shown in SEQ ID NO:27, and HCDR3 with the amino acid sequence shown in SEQ ID NO:30;

[0103] and,

[0104] (2) A second antigen-binding domain that specifically binds to B7H3, selected from any of the following:

[0105] (i) a second VL comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:41, HCDR2 with the amino acid sequence shown in SEQ ID NO:46, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49;

[0106] (ii) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:39, HCDR2 with the amino acid sequence shown in SEQ ID NO:44, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49;

[0107] (iii) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:40, HCDR2 with the amino acid sequence shown in SEQ ID NO:45, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49;

[0108] (iv) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:53, LCDR2 with the amino acid sequence shown in SEQ ID NO:56, and LCDR3 with the amino acid sequence shown in SEQ ID NO:59; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:42, HCDR2 with the amino acid sequence shown in SEQ ID NO:47, and HCDR3 with the amino acid sequence shown in SEQ ID NO:50; or,

[0109] (v) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:54, LCDR2 with the amino acid sequence shown in SEQ ID NO:57, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:43, HCDR2 with the amino acid sequence shown in SEQ ID NO:48, and HCDR3 with the amino acid sequence shown in SEQ ID NO:51.

[0110] In some embodiments of the antibody-drug conjugate, the bispecific antibody comprises:

[0111] (1) A first antigen-binding domain that specifically binds to PDL1, selected from any of the following:

[0112] (i) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:18, HCDR2 with the amino acid sequence shown in SEQ ID NO:23, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system;

[0113] (ii) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:19, HCDR2 with the amino acid sequence shown in SEQ ID NO:24, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system;

[0114] (iii) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:20, HCDR2 with the amino acid sequence shown in SEQ ID NO:25, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system;

[0115] (iv) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:32, LCDR2 with the amino acid sequence shown in SEQ ID NO:35, and LCDR3 with the amino acid sequence shown in SEQ ID NO:38; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:21, HCDR2 with the amino acid sequence shown in SEQ ID NO:26, and HCDR3 with the amino acid sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or

[0116] (v) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:33, LCDR2 with the amino acid sequence shown in SEQ ID NO:36, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:22, HCDR2 with the amino acid sequence shown in SEQ ID NO:27, and HCDR3 with the amino acid sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system;

[0117] and,

[0118] (2) A second antigen-binding domain that specifically binds to B7H3, selected from any of the following:

[0119] (i) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:41, HCDR2 with the amino acid sequence shown in SEQ ID NO:46, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system;

[0120] (ii) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:39, HCDR2 with the amino acid sequence shown in SEQ ID NO:44, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system;

[0121] (iii) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:40, HCDR2 with the amino acid sequence shown in SEQ ID NO:45, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system;

[0122] (iv) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:53, LCDR2 with the amino acid sequence shown in SEQ ID NO:56, and LCDR3 with the amino acid sequence shown in SEQ ID NO:59; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:42, HCDR2 with the amino acid sequence shown in SEQ ID NO:47, and HCDR3 with the amino acid sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or,

[0123] (v) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:54, LCDR2 with the amino acid sequence shown in SEQ ID NO:57, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:43, HCDR2 with the amino acid sequence shown in SEQ ID NO:48, and HCDR3 with the amino acid sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0124] In some embodiments of the antibody-drug conjugate, (i) the first VL comprises the amino acid sequence shown in SEQ ID NO:61, and / or, the first VH comprises the amino acid sequence shown in SEQ ID NO:60; and

[0125] (ii) The second VL contains an amino acid sequence as shown in SEQ ID NO:63, and / or the second VH contains an amino acid sequence as shown in SEQ ID NO:62.

[0126] In some embodiments of the antibody-drug conjugate, (i) the amino acid sequence of the first VL is as shown in SEQ ID NO:61, and / or, the amino acid sequence of the first VH is as shown in SEQ ID NO:60; and

[0127] (ii) The amino acid sequence of the second VL is as shown in SEQ ID NO:63, and / or the amino acid sequence of the second VH is as shown in SEQ ID NO:62.

[0128] In some embodiments of the antibody-drug conjugate, one of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the other is an scFv;

[0129] Optionally, the scFv is optionally linked to the C-terminus or N-terminus of the heavy chain of the full-length antibody via a peptide linker.

[0130] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain is scFv, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0131] (i) A peptide chain VA comprising a VL of the second antigen-binding domain and a light chain constant region (CL); optionally, the CL is a kappa light chain constant region;

[0132] (ii) A peptide chain VB comprising a VH region of the second antigen-binding domain, a heavy chain CH1 region, an Fc domain monomer, and the first antigen-binding domain; optionally, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1; optionally, the Fc domain monomer comprises a hinge region, CH2, and CH3; optionally, the first antigen-binding domain is connected to the C-terminus of the Fc domain monomer via a peptide linker; optionally, the peptide linker comprises an amino acid sequence such as SEQ ID NO: 66 or 68.

[0133] Optionally, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; optionally, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68;

[0134] Optionally, the scFv may or may not contain disulfide bonds between VH and VL;

[0135] Optionally, the bispecific antibody comprises two identical or different peptide chains VA and two identical or different peptide chains VB, wherein the two peptide chains VB form a dimer through their respective Fc domain monomers.

[0136] In some embodiments of the antibody-drug conjugate, the Fc domain monomers further each independently include modifications capable of altering effector function;

[0137] Optionally, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / G237A;

[0138] Optionally, the Fc domain monomer comprises a sequence selected from the following sequence: SEQ ID NO: 74 or 75.

[0139] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain is scFv, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0140] (i) Peptide chain III-A, which includes the VL of the second antigen-binding domain and a light chain constant region (CL); optionally, the CL is a kappa light chain constant region;

[0141] (ii) Peptide chain III-B, comprising the first antigen-binding domain, and a VH region of the second antigen-binding domain, a heavy chain CH1 region, and an Fc domain monomer; optionally, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1; optionally, the Fc domain monomer comprises a hinge region, CH2, and CH3; optionally, the first antigen-binding domain is connected to the N-terminus of the VH region of the second antigen-binding domain via a peptide linker; optionally, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:67;

[0142] Optionally, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; optionally, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68;

[0143] Optionally, the scFv may or may not contain disulfide bonds between VH and VL;

[0144] Optionally, the bispecific antibody comprises two identical or different peptide chains III-A and two identical or different peptide chains III-B, wherein the two peptide chains III-B form a dimer through their respective Fc domain monomers.

[0145] In some embodiments of the antibody-drug conjugate, the Fc domain monomers further each independently include modifications capable of altering effector function;

[0146] Optionally, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / G237A;

[0147] Optionally, the Fc domain monomer comprises the sequence shown in SEQ ID NO:74.

[0148] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain is scFv, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0149] (i) peptide chain IV-A, which includes the VL of the second antigen-binding domain and a light chain constant region (CL); optionally, the CL is a kappa light chain constant region;

[0150] (ii) Peptide chain IV-B, comprising the VH of the second antigen-binding domain, the CH1 region of the heavy chain, and a first Fc domain monomer (or a second Fc domain monomer); optionally, the Fc domain monomer comprises a hinge region, CH2, and CH3.

[0151] (iii) A peptide chain IV-C comprising the first antigen-binding domain, a VH of the second antigen-binding domain, a heavy chain CH1 region, and a monomer of the second Fc domain (or a monomer of the first Fc domain); optionally, the first antigen-binding domain is connected to the N-terminus or C-terminus of the VH of the second antigen-binding domain via a peptide linker; optionally, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO: 67 or 68.

[0152] Optionally, the first and second Fc domain monomers are Fc domain monomers of IgG, such as Fc domain monomers of IgG1; optionally, the Fc domain monomers include a hinge region, CH2 and CH3.

[0153] Optionally, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; optionally, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68;

[0154] Optionally, the scFv may or may not contain disulfide bonds between VH and VL;

[0155] Optionally, the bispecific antibody comprises two identical or different peptide chains IV-A, one peptide chain IV-B, and one peptide chain IV-C, wherein peptide chains IV-B and IV-C form a dimer through their respective Fc domain monomers.

[0156] In some embodiments of the antibody-drug conjugate, the first and second Fc domain monomers each independently contain modifications of one or more amino acids that promote dimerization of the first and second Fc domain monomers.

[0157] Optionally, 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 "knob-into-hole" modification.

[0158] Optionally, the “knob” is modified to be the EU-numbered T366W / S354C mutation;

[0159] Optionally, the “hole” is modified to be the EU numbered T366S / L368A / Y407V / Y349C mutation.

[0160] In some embodiments of the antibody-drug conjugate, the first and second Fc domain monomers of the Fc domain further each independently contain modifications capable of altering effector function.

[0161] Optionally, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / P329A;

[0162] Optionally,

[0163] (i) The first Fc domain monomer contains the sequence shown in SEQ ID NO: 72, and the second Fc domain monomer contains the sequence shown in SEQ ID NO: 73; or

[0164] (ii) The first Fc domain monomer contains the sequence shown in SEQ ID NO: 73, and the second Fc domain monomer contains the sequence shown in SEQ ID NO: 72.

[0165] In some embodiments of the antibody-drug conjugate, one of the first antigen-binding domain and the second antigen-binding domain is Fab, and the other is scFv or Fab; and the bispecific antibody 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 peptide linkers, respectively.

[0166] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain is scFv, the second antigen-binding domain is Fab, and the bispecific antibody comprises:

[0167] (i) A peptide chain IA comprising a first antigen-binding domain and a first Fc domain monomer; optionally, the first Fc domain monomer comprises a hinge region, CH2 and CH3; optionally, the first antigen-binding domain is connected to the N-terminus of the first Fc domain monomer via a peptide linker; optionally, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:64.

[0168] (ii) a peptide chain IB comprising a second antigen-binding domain VH, a heavy chain CH1 region, and a second Fc domain monomer; optionally, the second Fc domain monomer comprises a hinge region, CH2, and CH3; and

[0169] (iii) Peptide chain IC, which includes a second protobinding domain VL and a light chain constant region (CL).

[0170] Optionally, the second Fc domain monomer forms a dimer with the first Fc domain monomer;

[0171] Optionally, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; optionally, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68;

[0172] Optionally, the scFv may or may not contain disulfide bonds between VH and VL;

[0173] Optionally, the bispecific antibody comprises a peptide chain IA, a peptide chain IB, and a peptide chain IC, wherein peptide chains IA and IB form a dimer through their respective Fc domain monomers.

[0174] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain is a Fab, and the second antigen-binding domain is a Fab containing domain exchanges in the form of CrossMab.

[0175] Optionally, the CrossMab-style domain swapping is selected from:

[0176] (a) CrossMab Fab: In the Fab, the antibody light chain CL-VL and the antibody heavy chain CH1-VH are interchanged;

[0177] (b) CrossMab VH-VL: In the Fab, the VL and VH of the antibody are interchanged; or

[0178] (c)CrossMab CH1-CL: In the Fab, the CH and CL of the antibody are interchanged.

[0179] In some embodiments of the antibody-drug conjugate, the bispecific antibody comprises:

[0180] (i) Peptide chain II-A, which includes the VL of the first antigen-binding domain and a light chain constant region (CL); optionally, the CL is a kappa light chain constant region;

[0181] (ii) peptide chain II-B, which includes the VH of the first antigen-binding domain, the CH1 region of the heavy chain, and the monomer of the first Fc domain.

[0182] (iii) Peptide chain II-C, comprising the VH of the second antigen-binding domain, the light chain constant region (CL), and the second Fc domain monomer;

[0183] (iv) Peptide chain II-D, which includes the VL region of the second antigen-binding domain and the CH1 region of the heavy chain;

[0184] Optionally, the first Fc domain monomer and the second Fc domain monomer form a dimer;

[0185] Optionally, the bispecific antibody comprises a peptide chain II-A, a peptide chain II-B, a peptide chain II-C, and a peptide chain II-D, wherein peptide chains II-B and II-C form a dimer through their respective Fc domain monomers.

[0186] In some embodiments of the antibody-drug conjugate, the first and second Fc domain monomers each independently contain modifications of one or more amino acids that promote dimerization of the first and second Fc domain monomers.

[0187] Optionally, 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 "knob-into-hole" modification.

[0188] Optionally, the “knob” is modified to be the EU-numbered T366W / S354C mutation;

[0189] Optionally, the “hole” is modified to be the EU numbered T366S / L368A / Y407V / Y349C mutation.

[0190] In some embodiments of the antibody-drug conjugate, the first and second Fc domain monomers of the Fc domain further each independently contain modifications capable of altering effector function.

[0191] Optionally, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / P329A;

[0192] Optionally, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:72 and 73, or SEQ ID NO:73 and 72.

[0193] In some embodiments of the antibody-drug conjugate, one of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the other is VH / VL;

[0194] Optionally, the VH is connected to the N-terminus of the heavy chain of the full-length antibody via a peptide linker; and the VL is connected to the N-terminus of the light chain of the full-length antibody via a peptide linker.

[0195] Optionally, the VH is connected to the N-terminus of one of the heavy chains of the full-length antibody via a peptide linker; and the VL is connected to the N-terminus of one of the light chains of the full-length antibody via a peptide linker.

[0196] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain is VH / VL, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0197] (i) Peptide chain VI-A, comprising a VL of the first antigen-binding domain, a VL of the second antigen-binding domain, and a light chain constant region (CL); optionally, the CL is a kappa light chain constant region; optionally, the VL of the first antigen-binding domain is connected to the N-terminus of the VL of the second antigen-binding domain via a peptide linker.

[0198] (ii) Peptide chain VI-B, comprising a VH of the first antigen-binding domain, a VH of the second antigen-binding domain, a heavy chain CH1 region, and an Fc domain monomer; optionally, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1; optionally, the Fc domain monomer comprises a hinge region, CH2, and CH3; optionally, the VH of the first antigen-binding domain is connected to the N-terminus of the VH of the second antigen-binding domain via a peptide linker;

[0199] Optionally, each of the peptide linkers is 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, 4, 5, 6, 7, 8, 9 or 10.

[0200] Optionally, each of the peptide linkers independently comprises an amino acid sequence as shown in SEQ ID NO:68;

[0201] Optionally, the antibody-drug conjugate comprises two identical or different peptide chains VI-A and two identical or different peptide chains VI-B, wherein the two peptide chains VI-B form a dimer through their respective Fc domain monomers.

[0202] In some embodiments of the antibody-drug conjugate, the Fc domain monomer contains modifications that enable altered effector functions;

[0203] Optionally, the modification of the altered effector function includes the following mutations with EU numbers: L234A / L235A / G237A.

[0204] Optionally, the Fc domain monomer comprises the sequence shown in SEQ ID NO:74.

[0205] In some embodiments of the antibody-drug conjugate, the first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), the second antigen-binding domain is VH / VL, and the bispecific antibody comprises:

[0206] (i) Peptide chain VII-A, comprising a VL of the second antigen-binding domain, a VL of the first antigen-binding domain, and a light chain constant region (CL); optionally, the CL is a kappa light chain constant region; optionally, the VL of the second antigen-binding domain is connected to the N-terminus of the VL of the first antigen-binding domain via a peptide linker.

[0207] (ii) Peptide chain VII-B, comprising VH of the second antigen-binding domain, VH of the first antigen-binding domain, heavy chain CH1 region, and a second Fc domain monomer; optionally, VH of the second antigen-binding domain is connected to the N-terminus of VH of the first antigen-binding domain via a peptide linker.

[0208] (iii) A peptide chain VII-C, comprising the VL of the second antigen-binding domain and a light chain constant region (CL); optionally, the CL is a kappa light chain constant region; and

[0209] (iv) Peptide chain VII-D, comprising the VH of the second antigen-binding domain, the CH1 region of the heavy chain, and a monomer of the first Fc domain;

[0210] Optionally, the first and second Fc domain monomers are Fc domain monomers of IgG, such as Fc domain monomers of IgG1; optionally, the Fc domain monomers include a hinge region, CH2 and CH3.

[0211] Optionally, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68;

[0212] Optionally, the antibody-drug conjugate comprises a peptide chain VII-A, a peptide chain VII-B, a peptide chain VII-C, and a peptide chain VII-D, wherein peptide chains VII-B and VII-D form a dimer through their respective Fc domain monomers.

[0213] In some embodiments of the antibody-drug conjugate, the first and second Fc domain monomers each independently contain modifications of one or more amino acids that promote dimerization of the first and second Fc domain monomers.

[0214] Optionally, 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 "knob-into-hole" modification.

[0215] Optionally, the “knob” is modified to be the EU-numbered T366W / S354C mutation;

[0216] Optionally, the “hole” is modified to be the EU numbered T366S / L368A / Y407V / Y349C mutation.

[0217] In some embodiments of the antibody-drug conjugate, the first and second Fc domain monomers of the Fc domain further each independently contain modifications capable of altering effector function.

[0218] Optionally, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / P329A;

[0219] Optionally, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:72 and 73, or SEQ ID NO:73 and 72.

[0220] In some embodiments of the antibody-drug conjugate, the bispecific antibody or its antigen-binding fragment comprises:

[0221] (1) A peptide chain VA comprising the amino acid sequence shown in SEQ ID NO:2, and / or a peptide chain VB comprising the amino acid sequence shown in SEQ ID NO:5;

[0222] (2) peptide chain III-A comprising the amino acid sequence shown in SEQ ID NO:2, and / or peptide chain III-B comprising the amino acid sequence shown in SEQ ID NO:4;

[0223] (3) A peptide chain IV-A containing the amino acid sequence shown in SEQ ID NO:2, a peptide chain IV-B containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain IV-C containing the amino acid sequence shown in SEQ ID NO:12.

[0224] (4) A peptide chain IV-A containing the amino acid sequence shown in SEQ ID NO:2, a peptide chain IV-B containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain IV-C containing the amino acid sequence shown in SEQ ID NO:15.

[0225] (5) A peptide chain IA containing the amino acid sequence shown in SEQ ID NO:3, a peptide chain IB containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain IC containing the amino acid sequence shown in SEQ ID NO:2.

[0226] (6) A peptide chain II-A containing the amino acid sequence shown in SEQ ID NO:9, a peptide chain II-B containing the amino acid sequence shown in SEQ ID NO:8, a peptide chain II-C containing the amino acid sequence shown in SEQ ID NO:10, and / or a peptide chain II-D containing the amino acid sequence shown in SEQ ID NO:11.

[0227] (7) peptide chain VI-A comprising the amino acid sequence shown in SEQ ID NO:7, and / or peptide chain VI-B comprising the amino acid sequence shown in SEQ ID NO:6; or

[0228] (8) A peptide chain VII-A containing the amino acid sequence shown in SEQ ID NO:14, a peptide chain VII-B containing the amino acid sequence shown in SEQ ID NO:13, a peptide chain VII-C containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain VII-D containing the amino acid sequence shown in SEQ ID NO:2.

[0229] In some embodiments of the antibody-drug conjugate, the bispecific antibody or its antigen-binding fragment comprises:

[0230] (1) Peptide chain VA with amino acid sequence as shown in SEQ ID NO:2, and / or peptide chain VB with amino acid sequence as shown in SEQ ID NO:5;

[0231] (2) Peptide chain III-A with an amino acid sequence as shown in SEQ ID NO:2, and / or peptide chain III-B with an amino acid sequence as shown in SEQ ID NO:4;

[0232] (3) Peptide chain IV-A with amino acid sequence as shown in SEQ ID NO:2, peptide chain IV-B with amino acid sequence as shown in SEQ ID NO:1, and / or peptide chain IV-C with amino acid sequence as shown in SEQ ID NO:12;

[0233] (4) Peptide chain IV-A with amino acid sequence as shown in SEQ ID NO:2, peptide chain IV-B with amino acid sequence as shown in SEQ ID NO:1, and / or peptide chain IV-C with amino acid sequence as shown in SEQ ID NO:15;

[0234] (5) peptide chain IA with the amino acid sequence of SEQ ID NO:3, peptide chain IB with the amino acid sequence of SEQ ID NO:1, and / or peptide chain IC with the amino acid sequence of SEQ ID NO:2; or

[0235] (6) Peptide chain II-A with amino acid sequence as shown in SEQ ID NO:9, peptide chain II-B with amino acid sequence as shown in SEQ ID NO:8, peptide chain II-C with amino acid sequence as shown in SEQ ID NO:10, and / or peptide chain II-D with amino acid sequence as shown in SEQ ID NO:11.

[0236] (7) Peptide chain VI-A with the amino acid sequence shown in SEQ ID NO:7, and / or peptide chain VI-B with the amino acid sequence shown in SEQ ID NO:6; or

[0237] (8) Peptide chain VII-A with amino acid sequence as shown in SEQ ID NO:14, peptide chain VII-B with amino acid sequence as shown in SEQ ID NO:13, peptide chain VII-C with amino acid sequence as shown in SEQ ID NO:1, and / or peptide chain VII-D with amino acid sequence as shown in SEQ ID NO:2.

[0238] In some embodiments of antibody-drug conjugates, M comprises

[0239] Wherein, ring A is a 5-6 membered aliphatic heterocycle or a 5-20 membered aromatic ring system, wherein the aliphatic heterocycle and aromatic ring system are optionally selected independently by one or more groups selected from oxygen (=O), halogen, cyano, amino, carboxyl, mercapto, and C. 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C2-20 imidene group, C 2-20 Alynyl or amino group.

[0240] In some embodiments of antibody-drug conjugates, M comprises Wherein ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by one or more 6-membered heteroaromatic rings connected to a benzene ring via single bonds, or a polycyclic ring formed by multiple 6-membered heteroaromatic rings connected via single bonds, wherein the aliphatic heterocycle is optionally surrounded by one or more elements selected from oxygen (=O), halogens, and C. 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

[0241] In some embodiments of antibody-drug conjugates, M comprises Where ring A is selected from M1 is selected from single bond, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl or amino group.

[0242] In some embodiments of antibody-drug conjugates, M is selected from...

[0243] In some embodiments of antibody-drug conjugates, M is...

[0244] In some embodiments of antibody-drug conjugates, M is selected from...

[0245] In some embodiments of antibody-drug conjugates, M is selected from...

[0246] In some embodiments of the antibody-drug conjugate, L is selected from one or more of the following structures: C 1- 6. Alkyl group, -N(R')-, carbonyl group, -O-, selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) sNatural or non-natural amino acids and their analogues containing 1, 2, 3 or 4 amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG(SEQ)) ID NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81))),

[0247] Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20.

[0248] In some embodiments of the antibody-drug conjugate, the short peptide is selected from Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG(SEQ ID)) NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81)).

[0249] In some embodiments of the antibody-drug conjugate, L is selected from structures comprising one or more of the following: C 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly(GGFG(SEQ ID NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81)), Where s is selected from integers from 1 to 20.

[0250] In some embodiments of the antibody-drug conjugate, L is selected from one or more of the following structures:

[0251] In some embodiments of the antibody-drug conjugate, the L is selected from the following structures:

[0252] In some embodiments of the antibody-drug conjugate, the L is selected from the following structures:

[0253] In some embodiments of the antibody-drug conjugate, the L is selected from the following structures:

[0254] In some embodiments of the antibody-drug conjugate, the L is selected from the following structures:

[0255] In some embodiments of the antibody-drug conjugate, the L is selected from the following structures:

[0256] In some embodiments of the antibody-drug conjugate, E is a single bond or selected from the following structures:

[0257] -NHCH2-, -NHCH2-O-CH2-CO-, -CO-O-CH2-CO-,

[0258] In some embodiments of antibody-drug conjugates, E is a single bond, -NHCH2-, -NHCH2-O-CH2-CO-, etc.

[0259] In some embodiments of the antibody-drug conjugate, E is -NHCH2- or...

[0260] In some embodiments of antibody-drug conjugates, E is -NHCH2- or a single bond.

[0261] In some embodiments of the antibody-drug conjugate, E is -NHCH2- or -NHCH2-O-CH2-CO-.

[0262] In some embodiments of antibody-drug conjugates, E is...

[0263] In some implementation schemes of antibody-drug conjugates Selected from the following structures:

[0264] In some implementation schemes of antibody-drug conjugates Selected from the following structures:

[0265] In some embodiments of the antibody-drug conjugate, the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

[0266] In some embodiments of the antibody-drug conjugate, the microtubule inhibitor is an olistatin or maytansine compound.

[0267] In some embodiments of the antibody-drug conjugate, the olistatin compound is selected from the following:

[0268] In some embodiments of the antibody-drug conjugate, the DNA intercalating agent is pyrrolobenzodiazepine. (PBD)

[0269] In some embodiments of the antibody-drug conjugate, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0270] In some embodiments of the antibody-drug conjugate, the topoisomerase I inhibitor is selected from camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, rubotecan, and pharmaceutically acceptable salts, esters, or analogs thereof; and the topoisomerase II inhibitor is selected from doxorubicin, PNU-159682, docalimcin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, and pharmaceutically acceptable salts, esters, or analogs thereof.

[0271] In some embodiments of the antibody-drug conjugate, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analogue thereof.

[0272] In some embodiments of the antibody-drug conjugate, the cytotoxic drug is selected from compounds of Formula I and II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of Formula I and II.

[0273] Among them, R1 and R2 are each independently selected from C. 1-6 Alkyl and halogen;

[0274] R3 is selected from H and -CO-CH2OH;

[0275] R4 and R5 are each independently selected from H, halogens, and hydroxyl groups; or R4 and R5 are linked to form a 5-6 membered oxygen-containing heterocycle;

[0276] R6 is selected from hydrogen or -C. 1-4 Alkylene-NR a R b ;

[0277] R7 is selected from C 1-6 Alkyl, -C 1-4 Alkylene-NR a R b -C 1-4 Alkylene-SiR a R b R c -SiR a R b R c -C 1-4 Alkylene = N-OR a ;where R a R b and R c Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, -SO2-C 1-6 Alkyl and -CO-C 1-6 Alkyl; wherein R is optionally present a and R b It forms 5-6 member nitrogen-containing heterocycles with the connected atoms.

[0278] In some embodiments of the antibody-drug conjugate, the cytotoxic drug is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds:

[0279] The fragment of the cytotoxic drug obtained after the cytotoxic drug is linked to the linker is D in the above general formula.

[0280] In some embodiments of the antibody-drug conjugate, the cytotoxic drug is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds:

[0281] The fragment of the cytotoxic drug obtained after the cytotoxic drug is linked to the linker is D in the above general formula.

[0282] In some embodiments of the antibody-drug conjugate, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amine group on the cytotoxic drug.

[0283] In some embodiments of the antibody-drug conjugate, the antibody-drug conjugate is selected from:

[0284] Wherein, Ab or HA is a bispecific antibody or its antigen-binding fragment as defined above;

[0285] This indicates the specific linkage between the thiol group of the cysteine ​​residue in the bispecific antibody or its antigen-binding fragment and M in the antibody-drug conjugate; and x represents the amount of drug load.

[0286] In some embodiments of the antibody-drug conjugate, the thiol group in the bispecific antibody or its antigen-binding fragment forms a thioether bond with M in the antibody-drug conjugate through an addition reaction or a substitution reaction to obtain the antibody-drug conjugate.

[0287] In some embodiments of the antibody-drug conjugate, the Ab or HA is a bispecific antibody selected from bsAb01, bsAb02, bsAb03, bsAb04, bsAb05, bsAb06, bsAb07, and bsAb08, with bsAb01, bsAb02, bsAb03, and bsAb04 being optional. For a detailed description of bsAb01, bsAb02, bsAb03, bsAb04, bsAb05, bsAb06, bsAb07, and bsAb08 in some embodiments, please refer to Example 1 of this disclosure.

[0288] In some embodiments of the antibody-drug conjugate, the antibody-drug conjugate is selected from bsAb01-A-10, bsAb02-A-10, bsAb03-A-10, and bsAb04-A-10.

[0289] In a second aspect, this disclosure provides compositions comprising one or more antibody-drug conjugates as described in any embodiment of the first aspect.

[0290] In some embodiments of the composition, the DAR (drug-antibody conjugate ratio) of the composition is 1-10.

[0291] In some embodiments of the composition, the DAR value of the composition is 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.

[0292] In some embodiments of the composition, the DAR value of the composition is 3 to 9.

[0293] In some embodiments of the composition, the DAR value of the composition is 4 to 8.

[0294] In some embodiments of the composition, the DAR value of the composition is 3.0–3.5, 3.0–4.0, 3.0–4.5, 3.0–5.0, 3.0–5.5, 3.0–6.0, 3.5–4.0, 3.5–4.5, 3.5–5.0, 3.5–5.5, 3.5–6.0, 3.5–6.5, 3.5–7.0, 3.5–7.5, 3.5–8.0, 4.0–4.5, 4.0–5.0, 4.0–5.5, 4.0–6.0, 4.0–6.5, 4.0–7.0, 4.0–7.5, 4.0–8.0, 4.5–5.0. 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0~5.5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0295] In some embodiments of the composition, the composition has a DAR value of 5.99, 7.94, 7.99, or 8.0.

[0296] Thirdly, this disclosure provides a pharmaceutical composition comprising one or more antibody-drug conjugates as described in any embodiment of the first aspect or a composition as described in any embodiment of the second aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0297] In some embodiments of the pharmaceutical composition, the pharmaceutical composition further comprises one or more additional pharmaceutically active agents selected from: TROP2 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, B7H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0298] Fourthly, this disclosure provides a pharmaceutical combination comprising a first therapeutic agent and a second therapeutic agent, wherein the first therapeutic agent and the second therapeutic agent are administered simultaneously or sequentially, wherein the first therapeutic agent is selected from one or more antibody-drug conjugates as described in any embodiment of the first aspect or compositions as described in any embodiment of the second aspect; and the second therapeutic agent is selected from: TROP2 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, B7H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0299] Fifthly, this disclosure provides the use of antibody-drug conjugates as described in any embodiment of the first aspect, compositions as described in any embodiment of the second aspect, pharmaceutical compositions as described in any embodiment of the third aspect, or pharmaceutical combinations as described in the fourth aspect in the preparation of a medicament, wherein the medicament is used in a subject for the prevention and / or treatment of a disease associated with PDL1 and / or B7H3 and / or as an adjuvant for the treatment of such disease in a subject, and / or for inhibiting the activity of PDL1 and / or B7H3 in vitro or in a subject; optionally, the disease associated with PDL1 and / or B7H3 is a tumor; optionally, the tumor is B7H3 and / or PDL1 positive.

[0300] In some embodiments of the use, the tumor is lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Optionally, the tumor is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma.

[0301] In a sixth aspect, this disclosure provides a method for inhibiting the activity of PDL1 and / or B7H3 in cells, the method comprising contacting the cells with an antibody-drug conjugate as described in any embodiment of the first aspect, a composition as described in any embodiment of the second aspect, a pharmaceutical composition as described in any embodiment of the third aspect, or a pharmaceutical combination as described in the fourth aspect; wherein the cells are cells expressing PDL1 and / or B7H3.

[0302] In a seventh aspect, this disclosure provides a method for the prevention and / or treatment and / or adjuvant treatment of a disease associated with PDL1 and / or B7H3 in a subject, the method comprising administering to a subject in need an effective amount of an antibody-drug conjugate as described in any embodiment of the first aspect, a composition as described in any embodiment of the second aspect, a pharmaceutical composition as described in any embodiment of the third aspect, or a pharmaceutical combination as described in the fourth aspect, optionally, wherein the disease associated with PDL1 and / or B7H3 is a tumor; optionally, the tumor is B7H3 and / or PDL1 positive.

[0303] In some embodiments of the method described in this aspect, the tumor is lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Optionally, the tumor is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma.

[0304] In some embodiments of the method described in this aspect, the method further includes administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy may be administered simultaneously, separately, or sequentially.

[0305] Eighthly, this disclosure provides antibody-drug conjugates as described in any embodiment of the first aspect, compositions as described in any embodiment of the second aspect, pharmaceutical compositions as described in any embodiment of the third aspect, or pharmaceutical combinations as described in the fourth aspect for the prevention and / or treatment and / or as adjuvant therapy of diseases associated with PDL1 and / or B7H3 in subjects, wherein the disease associated with PDL1 and / or B7H3 is a tumor; optionally, the tumor is B7H3 and / or PDL1 positive.

[0306] In some embodiments of this aspect, the tumor is lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Optionally, the tumor is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma.

[0307] In some embodiments of this aspect, the prevention and / or treatment and / or adjunctive treatment of diseases associated with PDL1 and / or B7H3 in the subject further includes administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjunctive therapy, and any combination thereof; optionally, the second therapy may be administered simultaneously, separately, or sequentially. Attached Figure Description

[0308] Figure 1A: Effects of different antibody-drug conjugates on tumor volume in mice bearing human breast cancer cells MDA-MB-231.

[0309] Figure 1B: Effects of different antibody-drug conjugates on the body weight of mice bearing human breast cancer cells MDA-MB-231.

[0310] Figure 2A: Effects of different antibody-drug conjugates on tumor volume in human melanoma cell A375-bearing mice.

[0311] Figure 2B: Effects of different antibody-drug conjugates on the body weight of human melanoma cell A375-bearing mice.

[0312] Invention Details

[0313] Terminology Definition

[0314] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used in this disclosure are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0315] As used herein, the term "antibody" is used in the broadest sense to encompass a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can consist of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the 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 variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. While not directly involved in antibody-antigen binding, the constant domain exhibits various effector functions, such as mediating interactions between immunoglobulins and 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). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains 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.

[0316] The term "antibody" also includes embodiments in which the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into a pyroglutamate salt. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or contain N-terminal glutamine or glutamate, or have an N-terminal amino acid cyclized into pyroglutamate.

[0317] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0318] 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. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), the MacCallum numbering system (MacCallum et al., (1996) J Mol Biol 262:732-745), and see also Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” Antibody Engineering, Kontermann and Dübel, eds., Chapter The definitions can be found in the following systems: 31, pp. 422-439, Springer-Verlag, Berlin (2001)); the AHo numbering system (Honegger and Plückthun, A., J. Mol. Biol. 309: 657-670 (2001)); or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR 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).

[0319] In this document, the CDR contained in the antibodies or antigen-binding fragments thereof disclosed herein can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibodies or antigen-binding fragments thereof disclosed herein is determined using the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is determined using the Chothia numbering system.

[0320] V H The complete amino acid sequence is typically numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the aforementioned numbering systems. In some embodiments, V H The amino acid sites in the sequence can be numbered sequentially starting from amino acid site 1 until the end of the sequence, or they can be numbered according to Kabat. Unless otherwise stated, the V mentioned herein... H and V L The amino acid sites in the sequence are defined according to their sequential numbering.

[0321] The amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.

[0322] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0323] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, 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.

[0324] As used herein, the term "bispecific antibody" or "BsAb" refers to an antibody that has binding specificity to two different antigens (or epitopes) and comprises antigen-binding domains that are specific to the binding of different antigens (or epitopes), such as two antigen-binding domains that are specific to the binding of different antigens (or epitopes), thereby enabling binding to two different binding sites and / or target molecules. The individual antigen-binding domains of a bispecific antibody can be independently selected from full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., Fv, Fab, scFab, or scFv). In some cases, the individual antigen-binding domains are linked by peptide linkers.

[0325] As used herein, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0326] As used herein, the term "Fc fragment" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0327] 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 peptide linkers consist of a repeating GGGGS amino acid sequence (SEQ ID NO:76) or a variant thereof, such as the amino acid sequence (GGGGS)4 (SEQ ID NO:68), 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.

[0328] As used herein, the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains, typically consisting of one peptide chain containing VL and CL and another peptide chain containing VH and CH1. However, those skilled in the art will understand that the Fab domains may be arranged according to the aforementioned natural orientation, but may also contain domain substitutions or exchanges that facilitate proper VH and VL pairing (e.g., domain exchanges in the form of Crossmab). The term "scFab" refers to a single polypeptide chain containing VL, VH, CL, and CH1 domains, wherein adjacent domains are optionally linked by a linker. In a typical structure, the single polypeptide chain contained in scFab comprises, from the N-terminus to the C-terminus: (1) VL, CL, VH, and CH1, wherein CL and VH are typically linked by a peptide linker (e.g., a flexible peptide linker), or (1) VH, CH1, VL, and CL, wherein CH1 and VL are typically linked by a peptide linker (e.g., a flexible peptide linker).

[0329] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or fragment thereof derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, generally refer to antibodies or fragments thereof derived from a group of antibodies comprising at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any specific method.

[0330] As used herein, the term "CrossMab" refers to a method for constructing bispecific antibodies by exchanging the heavy and light chain domains within the antigen-binding fragment (Fab) of half of the bispecific antibody, allowing the light chain to correctly bind to its homologous heavy chain. This "exchange" preserves the antigen-binding affinity but makes the two arms so different that light chain mismatches no longer occur. Three possible forms of "CrossMab" are: CrossMab Fab CrossMab refers to the crossover or exchange of all VH-CH1 and VL-CL domain positions in half of a bispecific antibody. VH-VL This refers to the crossover or exchange of only the VH and VL domain positions in half of a bispecific antibody; and CrossMab CH1-CLThis refers to the crossover or exchange of the CH1 and CL domain positions within half of the Fab region of a bispecific antibody. CorssMab antibodies have been described or claimed in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010136172, WO2010145792 and WO2013026831. The term “CrossMab” antibody is recognized in the art; see, for example, Brinkmann and Kontennann, MAbs 9(2):182-212 (2017); Kontermann and Brinkmann, Drug Discovery Today 20(7):838-846 (2015); Schaefer et al., PNAS, 108 11187-1191 (2011); Kleinet et al., MAbs 8(6):1010-1020 (2016); and Klein et al., MAbs 4(6):653-663 (2012).

[0331] As used herein, the term "DVD-IgG" refers to a tetravalent molecule with two binding sites for each antigen, produced by fusing the VL and VH of another antibody to the N-terminus of the light and heavy chains of a normal antibody (e.g., IgG), respectively. Specifically, the second antibody VH is fused to the first antibody VH, and the second antibody VL is fused to the first antibody VL. Bifunctionality is achieved by binding to two targets through the variable regions of the two antibodies. These molecules have the same Fc region as existing antibodies and can therefore be produced using existing universal antibody technologies. The structure of the DVD-IgG bispecific antibody is described or claimed by Abbott in PCT / US2007 / 017340. The antibody structure using the term "DVD-IgG" is well known in the art; see, for example, DiGiammarino E, Ghayur T, Liu JJ. Design and generation of DVD-IgG. TM molecules for dual-specific targeting. Methods Mol Biol, 2012,899:145-156.

[0332] As used herein, the term "variant structure of DVD-IgG" refers to a trivalent molecule with two binding sites against one antigen by fusing the VL and VH of another antibody to the N-terminus of one light chain and one heavy chain of a normal antibody (e.g., IgG), respectively. Specifically, the second antibody VH is fused with the first antibody VH, and the second antibody VL is fused with the first antibody VL, resulting in a molecule that binds to one of the antigens. The term "variant structure of DVD-IgG" is asymmetric compared to the bispecific antibody structure of "DVD-IgG".

[0333] As used herein, the term “N-scFv(2)-IgG” refers to a tetravalent molecule with two binding sites for each antigen, produced by fusing the scFv of another antibody to the N-terminus of each of the two heavy chains of a normal antibody (e.g., IgG), i.e., the scFv of the second antibody to the N-terminus of the VH of the first antibody. The antibody structure of the term “N-scFv(2)-IgG” is well known in the art; see, for example, Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019 Aug; 18(8):585-608. doi:10.1038 / s41573-019-0028-1.PMID:31175342.

[0334] As used herein, the term “N-scFv(1)-IgG” refers to a molecule formed by linking the VH of one antibody and the scFv of another antibody to the N-terminus of the CH1 of one heavy chain of a normal antibody (e.g., IgG), which includes either: (1) the scFv of one antibody is fused to the N-terminus of the VH of another antibody; or (2) the scFv of one antibody is fused to the C-terminus of the VH of another antibody. That is, the antigen-binding site of one heavy chain from the N-terminus to the C-terminus comprises: (1) the scFv of one antibody - the VH of another antibody - CH1; or (2) the VH of another antibody - the scFv of one antibody - CH1. The other heavy chain is the heavy chain of a normal antibody (e.g., IgG), resulting in a trivalent molecule with two binding sites for one antigen and one binding site for the other antigen. The antibody structure of the term “N-scFv(1)-IgG” is well known in the art; see, for example, Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019 Aug; 18(8):585-608. doi:10.1038 / s41573-019-0028-1.PMID:31175342.

[0335] As used herein, the term "IgG-C-scFv(2)" refers to a tetravalent molecule with two binding sites for each antigen, produced by fusing the scFv of another antibody to the C-terminus of each of the two heavy chains of a normal antibody (e.g., IgG), i.e., the scFv of the second antibody to the C-terminus of the Fc of the first antibody. The antibody structure of the term "IgG-C-scFv(2)" is well known in the art; see, for example, Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019 Aug; 18(8):585-608. doi:10.1038 / s41573-019-0028-1.PMID:31175342.

[0336] 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 measured by the equilibrium dissociation constant (KD) or half-maximal effective concentration (EC50) of the interaction. 50 )express.

[0337] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. The "binding rate constant" (k...) a or k on ) and "dissociation rate constant" (k dis or k off Both can be calculated from concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). dis / k on The ratio is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). KD and k can be measured by any effective method. on and k dis The dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method) in some implementations. Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used.

[0338] 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 polyomaviruses (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.

[0339] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is the one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. As used herein, the term "expression vector" refers to a vector containing recombinant polynucleotides that include expression regulatory sequences effectively linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as entrapments, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0340] As used herein, the term "host cell" refers to cells 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, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0341] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the Needleman algorithm in the GAP program integrated into the GCG software package (available at www.gcg.com) can be used, employing a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0342] The twenty common amino acids involved in this disclosure are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this disclosure, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in this disclosure, 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.

[0343] 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, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers 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). Stabilizers have the meaning commonly understood by those skilled in the art; stabilizers are those that can stabilize the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate).

[0344] The term "linker" or "linker unit" refers to a structural segment that links a cytotoxic drug to an antibody or antigen-binding fragment. For example, it refers to the formula Ab-[MLED]. x The -MLE- structure fragment in the text.

[0345] The term "drug linker" refers to the structure of the cytotoxic drug and linker described in this disclosure before they are linked to an antibody or its antigen-binding fragment. For example, "drug linker" refers to MLED. The "drug linker" is covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugate (ADC, also known as an antibody-drug conjugate) described in this application.

[0346] The "drug-linker" also includes all pharmaceutically acceptable isotopically labeled compounds identical to those of the "drug-linker" compounds of this disclosure, except that one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number in nature. Examples suitable for inclusion in the isotopes of this disclosure include, but are not limited to, isotopes of hydrogen (e.g., deuterium (D or 1200 ppm)). 2 H), tritium ( 3 H or T); carbon isotopes (e.g. 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0347] As used herein, the terms “DAR” or “drug-antibody ratio” or “drug-antibody conjugate ratio” (as used interchangeably herein) refer to the average number of linker / payload portions present in the composition that are linked to each antibody. For compositions containing the ADCs of this disclosure, the DAR of the composition is the average of all DARs (linker-payload portions of all ADC molecules present in the composition), which may be expressed as a decimal or an integer. Thus, in some embodiments of the compositions comprising ADC disclosed herein, the DAR of the composition is a decimal or integer among 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. In additional embodiments, for compositions of the present disclosure containing an ADC, the DAR of the composition is 3.0–3.5, 3.0–4.0, 3.0–4.5, 3.0–5.0, 3.0–5.5, 3.0–6.0, 3.5–4.0, 3.5–4.5, 3.5–5.0, 3.5–5.5, 3.5–6.0, 3.5–6.5, 3.5–7.0, 3.5–7.5, 3.5–8.0, 4.0–4.5, 4.0–5.0, 4.0–5.5, 4.0–6.0, 4.0–6.5, 4.0–7.0, 4.0–7.5, 4.0–8.0, or 4.5–5. Integers or decimals from 0, 4.5–5.5, 4.5–6.0, 4.5–6.5, 4.5–7.0, 4.5–7.5, 4.5–8.0, 5.0–5.5, 5.0–6.0, 5.0–6.5, 5.0–7.0, 5.0–7.5, 5.0–8.0, 5.5–6.0, 5.5–6.5, 5.5–7.0, 5.5–7.5, 5.5–8.0, 6.0–6.5, 6.0–7.0, 6.0–7.5, 6.0–8.5, 6.5–7.0, 6.5–7.5, 6.5–8.5, 7.0–7.5, 7.0–9.0, or 7.5–9.0. As used above, the term "composition" is understood to include pharmaceutical compositions. Mean DAR can be determined by a variety of conventional methods, such as ultraviolet spectroscopy, mass spectrometry, ELISA, radiometrics, hydrophobic interaction chromatography (HIC), electrophoresis, and HPLC.

[0348] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also mean prolonged survival compared to expected survival (if no treatment was received).

[0349] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) has a tumor, or is at risk of having the disease described herein.

[0350] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, a preventive effective amount means an amount sufficient to prevent, stop, or delay the onset of a disease (e.g., a tumor); a therapeutic effective amount means an amount sufficient to cure or at least partially stop an existing disease and its complications. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0351] As used herein, the term "effector function" refers to the biological activities attributable to the antibody's Fc region (either the native sequence Fc region or the Fc region of an amino acid sequence variant), and which vary with the antibody's Fc region. Examples of antibody effector functions include, but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADCP), binding to cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, and the half-life / clearance of antibodies and antigen-antibody complexes. Methods for altering antibody effector functions are known in the art, for example, by introducing mutations into the Fc region.

[0352] As used herein, the term “antibody-dependent cell-mediated cytotoxicity (ADCC)” refers to a form of cytotoxicity in which Ig interacts with Fc receptors (FcRs) present on cytotoxic cells (such as natural killer (NK) cells, neutrophils, or macrophages) to specifically bind to antigen-attached target cells, and then kills the target cells by secreting cytotoxins.

[0353] In this document, combination therapy includes the use of the bispecific antibody or pharmaceutical composition of this disclosure in combination with one or more other active therapeutic agents of a second therapy (e.g., chemotherapeutic agents) or other preventive or therapeutic modalities (e.g., radiotherapy).

[0354] In such combination therapies, the various active agents often have different complementary mechanisms of action, and the combination therapy may lead to a synergistic effect. Combination therapies include therapeutic agents that affect the immune response (e.g., enhance or activate the response) and therapeutic agents that affect (e.g., inhibit or kill) tumor / cancer cells. Combination therapies can reduce the likelihood of drug-resistant cancer cells developing. Combination therapies may allow for a reduction in the dosage of one or more agents in the regimen to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies may have a synergistic therapeutic or preventative effect on underlying diseases, conditions, or symptoms.

[0355] As used herein, "combination" includes therapies that can be administered separately, such as those formulated separately for individual administration (e.g., those provided in a kit), and therapies that can be administered together as a single formulation (i.e., a "co-formulation"). In some embodiments, the bispecific antibody of this disclosure may be administered sequentially. In other embodiments, the bispecific antibody may be administered simultaneously. The bispecific antibody of this disclosure may be used in combination with at least one other (active) pharmaceutical agent in any manner.

[0356] The terms "cancer" and "tumor" are used interchangeably to refer to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0357] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, such as "C". 1-20 Alkyl", C 1- 10 Alkyl", C 1-6 Alkyl", C 1-4 Alkyl", C 1-3Alkyl groups, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0358] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight-chain or branched hydrocarbon group, such as "C". 1-20 Alkylene, C 1-10 Alkylene, C 3-10 Alkylene, C 5-8 Alkylene, C 1-6 Alkylene, C 1-4 Alkylene, C 1-3 "alkylene", etc., specific examples include but are not limited to: methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene, etc.

[0359] The term "alkenyl" refers to a divalent group formed by the loss of two hydrogen atoms from a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C". 2-20 "Ideinyl", "C" 3-10 "Ideinyl", "C" 5-8 Examples of these include, but are not limited to: vinylidene, 1-propenyne, 2-propenyne, 1-butenyne, 2-butenyne, 1,3-butadiene, 1-pentenyne, 2-pentenyne, 3-pentenyne, 1,3-pentadiene, 1,4-pentadiene, 1-hexenyne, 2-hexenyne, 3-hexenyne, 1,4-hexadiene, etc.

[0360] The term "acetylenic" refers to a divalent group formed by the loss of two hydrogen atoms from a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. This includes, for example, "C..." 2-20 "Immyne", "C" 3-10 "Immyne", "C" 5-8 Examples of "ethynyl" include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentynyl, 1,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexynyl, etc.

[0361] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one ring member selected from N, O, and S. Specific examples include, but are not limited to, 5-6 membered aliphatic heterocycles, 5-6 membered nitrogen-containing aliphatic heterocycles, and 5-6 membered oxygen-containing aliphatic heterocycles, such as tetrahydrofuran, pyrrolidine, piperidine, and tetrahydropyran.

[0362] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O, and S. Specific examples include, but are not limited to, 5-6 membered aromatic heterocycles, 5-6 membered nitrogen-containing aromatic heterocycles, and 5-6 membered oxygen-containing aromatic heterocycles, such as furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, 1,2,4,5-tetraazine, etc.

[0363] The term "aromatic ring system" refers to a monocyclic or polycyclic system containing at least one aromatic ring (e.g., a benzene ring) or a heteroaromatic ring (e.g., a pyrimidine ring). Two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be linked by a single bond (e.g., a diamyrimidinylphenyl ring). The aromatic ring system may be divalent or higher valence (e.g., trivalent or tetravalent), such as 5-20 member aromatic ring systems.

[0364] abbreviation

[0365] The abbreviations used in this article have the following meanings:

[0366] Antibody-drug conjugates

[0367] In one aspect, this application provides a solution having the formula Ab-[MLED] x The antibody-drug conjugate shown has the following structure:

[0368] Ab is a bispecific antibody or its antigen-binding fragment containing a first antigen-binding domain that specifically binds to PDL1 and a second antigen-binding domain that specifically binds to B7H3.

[0369] M is the linker site that is linked to the bispecific antibody or its antigen-binding fragment;

[0370] L is a structural segment connecting the joint portions M and E;

[0371] E is a structural segment connecting L and D;

[0372] D is a cytotoxic drug or a fragment thereof; and

[0373] x is any integer from 1 to 10.

[0374] Antibodies for ADC

[0375] In a particular embodiment, the bispecific antibody of the ADC is a bispecific antibody with high affinity for both PDL1 and B7H3. It can specifically recognize or bind to PDL1 and B7H3, and inhibit or block the binding of PDL1 to the PD1 receptor and the binding of B7H3 to its receptor, thereby downregulating or blocking PDL1.

[0376] Bispecific antibodies

[0377] In some embodiments, the ADC is a bispecific antibody comprising a first antigen-binding domain that specifically binds to PDL1 and a second antigen-binding domain that specifically binds to B7H3.

[0378] In some embodiments, the first antigen-binding domain includes a first light chain variable region (VL) and a first heavy chain variable region (VH), wherein the first VL and the first VH together form a domain capable of specifically binding PDL1; the second antigen-binding domain includes a second VL and a second VH, wherein the second VL and the second VH together form a domain capable of specifically binding B7H3.

[0379] The individual CDRs of the antigen-binding domains of the bispecific antibodies of the ADCs provided in this article can be determined according to any CDR numbering scheme known in the art.

[0380] In some implementations, each CDR of the antigen-binding domain of the bispecific antibody of the ADC provided herein is determined independently according to one of the Chothia, AbM, Kabat, Contact, or IMGT numbering schemes, or by structural analysis of the antigen-binding domain, wherein the structural analysis identifies residues in the variable region that are expected to contact the epitope region of PDL1 or B7H3.

[0381] I. The first antigen-binding domain that specifically binds to PDL1

[0382] In some embodiments, the first VL includes LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO:61; and / or, the first VH includes HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:60.

[0383] The CDRs are defined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system.

[0384] In some implementations, the first VL includes:

[0385] (i) LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system;

[0386] (ii) LCDR1 comprising the sequence shown in SEQ ID NO:32, LCDR2 comprising the sequence shown in SEQ ID NO:35, and LCDR3 comprising the sequence shown in SEQ ID NO:38; wherein the CDRs are defined by the Contact numbering system; or,

[0387] (iii) LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the IMGT numbering system.

[0388] In some implementations, the first VH includes:

[0389] (i) HCDR1 containing the sequence shown in SEQ ID NO:18, HCDR2 containing the sequence shown in SEQ ID NO:23, and HCDR3 containing the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system;

[0390] (ii) HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system;

[0391] (iii) HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system;

[0392] (iv) HCDR1 comprising the sequence shown in SEQ ID NO:21, HCDR2 comprising the sequence shown in SEQ ID NO:26, and HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or,

[0393] (v) HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system.

[0394] In some embodiments, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:18, HCDR2 comprising the sequence shown in SEQ ID NO:23, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system.

[0395] In some embodiments, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system.

[0396] In some embodiments, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system.

[0397] In some embodiments, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:32, LCDR2 comprising the sequence shown in SEQ ID NO:35, and LCDR3 comprising the sequence shown in SEQ ID NO:38; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:21, HCDR2 comprising the sequence shown in SEQ ID NO:26, and HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system.

[0398] In some embodiments, the first antigen-binding domain that specifically binds to PDL1 comprises: a first VL, the first VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and / or, a first VH, the first VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system.

[0399] In some embodiments, the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 61. In some embodiments, the first VL comprises the amino acid sequence shown in SEQ ID NO: 61. In some embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO: 61.

[0400] In some embodiments, the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the first VH consists of the amino acid sequence shown in SEQ ID NO: 60.

[0401] In some embodiments, the first VL contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 60, and the first VH contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the first VL comprises the amino acid sequence shown in SEQ ID NO:61, and the first VH comprises the amino acid sequence shown in SEQ ID NO:60. In some embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO:61, and the first VH consists of the amino acid sequence shown in SEQ ID NO:60.

[0402] II. Second antigen-binding domain that specifically binds to B7H3

[0403] In some embodiments, the second VL includes LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO:63; and / or, the second VH includes HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:62;

[0404] The CDRs are defined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system.

[0405] In some implementations, the second VL includes:

[0406] (i) LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system.

[0407] (ii) LCDR1 comprising the sequence shown in SEQ ID NO:53, LCDR2 comprising the sequence shown in SEQ ID NO:56, and LCDR3 comprising the sequence shown in SEQ ID NO:59; wherein the CDRs are defined by the Contact numbering system; or,

[0408] (iii) LCDR1 comprising the sequence shown in SEQ ID NO:54, LCDR2 comprising the sequence shown in SEQ ID NO:57, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the IMGT numbering system.

[0409] In some implementations, the second VH comprises:

[0410] (i) HCDR1 containing the sequence shown in SEQ ID NO:39, HCDR2 containing the sequence shown in SEQ ID NO:44, and HCDR3 containing the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system;

[0411] (ii) HCDR1 comprising the sequence shown in SEQ ID NO:40, HCDR2 comprising the sequence shown in SEQ ID NO:45, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system;

[0412] (iii) HCDR1 comprising the sequence shown in SEQ ID NO:41, HCDR2 comprising the sequence shown in SEQ ID NO:46, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system;

[0413] (iv) HCDR1 comprising the sequence shown in SEQ ID NO:42, HCDR2 comprising the sequence shown in SEQ ID NO:47, and HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or,

[0414] (v) HCDR1 comprising the sequence shown in SEQ ID NO:43, HCDR2 comprising the sequence shown in SEQ ID NO:48, and HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0415] In some embodiments, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:41, HCDR2 comprising the sequence shown in SEQ ID NO:46, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system.

[0416] In some embodiments, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:39, HCDR2 comprising the sequence shown in SEQ ID NO:44, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system.

[0417] In some embodiments, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:40, HCDR2 comprising the sequence shown in SEQ ID NO:45, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system.

[0418] In some embodiments, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:53, LCDR2 comprising the sequence shown in SEQ ID NO:56, and LCDR3 comprising the sequence shown in SEQ ID NO:59; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:42, HCDR2 comprising the sequence shown in SEQ ID NO:47, and HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system.

[0419] In some embodiments, the second antigen-binding domain that specifically binds to B7H3 comprises: a second VL, the second VL comprising: LCDR1 comprising the sequence shown in SEQ ID NO:54, LCDR2 comprising the sequence shown in SEQ ID NO:57, and LCDR3 comprising the sequence shown in SEQ ID NO:58; and / or, a second VH, the second VH comprising: HCDR1 comprising the sequence shown in SEQ ID NO:43, HCDR2 comprising the sequence shown in SEQ ID NO:48, and HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0420] In some embodiments, the second VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 63. In some embodiments, the second VL comprises the amino acid sequence shown in SEQ ID NO: 63. In some embodiments, the second VL consists of the amino acid sequence shown in SEQ ID NO: 63.

[0421] In some embodiments, the second VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the second VH consists of the amino acid sequence shown in SEQ ID NO: 62.

[0422] In some embodiments, the second VL contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 63, and the second VH contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the second VL comprises the amino acid sequence shown in SEQ ID NO:63, and the second VH comprises the amino acid sequence shown in SEQ ID NO:62. In some embodiments, the second VL consists of the amino acid sequence shown in SEQ ID NO:63, and the second VH consists of the amino acid sequence shown in SEQ ID NO:62.

[0423] III. First antigen-binding domain / Second antigen-binding domain

[0424] In some embodiments of the bispecific antibody for the ADC provided in this application:

[0425] (i) the first VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:61, and the first VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:60; and

[0426] (ii) The second VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:63, and the second VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:62.

[0427] The CDRs are defined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system.

[0428] In some embodiments of the bispecific antibody of the ADC in this application:

[0429] (a) The first VL includes:

[0430] (i) LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system;

[0431] (ii) LCDR1 comprising the sequence shown in SEQ ID NO:32, LCDR2 comprising the sequence shown in SEQ ID NO:35, and LCDR3 comprising the sequence shown in SEQ ID NO:38; wherein the CDRs are defined by the Contact numbering system; or,

[0432] (iii) LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the IMGT numbering system;

[0433] and,

[0434] (b) The first VH includes:

[0435] (i) HCDR1 containing the sequence shown in SEQ ID NO:18, HCDR2 containing the sequence shown in SEQ ID NO:23, and HCDR3 containing the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system;

[0436] (ii) HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system;

[0437] (iii) HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system;

[0438] (iv) HCDR1 comprising the sequence shown in SEQ ID NO:21, HCDR2 comprising the sequence shown in SEQ ID NO:26, and HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or,

[0439] (v) HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system;

[0440] and

[0441] (c) The second VL includes:

[0442] (i) LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system.

[0443] (ii) LCDR1 comprising the sequence shown in SEQ ID NO:53, LCDR2 comprising the sequence shown in SEQ ID NO:56, and LCDR3 comprising the sequence shown in SEQ ID NO:59; wherein the CDRs are defined by the Contact numbering system; or,

[0444] (iii) LCDR1 comprising the sequence shown in SEQ ID NO:54, LCDR2 comprising the sequence shown in SEQ ID NO:57, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the IMGT numbering system;

[0445] and,

[0446] (d) The second VH includes:

[0447] (i) HCDR1 containing the sequence shown in SEQ ID NO:39, HCDR2 containing the sequence shown in SEQ ID NO:44, and HCDR3 containing the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system;

[0448] (ii) HCDR1 comprising the sequence shown in SEQ ID NO:40, HCDR2 comprising the sequence shown in SEQ ID NO:45, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system;

[0449] (iii) HCDR1 comprising the sequence shown in SEQ ID NO:41, HCDR2 comprising the sequence shown in SEQ ID NO:46, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system;

[0450] (iv) HCDR1 comprising the sequence shown in SEQ ID NO:42, HCDR2 comprising the sequence shown in SEQ ID NO:47, and HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or,

[0451] (v) HCDR1 comprising the sequence shown in SEQ ID NO:43, HCDR2 comprising the sequence shown in SEQ ID NO:48, and HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0452] In some embodiments of the bispecific antibody for the ADC provided in this application, the bispecific antibody comprises:

[0453] (1) A first antigen-binding domain that specifically binds to PDL1, selected from any of the following:

[0454] (i) A first VL, comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 comprising the sequence shown in SEQ ID NO:18, HCDR2 comprising the sequence shown in SEQ ID NO:23, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system;

[0455] (ii) A first VL, comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system;

[0456] (iii) A first VL, comprising: LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system;

[0457] (iv) A first VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:32, an LCDR2 comprising the sequence shown in SEQ ID NO:35, and an LCDR3 comprising the sequence shown in SEQ ID NO:38; and a first VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:21, an HCDR2 comprising the sequence shown in SEQ ID NO:26, and an HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or

[0458] (v) A first VL, comprising: LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system;

[0459] and,

[0460] (2) A second antigen-binding domain that specifically binds to B7H3, selected from any of the following:

[0461] (i) a second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:52, an LCDR2 comprising the sequence shown in SEQ ID NO:55, and an LCDR3 comprising the sequence shown in SEQ ID NO:58; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:41, an HCDR2 comprising the sequence shown in SEQ ID NO:46, and an HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system;

[0462] (ii) A second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:52, an LCDR2 comprising the sequence shown in SEQ ID NO:55, and an LCDR3 comprising the sequence shown in SEQ ID NO:58; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:39, an HCDR2 comprising the sequence shown in SEQ ID NO:44, and an HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system;

[0463] (iii) A second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:52, an LCDR2 comprising the sequence shown in SEQ ID NO:55, and an LCDR3 comprising the sequence shown in SEQ ID NO:58; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:40, an HCDR2 comprising the sequence shown in SEQ ID NO:45, and an HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system;

[0464] (iv) A second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:53, an LCDR2 comprising the sequence shown in SEQ ID NO:56, and an LCDR3 comprising the sequence shown in SEQ ID NO:59; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:42, an HCDR2 comprising the sequence shown in SEQ ID NO:47, and an HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or,

[0465] (v) A second VL, comprising: an LCDR1 comprising the sequence shown in SEQ ID NO:54, an LCDR2 comprising the sequence shown in SEQ ID NO:57, and an LCDR3 comprising the sequence shown in SEQ ID NO:58; and a second VH, comprising: an HCDR1 comprising the sequence shown in SEQ ID NO:43, an HCDR2 comprising the sequence shown in SEQ ID NO:48, and an HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0466] In some embodiments of the bispecific antibody for the ADC provided in this application, the bispecific antibody comprises:

[0467] (1) A first antigen-binding domain that specifically binds to PDL1, selected from any of the following:

[0468] (i) a first VL, the first VL comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, the first VH comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:18, HCDR2 with the amino acid sequence shown in SEQ ID NO:23, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28;

[0469] (ii) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:19, HCDR2 with the amino acid sequence shown in SEQ ID NO:24, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28;

[0470] (iii) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:20, HCDR2 with the amino acid sequence shown in SEQ ID NO:25, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28;

[0471] (iv) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:32, LCDR2 with the amino acid sequence shown in SEQ ID NO:35, and LCDR3 with the amino acid sequence shown in SEQ ID NO:38; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:21, HCDR2 with the amino acid sequence shown in SEQ ID NO:26, and HCDR3 with the amino acid sequence shown in SEQ ID NO:29; or

[0472] (v) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:33, LCDR2 with the amino acid sequence shown in SEQ ID NO:36, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:22, HCDR2 with the amino acid sequence shown in SEQ ID NO:27, and HCDR3 with the amino acid sequence shown in SEQ ID NO:30;

[0473] and,

[0474] (2) A second antigen-binding domain that specifically binds to B7H3, selected from any of the following:

[0475] (i) a second VL comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:41, HCDR2 with the amino acid sequence shown in SEQ ID NO:46, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49;

[0476] (ii) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:39, HCDR2 with the amino acid sequence shown in SEQ ID NO:44, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49;

[0477] (iii) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:40, HCDR2 with the amino acid sequence shown in SEQ ID NO:45, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49;

[0478] (iv) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:53, LCDR2 with the amino acid sequence shown in SEQ ID NO:56, and LCDR3 with the amino acid sequence shown in SEQ ID NO:59; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:42, HCDR2 with the amino acid sequence shown in SEQ ID NO:47, and HCDR3 with the amino acid sequence shown in SEQ ID NO:50; or,

[0479] (v) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:54, LCDR2 with the amino acid sequence shown in SEQ ID NO:57, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:43, HCDR2 with the amino acid sequence shown in SEQ ID NO:48, and HCDR3 with the amino acid sequence shown in SEQ ID NO:51.

[0480] In some embodiments of the bispecific antibody for the ADC provided in this application, the bispecific antibody comprises:

[0481] (1) A first antigen-binding domain that specifically binds to PDL1, selected from any of the following:

[0482] (i) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:18, HCDR2 with the amino acid sequence shown in SEQ ID NO:23, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system;

[0483] (ii) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:19, HCDR2 with the amino acid sequence shown in SEQ ID NO:24, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system;

[0484] (iii) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:31, LCDR2 with the amino acid sequence shown in SEQ ID NO:34, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:20, HCDR2 with the amino acid sequence shown in SEQ ID NO:25, and HCDR3 with the amino acid sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system;

[0485] (iv) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:32, LCDR2 with the amino acid sequence shown in SEQ ID NO:35, and LCDR3 with the amino acid sequence shown in SEQ ID NO:38; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:21, HCDR2 with the amino acid sequence shown in SEQ ID NO:26, and HCDR3 with the amino acid sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or

[0486] (v) A first VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:33, LCDR2 with the amino acid sequence shown in SEQ ID NO:36, and LCDR3 with the amino acid sequence shown in SEQ ID NO:37; and a first VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:22, HCDR2 with the amino acid sequence shown in SEQ ID NO:27, and HCDR3 with the amino acid sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system;

[0487] and,

[0488] (2) A second antigen-binding domain that specifically binds to B7H3, selected from any of the following:

[0489] (i) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:41, HCDR2 with the amino acid sequence shown in SEQ ID NO:46, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system;

[0490] (ii) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:39, HCDR2 with the amino acid sequence shown in SEQ ID NO:44, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system;

[0491] (iii) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:52, LCDR2 with the amino acid sequence shown in SEQ ID NO:55, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:40, HCDR2 with the amino acid sequence shown in SEQ ID NO:45, and HCDR3 with the amino acid sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system;

[0492] (iv) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:53, LCDR2 with the amino acid sequence shown in SEQ ID NO:56, and LCDR3 with the amino acid sequence shown in SEQ ID NO:59; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:42, HCDR2 with the amino acid sequence shown in SEQ ID NO:47, and HCDR3 with the amino acid sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or,

[0493] (v) A second VL, comprising: LCDR1 with the amino acid sequence shown in SEQ ID NO:54, LCDR2 with the amino acid sequence shown in SEQ ID NO:57, and LCDR3 with the amino acid sequence shown in SEQ ID NO:58; and a second VH, comprising: HCDR1 with the amino acid sequence shown in SEQ ID NO:43, HCDR2 with the amino acid sequence shown in SEQ ID NO:48, and HCDR3 with the amino acid sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system.

[0494] In some embodiments of the bispecific antibody of the ADC in this application:

[0495] (i) the first VL contains the amino acid sequence shown in SEQ ID NO:61, and / or the first VH contains the amino acid sequence shown in SEQ ID NO:60; and

[0496] (ii) The second VL contains an amino acid sequence as shown in SEQ ID NO:63, and / or the second VH contains an amino acid sequence as shown in SEQ ID NO:62.

[0497] In some embodiments of the bispecific antibody for the ADC provided in this application:

[0498] (i) The amino acid sequence of the first VL is as shown in SEQ ID NO:61, and / or, the amino acid sequence of the first VH is as shown in SEQ ID NO:60; and

[0499] (ii) The amino acid sequence of the second VL is as shown in SEQ ID NO:63, and / or the amino acid sequence of the second VH is as shown in SEQ ID NO:62.

[0500] In some embodiments, this disclosure provides an ADC comprising a bispecific antibody that cross-competes with any antibody described herein for binding to PDL1 and / or B7H3. In some embodiments, this disclosure provides an ADC comprising a bispecific antibody that binds to the same or overlapping epitopes of PDL1 and / or B7H3 with the antibody described herein.

[0501] In some implementations, the epitopes of the antibody can be determined by, for example, NMR spectroscopy, surface plasmon resonance. X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange and mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping) are used to determine the crystals. For X-ray crystallography, crystallization can be performed using any method known in the art (e.g., Gigé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are incorporated herein by reference in their entirety). Antibodies: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) volumes 114 & 115, edited by Eds. Wyckoff HW et al.; U.S. Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr. D. Biocrystals 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323, all of which are incorporated herein by reference in their entirety). Mutagenic mapping studies can be performed using any method known to those skilled in the art. Descriptions of mutagenic techniques, including alanine scan mutagenesis, can be found, for example, in Champe M et al. (1995) and Cunningham BC and Wells JA (1989). In one specific embodiment, alanine scan mutagenesis is used to determine the epitopes of the antibody. Additionally, bispecific antibodies that recognize and bind to the same or overlapping epitopes of PDL1 and / or B7H3 can be identified using conventional techniques such as immunoassays, for example by demonstrating the ability of one antibody to block the binding of another antibody to the target antigen, i.e., a competitive binding assay. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity to the epitopes. Competitive binding can be determined in assays that specifically bind to a reference antibody inhibited by the tested immunoglobulin to a common antigen, such as PDL1 or B7H3.Several types of competitive binding assays are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli C et al., (1983) Method Enzyme Mole 9:242-253); solid-phase direct biotinylate EIA (see Kirkland TN et al., (1986) J immunl 137:3614-9); solid-phase direct labeling, solid-phase direct labeling sandwich assay (see Harlow E & Lane D, (1988) Antibody: Laboratory Handbook, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 labeling (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid-phase direct biotin-avidinase immunoassay (see Zhang RC et al., (1990) Virology 176:546-52); and directly labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference in their entirety. Typically, such assays involve the use of purified antigens (e.g., PDL1 or B7H3) bound to a solid surface or cells containing any of the above, unlabeled target immunoglobulin, and labeled reference immunoglobulin. Competitive inhibition can be determined by measuring the amount of labeling bound to the solid surface or cells in the presence of the target immunoglobulin. Typically, an excess of the target immunoglobulin is present. Typically, when a competitive antibody is present in excess, it will inhibit the specific binding of a reference antibody to the same antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured in a wide variety of different formats using labeled antigens or labeled antibodies. In a common version of this assay, the antigen is immobilized on a 96-well plate. Radioactive or enzyme labeling is then used to determine the ability of the unlabeled antibody to block the binding of the labeled antibody to the antigen. For further details, see, for example, Wagener C et al., (1983) J immunl 130:2308-2315; Weiner et al., (1984) Immunomethods 68:269-274; Kuroki M et al., (1990) Cancer Res 50:4872-4879; Kuroki M et al., (1992) Immunol Invest 21:523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D, eds., pp. 386-389, all of which are incorporated herein by reference in their entirety.

[0502] IV. Structure

[0503] The first antigen-binding domain and the second antigen-binding domain of the bispecific antibody of the ADC disclosed herein include a Fab structure, and the other includes a Fab structure, an scFv structure, or a VH / VL structure.

[0504] Unless otherwise explicitly stated, Fab domains may be arranged according to their natural orientation or may contain domain substitutions or exchanges that facilitate proper VH and VL pairing (e.g., domain exchanges of the form CrossMab).

[0505] CrossMab is a method for preventing light chain and heavy chain mismatch, see US Patent US9266967B2, which is incorporated herein by reference in its entirety. This method primarily utilizes the principle of mutual repulsion among the same parts of an antibody, i.e., VH vs. VH, CH1 vs. CH1, CL vs. CL, and VL vs. VL. It prevents light chain mismatch by exchanging regions of the heavy and light chains of one of the Fab components in a bispecific antibody (e.g., exchanging light chain CL-VL with heavy chain CH1-VH, VL and VH, or CH and CL).

[0506] In some implementations, the CrossMab-style domain swaps are selected from:

[0507] CrossMab Fab In the Fab, the antibody light chain CL-VL and the antibody heavy chain CH1-VH were interchanged;

[0508] CrossMab VH-VL In Fab, the VL and VH of the antibody were interchanged; and

[0509] CrossMab CH1-CL In Fab, the CH and CL of the antibody are interchanged.

[0510] In some embodiments, the bispecific antibody further comprises an Fc domain. In some embodiments, the Fc domain is an Fc domain comprising a first domain monomer and a second Fc domain monomer. In some embodiments, the first and second Fc domain monomers each independently comprise one or more amino acid modifications that promote heterodimerization of the first and second Fc domain monomers.

[0511] In some embodiments, the Fc domain comprises a first Fc domain monomer containing amino acid modifications capable of forming a knot structure and a second Fc domain monomer containing amino acid modifications capable of forming a hole structure, wherein the hole structure can pair with the knot structure to form the Fc domain.

[0512] Those skilled in the art will readily understand that the hole structure can pair with the knot structure to form a "knob in hole (KIH)". The "knob in hole" structure is a method for reducing heavy chain mismatches in bispecific antibodies by introducing amino acid modifications that form a "knob" and a "hole" structure at corresponding positions in the CH3 domains of two Fc domain monomers, respectively, to form a specific interaction interface between the two Fc domain monomers (see Ridgway et al., Protein Eng., 9: 617-621 (1996); WO 2006 / 028936; the full text of which is incorporated herein by reference). Because the "knob" and "hole" structures attract each other, and the "knob" structures repel each other, heavy chain mismatches in bispecific antibodies can be effectively reduced.

[0513] WO9850431 discloses amino acid modifications that promote heterodimer formation, specifically a KIH method for generating Fc domain monomer heterodimers. In this KIH method, one Fc domain monomer in the heterodimer pair contains an amino acid substitution that produces a protrusion (knob) extending outward from the surface of the Fc domain monomer. This protrusion is adapted to a depression (hole) created by an appropriate amino acid substitution in the other Fc domain monomer in the heterodimer pair. This promotes heterodimer formation more than homodimer formation. Examples of amino acid substitutions include S354C:T366W amino acid substitution of the first Fc domain monomer to form a knob, and Y349C:T366S:L368A:Y407V amino acid substitution of the second Fc domain monomer to form a hole (amino acids according to EU index numbers), wherein the first and second Fc domain monomers form a heterodimer pair. WO2014084607 discloses a KIH, wherein the Fc heterodimer comprises a first Fc domain monomer and a second Fc domain monomer, the first Fc domain monomer comprising a K409W amino acid substitution to form a knot, and the second Fc domain monomer comprising D399V and F405T amino acid substitutions to form a hole (amino acid numbers according to the EU index), wherein the first and second Fc domain monomers form a heterodimer pair. WO2013063702 discloses a KIH, wherein the Fc heterodimer comprises a first Fc domain monomer comprising amino acid modifications at positions T350, L351, F405, and Y407, and a second Fc domain monomer comprising amino acid modifications at positions T350, T366, K392, and T394 (amino acid numbers according to the EU index), wherein the first and second Fc domain monomers form a heterodimer pair.

[0514] In some embodiments, the Fc domain monomer is derived from the Fc domain of a human immunoglobulin and modified based on the Fc domain to obtain an Fc domain monomer capable of forming a knock or hole structure. For example, the amino acid sequence of a first Fc domain monomer is modified in a specific region to provide a knock structure, and the amino acid sequence of a second Fc domain monomer is modified in a corresponding region to provide a hole structure capable of accepting a knock structure on the first Fc domain monomer, thereby allowing the first and second Fc domain monomers to form heterodimer pairs while preventing them from forming homodimer pairs. In some embodiments, the human immunoglobulin is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain monomer contains one or more modifications that form a knock structure, wherein the one or more modifications contain S354C and / or T366W amino acid substitutions, numbered according to the Eu numbering scheme. In some embodiments, the Fc domain monomer contains S354C and T366W amino acid substitutions, numbered according to the Eu numbering scheme. In some embodiments, the Fc domain monomer comprises one or more modifications forming a hole structure, wherein said one or more modifications comprise amino acid substitutions of Y349C, T366S, L368A, and / or Y407V, wherein the substitutions are numbered according to the Eu numbering scheme.

[0515] In some embodiments, the Fc domain further possesses altered effector functions (e.g., enhanced ADCC activity) compared to the wild-type Fc region. These altered effector functions can be introduced, for example, by mutation or chemical modification of the Fc domain monomer in the Fc heterodimer.

[0516] In some embodiments of the ADC provided herein, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1, according to the EU numbering system) and / or the hinge region (residues 216-230, according to the EU numbering system) of the bispecific antibody of the ADC to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding and / or antigen-dependent cytotoxicity.

[0517] In a further embodiment, the first and second Fc domain monomers further comprise one or more amino acid substitutions that reduce or eliminate the function of the Fc region effectors. In certain specific embodiments, the first and second Fc domain monomers further comprise: (i) L234A / L235A / G237A (LALAGA) amino acid substitutions, wherein the amino acids are numbered according to the Eu numbering scheme; or (ii) L234A / L235A / P329A (LALAPA) amino acid substitutions, wherein the amino acids are numbered according to the Eu numbering scheme.

[0518] IgG-scFv

[0519] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the other is an scFv.

[0520] In some embodiments, the scFv is optionally linked to the C-terminus of the heavy chain of the full-length antibody via a peptide linker.

[0521] In some embodiments, the scFv is optionally linked to the N-terminus of the heavy chain of the full-length antibody via a peptide linker. In some embodiments, the scFv (preferably, two) is optionally linked to the N-terminus of the heavy chain of the full-length antibody via a peptide linker. In some embodiments, the scFv (preferably, one) is optionally linked to the N-terminus of the heavy chain of the full-length antibody via a peptide linker.

[0522] In some embodiments, the peptide linker is 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, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO: 66, 67, or 68.

[0523] In some embodiments, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide comprising (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68.

[0524] In some implementations, the VH and VL of the scFv do not contain disulfide bonds.

[0525] In some embodiments, a disulfide bond is present between the VH and VL of the scFv. Methods for introducing a disulfide bond between the VH and VL of the antibody are well known in the art, for example, by introducing a cysteine ​​residue onto each of the VH and VL residues to form a disulfide bond.

[0526] 1. IgG-C-scFv(2)

[0527] In some embodiments, the first antigen-binding domain is scFv, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0528] (i) A peptide chain VA, comprising a VL of the second antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region;

[0529] (ii) A peptide chain VB comprising a VH region of the second antigen-binding domain, a heavy chain CH1 region, an Fc domain monomer, and the first antigen-binding domain; preferably, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1; preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3; preferably, the first antigen-binding domain is connected to the C-terminus of the Fc domain monomer via a peptide linker; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO: 66 or 68;

[0530] Preferably, the scFv has a structure shown as [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, for example, containing (G4S). n The flexible peptide, where n is an integer not less than 0, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68;

[0531] Preferably, the VH and VL of the scFv may or may not contain disulfide bonds.

[0532] In some embodiments, the bispecific antibody comprises two identical or different peptide chains VA and two identical or different peptide chains VB, wherein the two peptide chains VB form a dimer through their respective Fc domain monomers.

[0533] In some embodiments, the bispecific antibody comprises two distinct peptide chains VA, wherein the VL and / or light chain constant region (CL) of the second antigen-binding domain are different.

[0534] In some embodiments, the bispecific antibody comprises different peptide chains VB, wherein the VH of the second antigen-binding domain, the CH1 region of the heavy chain, the Fc domain monomer, and / or the first antigen-binding domain are different.

[0535] In some implementations, the Fc domain monomers further include, independently, modifications that can alter the function of the effector.

[0536] Preferably, the modification that alters the effector function includes the following mutations in EU number: L234A / L235A / G237A;

[0537] Preferably, the Fc domain monomer comprises a sequence selected from the following sequence: SEQ ID NO: 74 or 75.

[0538] 2. N-scFv(2)-IgG

[0539] In some embodiments, the first antigen-binding domain is scFv, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0540] (i) peptide chain III-A, which includes the VL of the second antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region;

[0541] (ii) Peptide chain III-B, comprising the first antigen-binding domain, and a VH region of the second antigen-binding domain, a heavy chain CH1 region, and an Fc domain monomer; preferably, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1; preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3; preferably, the first antigen-binding domain is connected to the N-terminus of the VH region of the second antigen-binding domain via a peptide linker; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:67.

[0542] Preferably, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68;

[0543] Preferably, the scFv may or may not contain a disulfide bond between VH and VL;

[0544] In some embodiments, the bispecific antibody comprises two identical or different peptide chains III-A and two identical or different peptide chains III-B, wherein the two peptide chains III-B form a dimer through their respective Fc domain monomers.

[0545] In some embodiments, the bispecific antibody comprises two distinct peptide chains III-A, wherein the VL of the second antigen-binding domain and / or the constant region (CL) of the light chain are different.

[0546] In some embodiments, the bispecific antibody comprises two different peptide chains III-B, wherein the VH, heavy chain CH1 region, and / or Fc domain monomers of the first antigen-binding domain and the second antigen-binding domain are different.

[0547] In some implementations, the Fc domain monomers further include, independently, modifications that can alter the function of the effector.

[0548] Preferably, the modification that alters the effector function includes the following mutations in EU number: L234A / L235A / G237A;

[0549] Preferably, the Fc domain monomer contains the sequence shown in SEQ ID NO:74.

[0550] 3. N-scFv(1)-IgG

[0551] In some embodiments, the first antigen-binding domain is scFv, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0552] (i) peptide chain IV-A, which includes the VL of the second antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region;

[0553] (ii) Peptide chain IV-B, comprising the VH of the second antigen-binding domain, the CH1 region of the heavy chain, and a first Fc domain monomer (or a second Fc domain monomer); preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3.

[0554] (iii) A peptide chain IV-C comprising the first antigen-binding domain, a VH region of the second antigen-binding domain, a heavy chain CH1 region, and a monomer of the second Fc domain (or a monomer of the first Fc domain); preferably, the first antigen-binding domain is connected to the N-terminus or C-terminus of the VH region of the second antigen-binding domain via a peptide linker; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO: 67 or 68.

[0555] Preferably, the first and second Fc domain monomers are Fc domain monomers of IgG, such as Fc domain monomers of IgG1; preferably, the Fc domain monomers comprise a hinge region, CH2, and CH3.

[0556] Preferably, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68;

[0557] Preferably, the scFv may or may not contain a disulfide bond between VH and VL;

[0558] In some embodiments, the first and second Fc domain monomers each independently contain modifications of one or more amino acids that promote dimerization of the first and second Fc domain monomers;

[0559] 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 "knob-into-hole" modification;

[0560] Preferably, the "knob" modification is the EU-numbered T366W / S354C mutation;

[0561] Preferably, the “hole” is modified to be the EU numbered T366S / L368A / Y407V / Y349C mutation.

[0562] In some embodiments, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:72 and 73, SEQ ID NO:73 and 72.

[0563] In some embodiments, the bispecific antibody comprises two identical or different peptide chains IV-A, one peptide chain IV-B, and one peptide chain IV-C, wherein peptide chains IV-B and IV-C form a dimer through their respective Fc domain monomers.

[0564] In some embodiments, the bispecific antibody comprises two distinct peptide chains IV-A, wherein the VL of the second antigen-binding domain and / or the constant region (CL) of the light chain are different.

[0565] In some embodiments, the first and second Fc domain monomers of the Fc domain further each independently include modifications that can alter the function of the effector.

[0566] Preferably, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / P329A;

[0567] Preferably, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:72 and 73, or SEQ ID NO:73 and 72.

[0568] In some embodiments, the bispecific antibody comprises:

[0569] (1) A peptide chain VA comprising the amino acid sequence shown in SEQ ID NO:2, and / or a peptide chain VB comprising the amino acid sequence shown in SEQ ID NO:5;

[0570] (2) peptide chain III-A comprising the amino acid sequence shown in SEQ ID NO:2, and / or peptide chain III-B comprising the amino acid sequence shown in SEQ ID NO:4;

[0571] (3) A peptide chain IV-A containing the amino acid sequence shown in SEQ ID NO:2, a peptide chain IV-B containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain IV-C containing the amino acid sequence shown in SEQ ID NO:12.

[0572] (4) A peptide chain IV-A containing the amino acid sequence shown in SEQ ID NO:2, a peptide chain IV-B containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain IV-C containing the amino acid sequence shown in SEQ ID NO:15.

[0573] In some embodiments, the bispecific antibody comprises:

[0574] (1) Peptide chain VA with amino acid sequence as shown in SEQ ID NO:2, and / or peptide chain VB with amino acid sequence as shown in SEQ ID NO:5;

[0575] (2) Peptide chain III-A with an amino acid sequence as shown in SEQ ID NO:2, and / or peptide chain III-B with an amino acid sequence as shown in SEQ ID NO:4;

[0576] (3) Peptide chain IV-A with amino acid sequence as shown in SEQ ID NO:2, peptide chain IV-B with amino acid sequence as shown in SEQ ID NO:1, and / or peptide chain IV-C with amino acid sequence as shown in SEQ ID NO:12;

[0577] (4) Peptide chain IV-A with amino acid sequence as shown in SEQ ID NO:2, peptide chain IV-B with amino acid sequence as shown in SEQ ID NO:1, and / or peptide chain IV-C with amino acid sequence as shown in SEQ ID NO:15.

[0578] Fab-scFv / Fab

[0579] 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; and the bispecific antibody 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 peptide linkers, respectively.

[0580] 1. Fab-scFv

[0581] In some embodiments, the first antigen-binding domain is scFv, the second antigen-binding domain is Fab, and the bispecific antibody comprises:

[0582] (i) A peptide chain IA comprising a first antigen-binding domain and a first Fc domain monomer; preferably, the first Fc domain monomer comprises a hinge region, CH2 and CH3; preferably, the first antigen-binding domain is connected to the N-terminus of the first Fc domain monomer via a peptide linker; in some embodiments, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:64.

[0583] (ii) a peptide chain IB comprising a second antigen-binding domain VH, a heavy chain CH1 region, and a second Fc domain monomer; preferably, the second Fc domain monomer comprises a hinge region, CH2, and CH3; and

[0584] (iii) Peptide chain IC, which includes a second protobinding domain VL and a light chain constant region (CL).

[0585] In some implementations, the second Fc domain monomer forms a dimer with the first Fc domain monomer.

[0586] In some embodiments, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide comprising (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68.

[0587] Preferably, the VH and VL of the scFv may or may not contain disulfide bonds.

[0588] In some embodiments, the bispecific antibody comprises a peptide chain IA, a peptide chain IB, and a peptide chain IC, wherein peptide chains IA and IB form a dimer through their respective Fc domain monomers.

[0589] 2. Fab-Fab (Exchange)

[0590] 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.

[0591] In some implementations, the CrossMab-style domain swaps are selected from:

[0592] (a)CrossMabFab: In Fab, the antibody light chain CL-VL and antibody heavy chain CH1-VH are interchanged;

[0593] (b) CrossMabVH-VL: In the Fab, the VL and VH of the antibody were interchanged; or

[0594] (c)CrossMabCH1-CL: In Fab, the CH and CL of the antibody are interchanged.

[0595] In some embodiments, the bispecific antibody comprises:

[0596] (i) Peptide chain II-A, which includes the VL of the first antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region;

[0597] (ii) peptide chain II-B, which includes the VH of the first antigen-binding domain, the CH1 region of the heavy chain, and the monomer of the first Fc domain.

[0598] (iii) Peptide chain II-C, comprising the VH of the second antigen-binding domain, the light chain constant region (CL), and the second Fc domain monomer;

[0599] (iv) Peptide chain II-D, which includes the VL region of the second antigen-binding domain and the CH1 region of the heavy chain;

[0600] In some implementations, the first Fc domain monomer and the second Fc domain monomer form a dimer.

[0601] In some embodiments, the bispecific antibody comprises a peptide chain II-A, a peptide chain II-B, a peptide chain II-C, and a peptide chain II-D, wherein peptide chains II-B and II-C form a dimer through their respective Fc domain monomers.

[0602] In some embodiments, the first and second Fc domain monomers in any of the above Fab-scFv or Fab-Fab structures each independently contain modifications of one or more amino acids that promote dimerization of the first and second Fc domain monomers.

[0603] In some implementations, 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 "knob-into-hole" modification.

[0604] In some implementations, the “knob” is modified to be the EU-numbered T366W / S354C mutation;

[0605] In some implementations, the “hole” is modified to be the EU-numbered T366S / L368A / Y407V / Y349C mutation.

[0606] In some implementations, the first and second Fc domain monomers in any of the above Fab-scFv or Fab-Fab structures may further and independently include modifications that can change the function of the effector.

[0607] In some implementations, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / P329A.

[0608] In some embodiments, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:72 and 73, or SEQ ID NO:73 and 72.

[0609] In some embodiments, the bispecific antibody comprises:

[0610] (1) A peptide chain IA comprising the amino acid sequence shown in SEQ ID NO:3, a peptide chain IB comprising the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain IC comprising the amino acid sequence shown in SEQ ID NO:2; or

[0611] (2) A peptide chain II-A containing the amino acid sequence shown in SEQ ID NO:9, a peptide chain II-B containing the amino acid sequence shown in SEQ ID NO:8, a peptide chain II-C containing the amino acid sequence shown in SEQ ID NO:10, and / or a peptide chain II-D containing the amino acid sequence shown in SEQ ID NO:11.

[0612] In some embodiments, the bispecific antibody comprises:

[0613] (1) Peptide chain IA with the amino acid sequence as shown in SEQ ID NO:3, peptide chain IB with the amino acid sequence as shown in SEQ ID NO:1, and / or peptide chain IC with the amino acid sequence as shown in SEQ ID NO:2; or

[0614] (2) Peptide chain II-A with amino acid sequence as shown in SEQ ID NO:9, peptide chain II-B with amino acid sequence as shown in SEQ ID NO:8, peptide chain II-C with amino acid sequence as shown in SEQ ID NO:10, and / or peptide chain II-D with amino acid sequence as shown in SEQ ID NO:11.

[0615] DVD-IgG and its variant structures

[0616] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the other is VH / VL.

[0617] In some embodiments, the VH is attached to the N-terminus of the heavy chain of the full-length antibody via a peptide linker; and the VL is attached to the N-terminus of the light chain of the full-length antibody via a peptide linker.

[0618] In some embodiments, the VH is attached to the N-terminus of one of the heavy chains of the full-length antibody via a peptide linker; and the VL is attached to the N-terminus of one of the light chains of the full-length antibody via a peptide linker.

[0619] 1. DVD-IgG

[0620] In some embodiments, the first antigen-binding domain is VH / VL, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0621] (i) Peptide chain VI-A, comprising a VL of the first antigen-binding domain, a VL of the second antigen-binding domain, and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; preferably, the VL of the first antigen-binding domain is connected to the N-terminus of the VL of the second antigen-binding domain via a peptide linker.

[0622] (ii) Peptide chain VI-B, comprising a VH of the first antigen-binding domain, a VH of the second antigen-binding domain, a heavy chain CH1 region, and an Fc domain monomer; preferably, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1; preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3; preferably, the VH of the first antigen-binding domain is connected to the N-terminus of the VH of the second antigen-binding domain via a peptide linker.

[0623] In some embodiments, each peptide linker is independently selected from peptide linkers comprising one or more glycine (G) and / or serine (S), such as flexible peptides comprising (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, each peptide linker independently comprises an amino acid sequence as shown in SEQ ID NO:68.

[0624] In some implementations, the Fc domain monomer includes modifications that enable it to have altered effector functions.

[0625] In some implementations, the modification of the altered effector function includes the following mutations with EU numbers: L234A / L235A / G237A.

[0626] In some embodiments, the Fc domain monomer comprises the sequence shown in SEQ ID NO:74.

[0627] In some embodiments, the bispecific antibody comprises two identical or different peptide chains VI-A and two identical or different peptide chains VI-B, wherein the two peptide chains VI-B form a dimer through their respective Fc domain monomers.

[0628] In some embodiments, the bispecific antibody comprises two different peptide chains VI-A, wherein the VL of the first antigen-binding domain, the VL of the second antigen-binding domain, and / or the light chain constant region (CL) are different.

[0629] In some embodiments, the bispecific antibody comprises different peptide chains VI-B, wherein the VH of the first antigen-binding domain, the VH of the second antigen-binding domain, the heavy chain CH1 region, and / or the Fc domain monomers are different.

[0630] 2. Deformed structure of DVD-IgG

[0631] In some embodiments, the first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), the second antigen-binding domain is VH / VL, and the bispecific antibody comprises:

[0632] (i) Peptide chain VII-A, comprising a VL of the second antigen-binding domain, a VL of the first antigen-binding domain, and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; preferably, the VL of the second antigen-binding domain is connected to the N-terminus of the VL of the first antigen-binding domain via a peptide linker.

[0633] (ii) Peptide chain VII-B, comprising VH of the second antigen-binding domain, VH of the first antigen-binding domain, heavy chain CH1 region, and monomer of the second Fc domain; preferably, VH of the second antigen-binding domain is connected to the N-terminus of VH of the first antigen-binding domain via a peptide linker.

[0634] (iii) A peptide chain VII-C, comprising the VL of the second antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; and

[0635] (iv) Peptide chain VII-D, comprising the VH of the second antigen-binding domain, the CH1 region of the heavy chain, and a monomer of the first Fc domain;

[0636] Preferably, the first and second Fc domain monomers are Fc domain monomers of IgG, such as Fc domain monomers of IgG1; preferably, the Fc domain monomers comprise a hinge region, CH2, and CH3.

[0637] In some embodiments, the first and second Fc domain monomers each independently contain modifications of one or more amino acids that promote dimerization of the first and second Fc domain monomers.

[0638] In some implementations, 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 "knob-into-hole" modification.

[0639] In some implementations, the “knob” is modified to be the EU-numbered T366W / S354C mutation;

[0640] In some implementations, the “hole” is modified to be the EU-numbered T366S / L368A / Y407V / Y349C mutation.

[0641] In some implementations, the first and second Fc domain monomers may further each independently include modifications that can alter the function of the effector.

[0642] In some implementations, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / P329A.

[0643] In some embodiments, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:72 and 73, or SEQ ID NO:73 and 72.

[0644] In some embodiments, the bispecific antibody comprises a peptide chain VII-A, a peptide chain VII-B, a peptide chain VII-C, and a peptide chain VII-D, wherein peptide chains VII-B and VII-D form a dimer through their respective Fc domain monomers.

[0645] In some embodiments, the bispecific antibody comprises:

[0646] (1) peptide chain VI-A comprising the amino acid sequence shown in SEQ ID NO:7, and / or peptide chain VI-B comprising the amino acid sequence shown in SEQ ID NO:6; or

[0647] (2) A peptide chain VII-A containing the amino acid sequence shown in SEQ ID NO:14, a peptide chain VII-B containing the amino acid sequence shown in SEQ ID NO:13, a peptide chain VII-C containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain VII-D containing the amino acid sequence shown in SEQ ID NO:2.

[0648] In some embodiments, the bispecific antibody comprises:

[0649] (1) Peptide chain VI-A with the amino acid sequence as shown in SEQ ID NO:7, and / or peptide chain VI-B with the amino acid sequence as shown in SEQ ID NO:6; or

[0650] (2) Peptide chain VII-A with amino acid sequence as shown in SEQ ID NO:14, peptide chain VII-B with amino acid sequence as shown in SEQ ID NO:13, peptide chain VII-C with amino acid sequence as shown in SEQ ID NO:1, and / or peptide chain VII-D with amino acid sequence as shown in SEQ ID NO:2.

[0651] ADC connector

[0652] The ADC provided herein includes a linker-MLE- for linking a cytotoxic drug fragment D to the antibody.

[0653] On the one hand, this disclosure provides a connection unit having the following structure: -MLE-

[0654] Where: M is the linker site where the linker unit connects to the bispecific antibody or its antigen-binding fragment;

[0655] L is a structural segment connecting the joint portions M and E;

[0656] E is a structural segment that connects L and the toxin molecule.

[0657] In some implementations, M includes Wherein, ring A is a 5-6 membered aliphatic heterocycle or a 5-20 membered aromatic ring system, wherein the aliphatic heterocycle and aromatic ring system are optionally selected independently by one or more groups selected from oxygen (=O), halogen, cyano, amino, carboxyl, mercapto, and C. 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2- 20 Alynyl or amino group.

[0658] In some implementations, M includes Wherein ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by one or more 6-membered heteroaromatic rings connected to a benzene ring via single bonds, or a polycyclic ring formed by multiple 6-membered heteroaromatic rings connected via single bonds, wherein the aliphatic heterocycle is optionally surrounded by one or more elements selected from oxygen (=O), halogens, and C. 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

[0659] In some implementations, M includes Where ring A is selected from M1 is selected from single bond, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl or amino group.

[0660] In some implementations, M is selected from

[0661] In some implementation schemes, M is

[0662] In some implementations, M is selected from

[0663] In some implementations, M is selected from

[0664] In some implementations, L is selected from one or more of the following structures: C 1-6 Alkyl group, -N(R')-, carbonyl group, -O-, selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) s Natural or non-natural amino acids and their analogues containing 1, 2, 3 or 4 amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG(SEQ)) ID NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81))),

[0665] Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20.

[0666] In some embodiments, the short peptide is selected from Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG(SEQ ID)) NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81)).

[0667] In some embodiments, the L is selected from structures comprising one or more of the following: C 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly(GGFG(SEQ ID NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81)), Where s is selected from integers from 1 to 20.

[0668] In some implementations, L is selected from one or more of the following structures:

[0669] In some implementations, L is selected from the following structures:

[0670] In some implementations, L is selected from the following structures:

[0671] In some implementations, L is selected from the following structures:

[0672] In some implementations, L is selected from the following structures:

[0673] In some implementations, L is selected from the following structures:

[0674] In some implementations, E is a single bond or selected from the following structures:

[0675] -NHCH2-, -NHCH2-O-CH2-CO-, -CO-O-CH2-CO-,

[0676] In some implementations, E is a single bond, -NHCH2-, -NHCH2-O-CH2-CO-,

[0677] In some implementations, E is -NHCH2- or

[0678] In some implementations, E is -NHCH2- or a single bond.

[0679] In some implementations, E is -NHCH2- or -NHCH2-O-CH2-CO-.

[0680] In some implementations, E is

[0681] In some implementations, the connection unit is selected from the following structures:

[0682] In some implementations, the connection unit has the following structure:

[0683] In another aspect, this disclosure provides compounds comprising the aforementioned connecting units.

[0684] In some embodiments, the compound is selected from:

[0685] In another aspect, this disclosure provides the use of the above-described linker or compound in the preparation of the antibody-drug conjugate of this disclosure.

[0686] ADC drug-linker

[0687] The ADC provided herein comprises a bispecific antibody as described herein, and further comprises a cytotoxic drug fragment D, which is linked to the antibody via a linker-MLE-.

[0688] In some embodiments, the linker described in European Patent Publication EP 4349372 is incorporated herein by reference. In some embodiments, the linker (MLE) and the individual components M, L, and E as described in European Patent Publication EP 4349372 are incorporated herein by reference. In some embodiments, the cytotoxic drug described in European Patent Publication EP 4349372 is incorporated herein by reference. In some embodiments, the linker / cytotoxic drug described in European Patent Publication EP 4349372 is incorporated herein by reference.

[0689] In some implementations, M includes Wherein, ring A is a 5-6 membered aliphatic heterocycle or a 5-20 membered aromatic ring system, wherein the aliphatic heterocycle and aromatic ring system are optionally selected independently by one or more groups selected from oxygen (=O), halogen, cyano, amino, carboxyl, mercapto, and C. 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2- 20 Alynyl or amino group.

[0690] In some implementations, M includes Wherein ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by one or more 6-membered heteroaromatic rings connected to a benzene ring via single bonds, or a polycyclic ring formed by multiple 6-membered heteroaromatic rings connected via single bonds, wherein the aliphatic heterocycle is optionally surrounded by one or more elements selected from oxygen (=O), halogens, and C. 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

[0691] In some implementations, M includes Where ring A is selected from M1 is selected from single bond, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl or amino group.

[0692] In some implementations, M is selected from

[0693] In some implementations, M is

[0694] In some implementations, M is selected from

[0695] In some implementations, M is selected from

[0696] In some implementations, L is selected from one or more of the following structures: C 1-6 Alkyl group, -N(R')-, carbonyl group, -O-, selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) sNatural or non-natural amino acids and their analogues containing 1, 2, 3 or 4 amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG(SEQ)) ID NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81))),

[0697] Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20.

[0698] In some embodiments, the short peptide is selected from Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG(SEQ ID)) NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81)).

[0699] In some embodiments, the L is selected from structures comprising one or more of the following: C 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly(GGFG(SEQ ID NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81)), Where s is selected from integers from 1 to 20.

[0700] In some implementations, L is selected from one or more of the following structures:

[0701] In some implementations, L is selected from the following structures:

[0702] In some implementations, L is selected from the following structures:

[0703] In some implementations, L is selected from the following structures:

[0704] In some implementations, L is selected from the following structures:

[0705] In some implementations, L is selected from the following structures:

[0706] In some implementations, E is a single bond or selected from the following structures:

[0707] -NHCH2-, -NHCH2-O-CH2-CO-, -CO-O-CH2-CO-,

[0708] In some implementations, E is a single bond, -NHCH2-, -NHCH2-O-CH2-CO-,

[0709] In some implementations, E is -NHCH2- or

[0710] In some implementations, E is -NHCH2- or a single bond.

[0711] In some implementations, E is -NHCH2- or -NHCH2-O-CH2-CO-.

[0712] In some implementations, E is

[0713] In some implementation schemes, Selected from the following structures:

[0714] In some implementation schemes, Selected from the following structures:

[0715] In some embodiments, the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

[0716] In some embodiments, the microtubule inhibitor is an olistatin or maytansine compound.

[0717] In some embodiments, the olistatin compound is selected from the following:

[0718] In some embodiments, the DNA intercalating agent is pyrrolobenzodiazepine. (PBD)

[0719] In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0720] In some embodiments, the topoisomerase I inhibitor is selected from camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, rubotecan, and pharmaceutically acceptable salts, esters, or analogs thereof; the topoisomerase II inhibitor is selected from doxorubicin, PNU-159682, docalimcin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, and pharmaceutically acceptable salts, esters, or analogs thereof.

[0721] In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analogue thereof.

[0722] In some embodiments, the cytotoxic agent is selected from compounds of Formula I and II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of Formula I and II.

[0723] Among them, R1 and R2 are each independently selected from C. 1-6 Alkyl and halogen;

[0724] R3 is selected from H and -CO-CH2OH;

[0725] R4 and R5 are each independently selected from H, halogens, and hydroxyl groups; or R4 and R5 are linked to form a 5-6 membered oxygen-containing heterocycle;

[0726] R6 is selected from hydrogen or -C. 1-4 Alkylene-NR a R b ;

[0727] R7 is selected from C 1-6 Alkyl, -C 1-4 Alkylene-NR a R b -C 1-4 Alkylene-SiR a R b R c -SiR a R b R c -C 1-4 Alkylene = N-OR a ;where R a R b and R c Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, -SO2-C 1-6 Alkyl and -CO-C 1-6 Alkyl; wherein R is optionally present a and R b It forms 5-6 member nitrogen-containing heterocycles with the connected atoms.

[0728] In some embodiments, the cytotoxic agent is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds:

[0729] The fragment of the cytotoxic drug obtained after the cytotoxic drug is linked to the linker is D in the above general formula.

[0730] In some embodiments, the cytotoxic agent is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds:

[0731] The fragment of the cytotoxic drug obtained after the cytotoxic drug is linked to the linker is D in the above general formula.

[0732] D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amine group on the cytotoxic drug.

[0733] Connection between connector and drug (load)

[0734] In some embodiments, the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, -SH, -NH2, secondary amine group, or tertiary amine group.

[0735] In some embodiments, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amine group on the cytotoxic drug.

[0736] ADC Examples

[0737] In some embodiments, the antibody-drug conjugate is selected from ADC A-01 to ADC A-41, ADC B-01 to ADC B-09, ADC C-01 to ADC C-28, and ADC D-01 to ADC D-03, wherein the structure of the ADC is as follows:

[0738] Wherein, Ab or HA is a bispecific antibody or its antigen-binding fragment as defined above;

[0739] This indicates the specific connection method between the thiol group in the bispecific antibody or its antigen-binding fragment and other parts of the antibody-drug conjugate; and x represents the amount of drug load.

[0740] In some embodiments, the thiol group in the bispecific antibody or its antigen-binding fragment forms a thioether bond with the precursor of other parts of the antibody-drug conjugate through an addition reaction or substitution reaction to obtain the antibody-drug conjugate.

[0741] In some specific embodiments of the antibody-drug conjugates disclosed herein, Ab or HA is selected from bispecific antibodies bsAb01, bsAb02, bsAb03, bsAb04, bsAb05, bsAb06, bsAb07, and bsAb08. For a detailed description of bsAb01, bsAb02, bsAb03, bsAb04, bsAb05, bsAb06, bsAb07, and bsAb08 in some embodiments, please refer to Example 1 of this disclosure.

[0742] In some specific embodiments of the antibody-drug conjugate disclosed herein, Ab or HA is selected from bispecific antibodies bsAb01, bsAb02, bsAb03, bsAb04, Ab05, Ab06, Ab07, or Ab08.

[0743] In some embodiments of the antibody-drug conjugates disclosed herein, the antibody-drug conjugates are selected from bsAb01-A-10, bsAb02-A-10, bsAb03-A-10, bsAb04-A-10, bsAb05-A-10, bsAb06-A-10, bsAb07-A-10, and bsAb08-A-10.

[0744] In some specific embodiments of the antibody-drug conjugate disclosed herein, the antibody-drug conjugate is bsAb01-A-10, and its structural formula is shown below:

[0745] Wherein, X can be an integer between 1 and 10, preferably an integer between 3 and 10, or an integer between 5 and 10. For example, X can be 5, 6, 7, 8, 9, or 10.

[0746] In some specific embodiments of the antibody-drug conjugate, the antibody-drug conjugate is bsAb02-A-10, and its structural formula is shown below:

[0747] Wherein, X can be an integer between 1 and 10, preferably an integer between 3 and 10, or an integer between 5 and 10. For example, X can be 5, 6, 7, 8, 9, or 10.

[0748] In some specific embodiments of the antibody-drug conjugate, the antibody-drug conjugate is bsAb03-A-10, and its structural formula is shown below:

[0749] Wherein, X can be an integer between 1 and 10, preferably an integer between 3 and 10, or an integer between 5 and 10. For example, X can be 5, 6, 7, 8, 9, or 10.

[0750] In some specific embodiments of the antibody-drug conjugate, the antibody-drug conjugate is bsAb04-A-10, and its structural formula is shown below:

[0751] Wherein, X can be an integer between 1 and 10, preferably an integer between 3 and 10, or an integer between 5 and 10. For example, X can be 5, 6, 7, 8, 9, or 10.

[0752] In some specific embodiments of the antibody-drug conjugate, the antibody-drug conjugate is bsAb05-A-10, and its structural formula is shown below:

[0753] Wherein, X can be an integer between 1 and 10, preferably an integer between 3 and 10, or an integer between 5 and 10. For example, X can be 5, 6, 7, 8, 9, or 10.

[0754] In some specific embodiments of the antibody-drug conjugate, the antibody-drug conjugate is bsAb06-A-10, and its structural formula is shown below:

[0755] Wherein, X can be an integer between 1 and 10, preferably an integer between 3 and 10, or an integer between 5 and 10. For example, X can be 5, 6, 7, 8, 9, or 10.

[0756] In some specific embodiments of the antibody-drug conjugate, the antibody-drug conjugate is bsAb07-A-10, and its structural formula is shown below:

[0757] Wherein, X can be an integer between 1 and 10, preferably an integer between 3 and 10, or an integer between 5 and 10. For example, X can be 5, 6, 7, 8, 9, or 10.

[0758] In some specific embodiments of the antibody-drug conjugate, the antibody-drug conjugate is bsAb08-A-10, and its structural formula is shown below:

[0759] Wherein, X can be an integer between 1 and 10, preferably an integer between 3 and 10, or an integer between 5 and 10. For example, X can be 5, 6, 7, 8, 9, or 10.

[0760] In specific embodiments of the antibody-drug conjugates disclosed herein, the heavy chain constant region of the bispecific antibody of the antibody-drug conjugate may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments, the N-terminal amino acid of the variable region of the bispecific antibody of the antibody-drug conjugate may contain a glutamic acid or glutamine residue, or the glutamic acid or glutamine residue may be cyclized to pyroglutamic acid.

[0761] In some embodiments, the N-terminal amino acid of the bispecific antibody or its antigen-binding fragment variable region of the antibody-drug conjugate may be cyclized to pyroglutamic acid. Therefore, in the antibody-drug conjugate compositions provided in this disclosure, each antibody-drug conjugate may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or contain an N-terminal glutamine or glutamate residue, or have an N-terminal glutamine or glutamate cyclized to a pyroglutamate salt.

[0762] Therefore, in some embodiments, this application further provides compositions comprising the antibody-drug conjugates described herein, wherein the major antibody-drug conjugate in the composition comprises (i) an antibody lacking a lysine residue at the C-terminus of the heavy chain; (ii) an antibody containing a glutamine, glutamic acid, or pyroglutamic acid residue at the N-terminus of the heavy chain; (iii) an antibody lacking a lysine residue at the C-terminus of the heavy chain and having a glutamine, glutamic acid, or pyroglutamic acid residue at the N-terminus of the heavy chain; (iv) an antibody lacking a lysine residue at the C-terminus of the heavy chain and having a pyroglutamic acid residue at the N-terminus of the heavy chain; or, (v) an antibody lacking a lysine residue at the C-terminus of the heavy chain and having a glutamine or glutamic acid residue at the N-terminus of the heavy chain.

[0763] 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.

[0764] In some embodiments, the DAR value of the composition is 3-9, or 4-8, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5-6.0, 3.5-6.5, 3.5-7.0, 3.5-7.5, 3.5-8.0, 4.0-4.5, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.0-6.5, 4.0-7.0, 4.0-7.5, 4.0-8.0, 4.5- 5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0~5.5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0765] In any of the above embodiments of the compositions disclosed herein, the antibody-drug conjugates comprising the compositions are bsAb01-A-10, bsAb02-A-10, bsAb03-A-10, bsAb04-A-10, bsAb05-A-10, bsAb06-A-10, bsAb07-A-10, and bsAb08-A-10.

[0766] Couplet

[0767] In another aspect, this application provides a method for preparing the antibody-drug conjugate (ADC) described herein by combining the adapter-load described herein with the antibody described herein.

[0768] In some implementations, the antibody described herein binds to the adapter-load described herein by binding to lysine residues in the antibody.

[0769] In some embodiments, the antibody described herein binds to the linker-loador described herein by binding to cysteine ​​residues in the antibody. In some embodiments, the cysteine ​​residues are derived from reduced intrachain disulfide bonds in the antibody. In some embodiments, the cysteine ​​residues are derived from reduced interchain disulfide bonds in the antibody.

[0770] In some embodiments, the antibody is conjugated to the linker-payload by conjugation to reduced interchain disulfide bonds in the antibody. For example, an IgG1 antibody consists of four polypeptide chains, two heavy chains containing VH, CH1, and Fc (e.g., hinge region, CH2, and CH3) domains, and two light chains containing VL and CL domains, linked by interchain cysteine ​​disulfide bonds (-SS-) (e.g., two heavy-to-light chain interchain disulfide bonds and two hinge-to-heavy chain interchain disulfide bonds). In some embodiments, when these disulfide bonds break under reducing conditions, eight (8) reactive cysteine ​​thiol moieties are generated. In some embodiments, each of the eight reactive cysteine ​​thiol moieties is a linker-drug conjugate site, thus allowing up to eight (x=8) linker-payloads to be conjugated to the reduced antibody. In some embodiments, any one of the four disulfide bonds breaks under reducing conditions, generating two (2) reactive cysteine ​​thiol moieties. In a further embodiment, each of the two reactive cysteine ​​thiol moieties is a linker-drug junction, thus allowing two (x=2) linker-drugs to the reducing antibody. In some embodiments, any two of the four disulfide bonds break under reducing conditions, resulting in four (4) reactive cysteine ​​thiol moieties. In a further embodiment, each of the four reactive cysteine ​​thiol moieties is a linker-drug junction, thus allowing four (x=4) linker-drugs to the reducing antibody. In some embodiments, any three of the four disulfide bonds break under reducing conditions, resulting in six (6) reactive cysteine ​​thiol moieties. In a further embodiment, each of the six reactive cysteine ​​thiol moieties is a linker-drug junction, thus allowing six (x=6) linker-drugs to the reducing antibody.

[0771] In some embodiments, the interchain disulfide bond is located between two cysteine ​​residues that cleave under reducing conditions to produce two reactive cysteine ​​thiol moieties. In further embodiments, the interchain disulfide bond in the antibody is located between a heavy chain and a light chain, for example, between C220 of the heavy chain according to EU designation and C214 of the kappa light chain according to EU and / or Kabat designation, or between C220 of the heavy chain according to EU designation and C214 of the lambda light chain according to EU and / or Kabat designation. And / or, the interchain disulfide bond in the antibody is located between two heavy chains, for example, between C226 and / or C229 of the first heavy chain according to EU designation and C226 and / or C229 of the second heavy chain. In some embodiments, the cysteine ​​residues are located in the hinge region of the antibody. In some embodiments, the cysteine ​​residue is located at one or more of positions 220, 226, or 229 in the heavy chain according to EU numbering (also referred to herein as C220, C226, or C229, respectively). In some embodiments, the cysteine ​​residue is located at position 214 in the light chain according to EU and / or Kabat numbering (also referred to herein as C214, e.g., position 214 in the kappa light chain according to EU and Kabat numbering or position 214 in the lambda light chain according to EU and Kabat numbering). In one embodiment, the cysteine ​​residue is located at each of positions 220, 226, and 229 in the heavy chain according to EU numbering and position 214 in the light chain according to EU or Kabat numbering. In one embodiment, the cysteine ​​residue is located at each of positions 220, 226, and 229 in the heavy chain according to EU numbering and position 214 in the kappa light chain according to EU and Kabat numbering. In one embodiment, the cysteine ​​residue is located at each of positions 220, 226, and 229 in the heavy chain according to EU numbers, and at position 214 in the lambda light chain according to EU and Kabat numbers. In one embodiment, the cysteine ​​residue is located at any one or more of the following positions:

[0772] (i) Any one, two, or three of the 220, 226, and 229 positions in the first heavy chain of the EU number;

[0773] (ii) Any one, two, or three of the 220, 226, and 229 positions in the second chain of the EU number;

[0774] (iii) 214th bit in the first light chain according to the Kabat number and / or EU number; and / or

[0775] (iv) 214th bit in the second light chain according to the Kabat number and / or EU number.

[0776] As used herein, C220, C226, and C229 refer to amino acid residues (cysteine, Cys, C) of immunoglobulins identified according to EU numbers. Those skilled in the art will understand that such numbers correspondingly represent amino acid residues of peptides aligned with amino acid residues identified in immunoglobulins, such as those shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0777] As used herein, the cysteine ​​residue at position 214 of the κ light chain refers to the amino acid residue (cysteine, Cys, C) of the immunoglobulin identified by Eu or Kabat numbering. Those skilled in the art will understand that such numbering correspondingly represents amino acid residues of a polypeptide aligned with amino acid residues identified in the immunoglobulin, such as those shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html.

[0778] As used herein, the cysteine ​​residue at position 214 of the λ light chain refers to the amino acid residue (cysteine, Cys, C) of the immunoglobulin identified by Eu or Kabat numbering. Those skilled in the art will understand that such numbering correspondingly indicates amino acid residues of polypeptides aligned with the amino acid residues identified in the immunoglobulin, such as the amino acid residues shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGLCnber.html.

[0779] In some embodiments, the antibody described herein contains four interchain disulfide bonds in the hinge region that can be reduced to break the bonds and expose a reactive thiol moiety that can bind to the maleimide portion on the linker-payload (e.g., the maleimide portion on the linker-payload described herein).

[0780] In some embodiments, the antibodies described herein comprise lysine residues, wherein the reactive amine side chains of the lysine residues can bind to the adapter-load, such as the maleimide portion of the adapter-load described herein.

[0781] In one embodiment, this disclosure provides a method for preparing the ADC described herein, comprising the steps of: a) providing a solution containing an antibody; b) contacting the solution of a) with a reducing agent; and c) contacting the solution of b) with a solution containing a linker-payload or a salt thereof, as described herein, to prepare the ADC.

[0782] In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0783] Pharmaceutical compositions or combinations

[0784] In another aspect, this application provides a pharmaceutical composition comprising one or more antibody-drug conjugates or compositions as described in any one of the preceding claims, and a pharmaceutically acceptable carrier and / or excipient.

[0785] 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. In some embodiments, the additional pharmaceutically active agent is selected from: TROP2 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, B7H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof. In some embodiments, the ADC or composition as described herein and the additional pharmaceutically active agent are provided as separate components or as a mixture of components.

[0786] In some embodiments, the ADC or composition in the pharmaceutical composition of this disclosure is sufficient (e.g., in a subject) to exert a tumor-suppressive effect (e.g., superior to the tumor-suppressive effect of monospecific anti-PDL1 antibody and / or monospecific anti-B7H3 antibody, wherein the amino acid sequence of the CDR of the monospecific anti-PDL1 antibody is identical to the amino acid sequence of the CDR of the first antigen-binding domain, and the amino acid sequence of the CDR of the monospecific anti-B7H3 antibody is identical to the amino acid sequence of the CDR of the second antigen-binding domain).

[0787] In some embodiments, the antibody-drug conjugates described herein are typically formulated into unit injection forms, such as intravenous boluses, intravenous injections, intratumoral injections, etc., together with a pharmaceutically acceptable parenteral carrier for parenteral use. Optionally, antibody-drug conjugates of desired purity are mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers to form lyophilized or solution forms (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A.Ed.). The antibody-drug conjugates described herein, or pharmaceutical compositions comprising said antibody-drug conjugates, may be administered to subjects via any suitable route.

[0788] Therapeutic applications of ADC

[0789] On the other hand, this disclosure provides the use of the antibody-drug conjugates, compositions, pharmaceutical compositions, or combinations thereof in the preparation of a medicament for use in subjects to prevent and / or treat and / or as adjunctive therapy for diseases associated with PDL1 and / or B7H3, and / or for use in vitro or in subjects to inhibit the activity of PDL1 and / or B7H3; optionally, the disease associated with PDL1 and / or B7H3 is a tumor; optionally, the tumor is B7H3 and / or PDL1 positive.

[0790] In some embodiments, the tumor is selected from lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Preferably, the tumor is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma.

[0791] In some embodiments, the antibody-drug conjugate or pharmaceutical composition is administered in combination with an additional pharmaceutically active agent, for example, simultaneously, separately, or sequentially. In some embodiments, the additional pharmaceutically active agent is a drug with antitumor activity. In some embodiments, the additional pharmaceutically active agent is selected from: TROP2 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, B7H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0792] In another aspect, this disclosure provides a method for inhibiting the activity of PDL1 and / or B7H3 in cells, comprising contacting the cells with the antibody-drug conjugate or pharmaceutical composition described herein. The cells are cells expressing PDL1 and / or B7H3, such as tumor cells.

[0793] In another aspect, this disclosure provides a method for preventing, treating, and / or treating, as adjunctive medicine, diseases associated with PDL1 and / or B7H3 in a subject, the method comprising administering to a subject in need an effective amount of any of the preceding antibody-drug conjugates, compositions, and / or pharmaceutical compositions.

[0794] In some embodiments, the disease associated with PDL1 and / or B7H3 is a tumor. In some embodiments, the tumor is B7H3 and / or PDL1 positive.

[0795] In some embodiments, the tumor is selected from lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Preferably, the tumor is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma.

[0796] In some implementations, the method further includes administering a second therapy to the subject, selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0797] In some implementations, the second therapy may be administered simultaneously, alone, or sequentially with the methods described above.

[0798] In certain embodiments, this disclosure provides an ADC, composition, or pharmaceutical composition as described herein for use in subjects to prevent, treat, and / or as adjunctive medicine for diseases associated with PDL1 and / or B7H3.

[0799] In some embodiments, the disease associated with PDL1 and / or B7H3 is a tumor. In some embodiments, the tumor is B7H3 and / or PDL1 positive.

[0800] In some embodiments, the tumor is selected from lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Preferably, the tumor is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma.

[0801] In some embodiments, the prevention, treatment, and / or adjunctive therapy of diseases associated with PDL1 and / or B7H3 in the subject further includes administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjunctive therapy, and any combination thereof. In some embodiments, the second therapy may be administered simultaneously, separately, or sequentially.

[0802] The ADC and pharmaceutical compositions 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 solutions for injection, sterile powders for injection, and concentrates for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions disclosed herein should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by adding the desired dose of the ADC or pharmaceutical composition disclosed herein to a suitable solvent, and optionally simultaneously adding other desired ingredients (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Furthermore, for ease of storage and use, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying). This sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0803] Furthermore, the ADC disclosed herein can be present in the pharmaceutical composition in unit dose form for convenient administration.

[0804] The ADC and pharmaceutical composition of this disclosure can be administered by any suitable method known in the art, including but not limited to oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, groin, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some preferred embodiments, the ADC and pharmaceutical composition of this disclosure are administered by intravenous injection or injection.

[0805] The pharmaceutical compositions disclosed herein may comprise a "therapeutic effective amount" or a "preventive effective amount" of the bispecific antibody disclosed herein. A "preventive effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of disease. A "therapeutic effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutic effective amount of the bispecific antibody disclosed herein may vary depending on factors such as the severity of the disease to be treated, the general state of the patient's immune system, the patient's general condition, such as age, weight, and sex, the route of administration of the drug, and other concurrent treatments.

[0806] In this disclosure, the dosing regimen can be adjusted to obtain the optimal desired response (e.g., therapeutic or preventative response). For example, a single dose can be administered, multiple doses can be administered over time, or the dose can be reduced or increased proportionally depending on the urgency of the treatment situation.

[0807] In this disclosure, the subjects may be mammals, such as humans.

[0808] Reagent test kit

[0809] Kits comprising one or more ADCs or pharmaceutical compositions thereof described herein are also provided. In certain embodiments, a pharmaceutical package or kit is provided herein comprising one or more containers containing one or more components of the pharmaceutical compositions described herein, such as one or more ADCs provided herein. In some embodiments, the kit comprises the pharmaceutical compositions described herein and any preventative or therapeutic agent, such as those described herein. In some embodiments, the kit may include T-cell mitogens, such as phytohemagglutinin (PHA) and / or phorbol myristate (PMA), or TCR complex stimulating antibodies, such as anti-CD3 antibodies and anti-CD28 antibodies. Optionally, associated with such containers may be a notification in the form of a government agency directing the manufacture, use, or sale of a pharmaceutical or biological product, reflecting the agency's approval for the manufacture, use, or sale for human administration.

[0810] Kits for use with the methods described herein are also provided. In some embodiments, the kit includes the ADC described herein, preferably a purified ADC, housed in one or more containers. In a particular embodiment, the kit described herein includes substantially isolated PDL1 and / or B7H3 antigens as controls. In another particular embodiment, the kit described herein also includes a control antibody that does not react with the PDL1 or B7H3 antigen. In yet another particular embodiment, the kit described herein includes one or more elements for detecting the binding of the antibody to the PDL1 and / or B7H3 antigen (e.g., the antibody may be conjugated to a detectable substrate (e.g., a fluorescent compound, enzyme substrate, radioactive compound, or luminescent compound), or a second antibody recognizing the first antibody may be conjugated to the detectable substrate). In a particular embodiment, the kits provided herein may include recombinantly generated or chemically synthesized PDL1 and / or B7H3 antigens. The antigens provided in the kit may also be attached to a solid support. In more specific embodiments, the detection means of the above-described kits includes a solid-phase carrier with PDL1 and / or B7H3 antigens attached. Such kits may also include anti-human antibodies or anti-mouse / rat antibodies without attached reporter labels. In this implementation, the binding of the bispecific antibody to the antigen can be detected by the binding of the reporter label antibody.

[0811] All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein in their entirety for all purposes, to the same extent that each individual reference (e.g., publication, patent, or patent application) is specifically and individually indicated to be incorporated herein in its entirety for all purposes. Other embodiments are within the scope of the following claims.

[0812] Sequence information

[0813] The descriptions of the sequences involved in this application are provided in the table below.

[0814] I. Antibody Preparation

[0815] Example 1: Preparation of anti-PDL1-B7H3 bispecific antibody

[0816] 1.1 Structure and sequence of bispecific antibodies

[0817] Based on published patents CN107151269B and WO2024114525A1, the sequences of anti-PDL1 antibody Ab01 and anti-B7H3 antibody Ab02 were determined. Specifically, the heavy chain sequence of antibody Ab01 is SEQ ID NO:70; the light chain sequence is SEQ ID NO:9; the heavy chain sequence of antibody Ab02 is SEQ ID NO:71; and the light chain sequence is SEQ ID NO:2.

[0818] Based on the sequences of anti-PDL1 antibody Ab01 and anti-B7H3 antibody Ab02, this disclosure provides bispecific antibodies bsAb01 to bsAb08 capable of simultaneously binding to PDL1 and B7H3. These eight bispecific antibodies employ structures of “Fab-scFv / Fab”, “IgG-scFv”, and “DVD-IgG and its variants”, respectively. Specific descriptions are as follows.

[0819] (1) Fab-scFv / Fab bispecific antibody

[0820] The bispecific antibody bsAb01 employs a "Fab-scFv" antibody structure. This structure consists of three polypeptide chains, comprising two distinct heavy chains and one light chain. Knob-into-hole (KIH) point mutations are introduced into the CH3 domains of the two heavy chains to prevent mismatches, and L234A / L235A / P329A mutations are further introduced into the Fc domain to eliminate ADCC activity. Specifically, the amino acid mutations used in bsAb01 to form the "knob" structure are S354C and T366W; the amino acid mutations used to form the "hole" structure are Y349C, T366S, L368A, and Y407V. The positions of these amino acids are assigned according to EU numbers.

[0821] The amino acid sequence composition of the bsAb01 bispecific antibody is shown in Table 1.

[0822] Table 1: Polypeptide chain composition of bispecific antibody bsAb01

[0823] The bispecific antibody bsAb05 uses a "Fab-Fab" antibody structure (also known as CrossMab). CH1-CL The structure consists of four polypeptide chains, including two different heavy chains and two different light chains. Knob-into-hole (KIH) point mutations were introduced into the CH3 domains of the two heavy chains to prevent mismatches, and L234A / L235A / P329A mutations were further introduced into the Fc domain. Specifically, the amino acid mutations used in bsAb05 to form the "knob" structure are S354C and T366W; the amino acid mutations used to form the "hole" structure are Y349C, T366S, L368A, and Y407V. The positions of these amino acids are according to EU numbers. In the Anti-PDL1 portion, the CH1 and CL domains of the Fab retain the original antibody sequence (VH-CH1 and VL-CL Fab structures), while B7H3 binds to and exchanges the CH1 and CL domains in the Fab, resulting in VH-CL and VL-CH1 Fab structures, to promote the correct assembly of the two different light chains.

[0824] The amino acid sequence composition of the bsAb05 bispecific antibody is shown in Table 2.

[0825] Table 2: CrossMab CH1-CL Bispecific antibody amino acid sequence

[0826] (2) IgG-scFv bispecific antibody

[0827] The bispecific antibody bsAb02 adopts an "IgG-N-scFv" antibody structure, containing two scFvs. This structure consists of four polypeptide chains, including two identical heavy chains and two identical light chains. Additionally, amino acids (according to EU designations) are introduced into the Fc domains of the two heavy chains to replace L234A / L235A / G237A, thereby removing the antibody's ADCC activity.

[0828] The amino acid sequence composition of the bsAb02 bispecific antibody is shown in Table 3.

[0829] Table 3: Amino acid sequence of N-(scFv)2-IgG bispecific antibody

[0830] Bispecific antibodies bsAb06 and bsAb08 both employ an "IgG-N-scFv" bispecific antibody structure, containing one scFv. This structure consists of four polypeptide chains, including two different heavy chains and two identical light chains. Knob-into-hole (KIH) point mutations are introduced into the CH3 domain of both heavy chains to prevent mismatches between the two heavy chains; the specific mutation types are as described previously. Furthermore, L234A / L235A / P329A mutations are introduced into the Fc domain.

[0831] In the second heavy chain of the bsAb06 bispecific antibody, the anti-PDL1 scFv portion from the N-terminus to the C-terminus contains the following fragments: VL-linker 1-VH; the N-terminus of the anti-B7H3 VH-CH1-Fc(hole) portion (arranged from the N-terminus to the C-terminus) is connected to the anti-PDL1 scFv portion via linker 2. Linker 1 is the peptide linker (GGGGS) 4 as shown in SEQ ID NO:68, and linker 2 is the peptide linker (GGGGS) 3 as shown in SEQ ID NO:67.

[0832] In the second chain of the bsAb08 bispecific antibody, the anti-PDL1 scFv portion from the N-terminus to the C-terminus includes the following fragments: VL-linker 3-VH; the C-terminus of anti-B7H3 VH is linked to the anti-PDL1 scFv portion via linker 4. Both linker 3 and linker 4 are peptide linkers (GGGGS)4 as shown in SEQ ID NO:68.

[0833] The amino acid sequence composition of the bsAb06 and bsAb08 bispecific antibodies is shown in Table 4.

[0834] Table 4: Amino acid sequence of N-(scFv)1-IgG bispecific antibody

[0835] The bispecific antibody bsAb03 adopts an "IgG-C-scFv" antibody structure. This structure consists of four polypeptide chains, including two identical heavy chains and two identical light chains. Additionally, amino acids (according to EU designations) are introduced into the Fc domains of the two heavy chains to replace L234A / L235A / G237A.

[0836] In the heavy chain of the bsAb03 bispecific antibody, the anti-PDL1 scFv portion is composed of the VH and VL of the Ab01 antibody, and from the N-terminus to the C-terminus, it includes the following: VL-linker 5-VH; the C-terminus of anti-B7H3 VH-CH1-Fc (arranged from the N-terminus to the C-terminus) is linked to anti-PDL1 scFv via linker 6. Linker 5 is the peptide linker (GGGGS)4 as shown in SEQ ID NO:68, and linker 6 is the peptide linker (GGGGS)2 as shown in SEQ ID NO:66.

[0837] The amino acid sequence composition of the bsAb03 bispecific antibody is shown in Table 5.

[0838] Table 5: Amino acid sequence of IgG-C-(scFv)2 bispecific antibody

[0839] (3) DVD-IgG and its variant bispecific antibodies

[0840] The bispecific antibody bsAb04 adopts a "DVD-IgG" antibody structure. This structure consists of four polypeptide chains, including two identical heavy chains and two identical light chains. Amino acids (according to EU designations) are introduced into the Fc domains of the two heavy chains to replace L234A / L235A / G237A.

[0841] The amino acid sequence composition of the bsAb04 bispecific antibody is shown in Table 6.

[0842] Table 6: Amino acid sequence of DVD-IgG bispecific antibody

[0843] The bispecific antibody bsAb07 employs a "DVD-IgG" variant bispecific antibody structure. This structure consists of four polypeptide chains, including two different heavy chains and two different light chains. Knob-into-hole (KIH) point mutations are introduced into the CH3 domain of both heavy chains to prevent mismatches between the two heavy chains; the specific mutation types are as described previously. Furthermore, L234A / L235A / P329A mutations are introduced into the Fc domain.

[0844] The amino acid sequence composition of the bsAb07 bispecific antibody is shown in Table 7.

[0845] Table 7: Bispecific antibody amino acid sequence of DVD-IgG deformed structure

[0846] 1.2 Gene Synthesis and Bispecific Antibody Expression

[0847] The nucleotide sequences encoding the aforementioned bispecific antibodies were sent to GenScript Biotech for codon optimization and DNA synthesis. The DNA encoding the heavy and light chains of the different bispecific antibody forms was ligated into plasmid pTT5 (contracted to GenScript). The pTT5 plasmids corresponding to the heavy and light chain nucleotides of the fully human antibody were simultaneously transfected into CHO-S cells, and the transfected cells were cultured in cell culture medium to express the bispecific antibodies. After allowing the bispecific antibodies to express for a period of time, the supernatant was collected by centrifugation, yielding the recombinant bispecific antibodies in the supernatant. These antibodies were then purified using Protein A (MabSelect SuRe, GE) to obtain the aforementioned bispecific antibodies with different structures.

[0848] II. Synthesis of Drug-Linker Compounds

[0849] Example 2: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonoxy-3,9-diazapentazatriacylamino)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indo[1,2-b]quinoline-9-yl) carbonate (B-03)

[0850] Step 1: Preparation of ethyl acetate 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)amino)-2-oxoethyl acetate (B-03-1)

[0851] (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]-indo[1,2-b]quinoline-10,13-dione (2 g, 3.65 mmol) was dissolved in DMF (50 mL), and DIPEA (1.18 g, 9.12 mmol, 1.59 mL) was added dropwise. Acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) was added dropwise while stirring in an ice bath, and the reaction was continued for 1 hour. The reaction solution was added to 0.1 M dilute hydrochloric acid aqueous solution to precipitate a solid, which was then filtered. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was then purified by silica gel column chromatography (methanol / dichloromethane = 0%–5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol).

[0852] Its structural characterization data are as follows:

[0853] ESI-MS (m / z): 552.2 [M+1] + .

[0854] Step 2: Preparation of 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentabenzotriacylamino)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzozazin[1,2-b]quinoline-1-yl)amino)-2-oxoethyl acetate (B-03-2)

[0855] Ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)amino)-2-oxoethyl acetate (500 mg, 0.905 mmol) and DMAP (885.33 mg, 7.25 mmol) were dissolved in dry dichloromethane (5 mL), cooled to 0 °C under nitrogen protection, and a dichloromethane solution of triphosgene (268.81 mg, 0.905 mmol) (5 mL) was added dropwise. The mixture was kept warm and stirred for 0.5 hours. A solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatrinitroamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanoamide (1.44 g, 1.36 mmol) in dichloromethane was slowly added dropwise, and the reaction was allowed to return to room temperature for 4 hours. The reaction was quenched with water, and the mixture was extracted three times with dichloromethane (100 ml x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (MeOH / DCM = 0%–5%) yielded the title compound (498 mg, 0.304 mmol).

[0856] Its structural characterization data are as follows:

[0857] ESI-MS (m / z): 1352.8 [M+1] + .

[0858] Step 3: Preparation of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonoxy-3,9-diazapentazatriacylamino)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]azoindo[1,2-b]quinoline-9-yl) carbonate (B-03-3)

[0859] 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentabenzotriacylamino)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,1 Ethyl 0,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazosin[1,2-b]quinoline-1-yl)amino)-2-oxoethyl acetate (200 mg, 0.122 mmol) was dissolved in THF (3 mL) and MeOH (3 mL). Sodium carbonate (25.88 mg, 0.224 mmol) aqueous solution (1 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. Dilute hydrochloric acid was added dropwise to the reaction solution to neutralize the reaction. After concentration under reduced pressure, the reaction proceeded directly to the next step.

[0860] Its structural characterization data are as follows:

[0861] ESI-MS (m / z): 1596.7 [M+1] + .

[0862] Step 4: Preparation of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentabenzotriacylamino)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indo[1,2-b]quinoline-9-yl) carbonate (B-03-4)

[0863] 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonoxy-3,9-diazapentazatriacylamino)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]azaindo[1,2-b]quinoline-9-yl) carbonate (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), and the reaction was continued for 1 hour after adding trifluoroacetic acid (0.5 mL). After neutralizing the reaction solution with a saturated sodium bicarbonate aqueous solution, the mixture was separated, and the organic phase was concentrated to obtain a crude product. The crude product was purified by reversed-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0%–50%) and then freeze-dried to obtain the title compound (95 mg, 69.35 μmol).

[0864] Its structural characterization data are as follows:

[0865] ESI-MS (m / z): 1323.6 [M+1] + .

[0866] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonoxy-3,9-diazapentazatriacrylamide)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-9-yl) carbonate (B-03)

[0867] 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentabenzotriacylamino)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[ [3',4':6,7] Indo[1,2-b]quinoline-9-yl) carbonate (90 mg, 0.066 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-yneamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL). Cuprous bromide (9.42 mg, 0.066 mmol) was added, and the mixture was stirred for 2 hours. The reaction solution was directly filtered, and the crude product concentrated by direct filtration was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

[0868] The structural characterization data are as follows:

[0869] ESI-MS (m / z): 1628.7 [M+1] + .

[0870] The preparative high performance liquid chromatography method is as follows:

[0871] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0872] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0873] Example 3: N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxy-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide (A-05)

[0874] Under nitrogen protection, 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetic acid ester (IM-2, 0.66 g, 1.80 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxo-5,8,11,14-tetraazahexadecanoic acid (IM-3, 0.75 g, 1.77 mmol) were added to DMF (19 mL), and the mixture was heated to 35 °C and reacted for 16 hours. Then, (1S,9S)- 1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-10,13-dione (1-4, 1.00 g, 1.77 mmol) was cooled to 5–15 °C with ice water, and DMTMM (0.98 g, 3.53 mmol) was added, followed by dropwise addition of DIPEA (1.14 g, 8.84 mmol). The reaction mixture was reacted at 25 °C for 16 hours. The reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes. The DCM phase was separated, washed with brine (100 mL), and concentrated to obtain the crude product. After purification by preparative high-performance liquid chromatography, the crude product was freeze-dried to obtain 0.98 g of compound A-05.

[0875] The separation and purification method for A-05 is as follows:

[0876] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

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

[0878] The structural characterization data for A-05 are as follows:

[0879] MS m / z(ESI): 1107.3 [M+H]+

[0880] 1H NMR (400MHz, DMSO) δ9.10 (s, 2H), 8.66-8.63 (m, 1H), 8.51 (d, J = 8.8Hz, 1H), 8.34-8.31 (m, 1H), 8.21-8.19 (m, 1H), 8.17-8.09 (m, 2 H),8.08-8.04(m,1H),7.30(s,1H),7.26-7.15(m,5H),6.55(s,1H),5.56-5.55(m,1H),5.48-5.35(m,2H),5.25-5.10(m,2H),4.6 4(d,J=6.4Hz,2H),4.45-4.44(m,1H),4.06-3.98(m,2H),3.77-3.52(m,6H),3.41(s,3H),3.25-3.12(m,2H),3.03-3.00(m,1H),2 .83-2.72(m,1H),2.58-2.56(m,2H),2.48(s,3H),2.33-2.30(m,2H),2.21-2.13(m,2H),1.91-1.76(m,4H),0.87(t,J=7.2Hz,3H).

[0881] Example 4: N-((S)-10-benzyl-1-(((1S,9S)-5-fluoro-9-ethyl-9-hydroxy-4-chloro-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxy-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide (A-07)

[0882] Under nitrogen protection, 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetic acid ester (IM-2, 21.6 mg, 0.059 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxo-5,8,11,14-tetraazahexadecanoic acid (IM-3, 24.5 mg, 0.058 mmol) were added to DMF (1 mL). After reacting at 35 °C for 16 hours, (1) S,9S)-1-amino-5-fluoro-9-ethyl-9-hydroxy-4-chloro-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-10,13-dione trifluoroacetate (30.0 mg, 0.053 mmol), HATU (30 mg, 0.079 mmol), and DIPEA (27.2 mg, 0.21 mmol) were reacted at 25 °C for 16 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 26.4 mg of compound A-07.

[0883] The separation and purification method for A-07 is as follows:

[0884] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0885] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0886] The structural characterization data for A-07 are as follows:

[0887] ESI-MS (m / z): 1111.3 [M+H] + .

[0888] Example 5: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazatetradecane-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-amide (A-10)

[0889] Step 1:

[0890] Compound IM-4 (657 mg, 1.22 mmol) and compounds 1-4 (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the addition of HATU (630.67 mg, 1.66 mmol) and N,N-diisopropylethylamine (428 mg, 3.32 mmol). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was purified directly by preparative high-performance liquid chromatography and then freeze-dried to obtain 700 mg of compound IM-5.

[0891] The preparation method for high performance liquid chromatography is as follows:

[0892] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0893] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0894] Step Two:

[0895] Compound IM-5 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (75.05 mg, 1.03 mmol) was added. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 307 mg of compound IM-6.

[0896] The preparation method for high performance liquid chromatography is as follows:

[0897] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0898] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0899] Step 3:

[0900] IM-6 (170 mg, 0.226 mmol) and compound IM-2 (90.83 mg, 0.249 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (29.21 mg, 0.226 mmol) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 50.56 mg of compound A-10.

[0901] The structural characterization data are as follows:

[0902] MS m / z(ESI): 1002.4 [M+H]+

[0903] The preparation method for high performance liquid chromatography is as follows:

[0904] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0905] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0906] 1 H NMR (400MHz, DMSO) δ9.11(s,2H),8.68(t,J=6.4Hz,1H),8.49(d,J=8.8Hz,1H),8.16(s,1H),8.10(d,J=7.2H z,1H),8.01(d,J=7.2Hz,1H),7.91(d,J=6.8Hz,1H),7.31(s,1H),6.55(s,1H),5.65-5.55(m,1H),5.43(s,2H ),5.21(s,2H),4.67-4.55(m,2H),4.29-4.15(m,3H),3.98(s,2H),3.41(s,3H),3.25-3.15(m,2H),2.57-2.5 6(m,2H),2.35-2.27(m,2H),2.22-2.12(m,2H),1.91-1.75(m,4H),1.23-1.09(m,9H),0.87(t,J=7.2Hz,3H).

[0907] Example 6: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl) carbonate (B-01)

[0908] Step 1:

[0909] At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis reference patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (72.95 mg, 0.503 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynylamide (95.10 mg, 0.302 mmol) were added. After stirring for 1 h, the mixture was filtered, and the filtrate was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 30.00 mg of compound B-01-2.

[0910] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0911] Mobile phase A: Acetonitrile; Mobile phase B: Water

[0912] Step Two:

[0913] Compound B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL) and trifluoroacetic acid (0.2 mL) was added. The reaction was carried out at room temperature for 30 min. The reaction solution was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (under the following conditions) to obtain 20.00 mg of trifluoroacetate of compound B-01.

[0914] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0915] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0916] The structural characterization data are as follows: ESI-MS (m / z): 1631.7 [M+H] + 816.0 [M / 2+H] + .

[0917] Example 7: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxy-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatriapentadecanoamide)benzyl((1S,9R)-9-ethyl-5-fluoro-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) carbonate (B-02)

[0918] Step 1:

[0919] At 25°C, 1-1 methanesulfonate (30.00 mg, 56.44 μmol) was dissolved in N,N-dimethylformamide (1 mL), followed by the sequential addition of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (58.74 mg, 112.88 μmol), N,N-diisopropylethylamine (43.76 mg, 338.63 μmol), and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (26.62 mg, 84.66 μmol). The reaction was maintained at 25°C for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was separated by thin-layer chromatography (dichloromethane:methanol = 15:1) to obtain 27.00 mg of compound B-02-1.

[0920] Step Two:

[0921] At 0°C, B-02-1 (20 mg, 27.33 μmol) was dissolved in dichloromethane (2 mL), followed by the addition of 0.5 mL solutions of 4-dimethylaminopyridine (26.71 mg, 218.61 μmol) and triphosgene (8.11 mg, 27.33 μmol) in dichloromethane. The reaction was maintained at 0°C for 0.5 hours. After replacing the residual triphosgene with nitrogen, (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,2) was added dropwise. A solution (1 mL) of 7,30-nonoxa-3,9-diazatriapentadecanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexamethyleneamide (43.46 mg, 40.99 μmol) in dichloromethane was prepared and reacted at 0 °C for 0.5 h. The reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by thin-layer chromatography (dichloromethane:methanol = 15:1) to obtain 30.00 mg of compound B-02-2.

[0922] Step 3:

[0923] At 25°C, B-02-2 (250.00 mg, 137.51 μmol) was dissolved in a mixed solvent of DMSO (2 mL) and water (0.4 mL), and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynylamide (62.98 mg, 206.26 μmol) and cuprous bromide (39.45 mg, 275.01 μmol) were added. The reaction was maintained at 25°C for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was lyophilized to obtain 150.00 mg of compound B-02-3.

[0924] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0925] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0926] Step Four:

[0927] At 25°C, B-02-3 (150 mg, 49.45 μmol) was dissolved in tetrahydrofuran (1 mL), and a mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) / glacial acetic acid (v / v = 13 / 1) (50 μL) was added dropwise. The reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was lyophilized to obtain 50.00 mg of compound B-02-4.

[0928] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0929] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0930] Step 5:

[0931] At 25°C, B-02-4 (50 mg, 26.52 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (60.49 mg, 530.49 μmol) was added. The reaction was maintained at 25°C for 0.5 hours. The reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by preparative high performance liquid chromatography (under the following conditions). The preparative solution was lyophilized to obtain 23.69 mg of compound B-02.

[0932] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0933] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0934] The structural characterization data of B-02 are as follows:

[0935] ESI-MS (m / z): 1613.6 [M+H] + .

[0936] Example 8: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolazine[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentane-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzamide (C-07)

[0937] Step 1:

[0938] The starting material C-07-1 (4.80 g, 16.33 mmol), tributyltin (16.27 g, 39.19 mmol), and palladium dichloride (2.29 g, 3.27 mmol) were dissolved in 1,4-dioxane (100 mL). The reaction system was stirred at 110 °C for 5 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The reaction system was concentrated and purified by column chromatography (EA / PE = 0-50%) to obtain 1.36 g of compound C-07-2.

[0939] Step Two:

[0940] Compound C-07-2 (510 mg, 1.33 mol) and NaOH (212.24 mg, 5.31 mmol) were dissolved in THF (12.5 mL), MeOH (12.5 mL), and H₂O (2.5 mL). The reaction was stirred at 25 °C for 2 hours. The reaction was monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 3N HCl. A large amount of solid precipitated. The solid was filtered, the filter cake was collected, and dried to obtain 380 mg of compound C-07-3.

[0941] Step 3:

[0942] Compound C-07-3 (315 mg, 850.32 μmol), 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxyacetic acid tert-butyl ester (246.31 mg, 935.35 μmol), HATU (484.99 mg, 1.28 mmol), and DIPEA (329.69 mg, 2.55 mmol) were added to DMF (3 mL), and the mixture was reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain 40 mg of compound C-07-3.

[0943] The preparation method for high performance liquid chromatography is as follows:

[0944] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0945] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0946] Step Four:

[0947] Compound C-07-4 (40 mg, 64.96 μmol) was dissolved in DCM (3 mL) and TFA (1.5 mL), and reacted at 25 °C for 1.5 h. The reaction was monitored by LC-MS, and the reaction system was concentrated to dryness to obtain 36 mg of compound C-07-5.

[0948] Step 5:

[0949] Compound C-07-5 (26 mg, 46.46 μmol), sodium periodate (99.37 mg, 464.57 μmol), and RuCl3·H2O (9.64 mg, 46.46 μmol) were dissolved in ACN (15 mL) and water (7.5 mL). The mixture was reacted at 25 °C for 40 min. The reaction was monitored by LC-MS. The mixture was extracted with water and ethyl acetate. The ethyl acetate layer was concentrated to give 28 mg of compound C-07-6.

[0950] Step Six:

[0951] Compound ecetane mesylate (600 mg, 1.13 mmol), (5S,8S,11S)-1-(9H-fluorene-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxy-2,15-dioxy-4,7,10,13-tetraazaheptan-17-acid (IM-4, 610.17 mg, 1.13 mmol), HATU (643.81 mg, 1.69 mmol), and DIPEA (437.65 mg, 3.39 mmol) were added to DMF (6 mL), and the reaction was carried out at 25 °C for 16 h. The reaction was monitored by LC-MS. Water was added to the reaction solution, and a large amount of solid precipitated. The solid was filtered, collected, dissolved in DCM, concentrated to obtain crude product, and purified by column chromatography (DCM / MeOH = 0-10%) to give 660 mg of compound C-07-7.

[0952] Step Seven:

[0953] Compound C-07-7 (660 mg, 688.94 μmmol) was dissolved in N,N-dimethylformamide (6 mL), and diethylamine (251.94 mg, 3.44 mmol) was added. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 325 mg of compound C-07-8.

[0954] The preparation method for high performance liquid chromatography is as follows:

[0955] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0956] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0957] Step 8:

[0958] Compounds C-07-6 (15.95 mg, 25.58 μmol), C-07-8 (20 mg, 25.58 μmol), HATU (14.59 mg, 38.37 μmol), and DIPEA (9.92 mg, 76.75 μmol) were added to DMF (3 mL) and reacted at 25 °C for 2 h. The reaction mixture was monitored by LC-MS. The reaction solution was purified by preparative high-performance liquid chromatography and then freeze-dried to obtain 7 mg of compound C-07.

[0959] The structural characterization data are as follows:

[0960] ESI-MS (m / z): 1342.4 [M+H] + .

[0961] The preparation method for high performance liquid chromatography is as follows:

[0962] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0963] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0964] Example 9: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentane-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-10)

[0965] Step 1:

[0966] The starting material C-10-1 (720 mg, 2.45 mmol), 2-methylthiopyrimidine-5-boronic acid (874 mg, 5.14 mmol), XPhosPd G3 (207 mg, 245 μmol), and K3PO4 (1.56 g, 7.35 mmol) were added to dioxane (12 mL) and H2O (4 mL). The reaction system was stirred at 90 °C for 3 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was filtered through diatomaceous earth, and water and ethyl acetate were added to the filtrate for extraction and concentration to obtain the crude product. The crude product was purified by column chromatography (EA / PE = 0-25%) to obtain 710 mg of compound C-10-1.

[0967] Step Two:

[0968] Compound C-10-1 (650 mg, 1.69 mol) and lithium hydroxide (121 mg, 5.07 mmol) were dissolved in THF (2 mL), MeOH (2 mL), and H2O (2 mL). The reaction was stirred at 25 °C for 2 hours. The reaction was monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 1 N HCl. A large amount of solid precipitated. The filter cake was collected by filtration and dried to obtain 560 mg of compound C-10-2.

[0969] Step 3:

[0970] Compound C-10-2 (450.80 mg, 1.22 mmol) was dissolved in DCM (10 mL), and m-CPBA (2.46 g, 12.1 mmol, purity 85%) was added to the reaction system. The reaction was carried out at 25 °C for 12 hours, and the reaction was monitored by LC-MS. The solvent was dried by a nitrogen stream to obtain the crude product, which was purified by preparative high performance liquid chromatography and then freeze-dried to obtain 153 mg of compound C-10-3.

[0971] The preparation method for high performance liquid chromatography is as follows:

[0972] Chromatographic column: Phenomenex Luna C18 200*40mm*10um.

[0973] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% hydrochloric acid)

[0974] Mobile phase: [water(HCl)-ACN]; B%: 13%-43%, 10 min).

[0975] Step Four:

[0976] Compound C-10-3 (140 mg, 322.25 μmol), 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxyacetate tert-butyl ester (84.86 mg, 322.25 μmol), HATU (183.80 mg, 483.37 μmol), and DIPEA (124.94 mg, 966.75 μmol) were added to DMF (4 mL), and the reaction was carried out at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain 51 mg of compound C-10-4.

[0977] The preparation method for high performance liquid chromatography is as follows:

[0978] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0979] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0980] Step 5:

[0981] Compound C-10-4 (50 mg, 73.56 μmol) was added to DCM (2 mL) and TFA (1 mL), and the reaction was carried out at 25 °C for 1 h. The reaction was monitored by LC-MS, and the reaction system was concentrated to dryness to obtain 45 mg of compound C-10-5.

[0982] Step Six:

[0983] Compounds C-10-5 (31.91 mg, 51.17 μmol), C-07-8 (40 mg, 51.17 μmol), HATU (29.18 mg, 76.75 μmol), and DIPEA (19.84 mg, 153.50 μmol) were added to 3 mL of DMF and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain 13 mg of compound C-10.

[0984] The structural characterization data are as follows:

[0985] ESI-MS (m / z): 1342.5 [M+H]+.

[0986] The preparation method for high performance liquid chromatography is as follows:

[0987] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0988] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0989] Example 10: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentane-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-17)

[0990] Step 1:

[0991] C-10-2 (3.00 g, 8.10 mmol) and tert-butyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-propionate (2.25 g, 8.10 mmol) were added to DMF (3 mL), followed by HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL). The mixture was heated to 60 °C and reacted for 2 hours. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude C-17-1 (3.8 g, 4.75 mmol), which was used directly in the next step without purification.

[0992] Step Two:

[0993] C-17-1 (3.40 g, 5.40 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (10.8 g, 94.2 mmol, 7 mL) was added. The reaction solution was stirred at 25 °C for 2 hours. The reaction solution was directly concentrated, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain C-17-2 (2.09 g, 3.64 mmol).

[0994] The preparation method for high performance liquid chromatography is as follows:

[0995] Column: Phenomenex luna C18 (250mm*70mm*10μm)

[0996] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0997] Step 3:

[0998] C-17-2 (56 mg, 97.61 μmol) was added to acetonitrile (6 mL) and water (3 mL), followed by sodium periodate (208.79 mg, 976.15 μmol) and ruthenium trichloride hydrate (8.10 mg, 39.05 μmol). The mixture was stirred at 25 °C for 30 minutes, and the reaction was monitored by LC-MS. The mixture was then extracted with water and ethyl acetate and concentrated to obtain C-17-3 (60 mg).

[0999] Step Four:

[1000] IM-6 (20 mg, 25.06 μmol), C-17-3 (16 mg, 25.06 μmol), HATU (19.05 mg, 50.11 μmol), and DIPEA (16.19 mg, 125.28 μmol) were added sequentially to DMF (3 mL), and the reaction system was reacted at 25 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain C-17 (16 mg).

[1001] The structural characterization data are as follows:

[1002] ESI-MS (m / z): 1371.4 [M+H] + .

[1003] The preparation method for high performance liquid chromatography is as follows:

[1004] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1005] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1006] Example 11: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentoxo-3,18,21,24-tetraoxo-5,8,11,14-tetraazahexaalkyl-26-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-19)

[1007] Step 1:

[1008] C-19-1 (5.00 g, 16.9 mmol), (2-(methylthio)pyrimidin-5-yl)boronic acid (6.34 g, 37.3 mmol), XPhos Pd G3 (1.44 g, 1.70 mmol), and potassium phosphate (10.80 g, 50.9 mmol) were added to 1,4-dioxane (51.0 mL) and water (17.0 mL). The reaction system was purged with nitrogen three times and then reacted at 100 °C for 5 hours. After the reaction system cooled to room temperature, water (50.0 mL) was added to the reaction solution, and the mixture was filtered. The filtrate was concentrated to obtain a crude product, which was then slurried with petroleum ether and filtered again. The filter cake was dried under vacuum to obtain C-19-2 (5.65 g).

[1009] Step Two:

[1010] C-19-2 (5.26 g, 13.7 mmol) was dissolved in THF (30.0 mL), MeOH (30.0 mL), and water (30.0 mL). LiOH·H2O (1.72 g, 40.9 mmol) was added, and the mixture was stirred at 25 °C for 2 hours. The pH of the reaction solution was adjusted to 3 with 1 N hydrochloric acid aqueous solution, and a solid precipitated out. The solid was filtered, and the filter cake was dried under vacuum to obtain C-19-3 (4.20 g).

[1011] Step 3:

[1012] C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 3-(2-(2-aminoethoxy)ethoxyethoxyethyl)propionate (1.12 g, 4.04 mmol) were dissolved in DMF (20.0 mL). HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol), and DIPEA (2.09 g, 16.2 mmol) were added sequentially, and the mixture was heated to 60 °C and stirred for 2 hours. After the reaction system cooled to room temperature, water (10.0 mL) and ethyl acetate (20.0 mL) were added to the reaction solution. The aqueous phase was extracted twice with ethyl acetate (25.0 mL * 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude C-19-4 (2.50 g), which was used directly in the next step without purification.

[1013] Step Four:

[1014] C-19-4 (2.50 g, 3.96 mmol) was dissolved in dichloromethane (3.00 mL), and TFA (4.61 g, 40.4 mmol) was added. The reaction mixture was stirred at 25 °C for 12 hours. The reaction solution was directly concentrated, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain C-19-5 (1.20 g).

[1015] The preparation method for high performance liquid chromatography is as follows:

[1016] Column: Phenomenex luna C18 (150mm*25mm*10μm)

[1017] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1018] Step 5:

[1019] C-19-5 (1.10 g, 1.91 mmol) was dissolved in a mixed solvent of acetonitrile (30 mL) and water (15 mL). Ruthenium trichloride hydrate (39.70 mg, 0.19 mmol) and sodium periodate (4.09 g, 19.14 mmol) were added. The reaction system was reacted at 25 °C for 1 hour. After extraction with water (50 mL) and ethyl acetate (80 mL), the organic phase was concentrated to obtain the crude product. The crude product was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to obtain C-19-6 (130 mg).

[1020] Step Six:

[1021] IM-6 (20.0 mg, 0.025 mmol) and C-19-6 (16.0 mg, 0.025 mmol) were added to DMF (1 mL) and stirred to dissolve. HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added, and the mixture was reacted at room temperature for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain C-19 (20.4 mg).

[1022] The structural characterization data are as follows:

[1023] ESI-MS (m / z): 1372.4 [M+H] + .

[1024] The preparation method for high performance liquid chromatography is as follows:

[1025] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1026] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1027] Example 12: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentoxo-3,18,21,24,27,30,33,36,39-nonoxy-5,8,11,14-tetraazatetral-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-21)

[1028] Step 1:

[1029] C-10-2 (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-oate (4.03 g, 8.10 mmol) were added to DMF (40 mL), followed by HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL). The reaction mixture was stirred at 60 °C for 2 hours. Water (100 mL) and ethyl acetate (60 mL x 3) were added to the reaction mixture for extraction. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain C-21-1 (4.20 g, 4.14 mmol), which was used directly in the next step without purification.

[1030] Step Two:

[1031] C-21-1 (3.60 g, 4.24 mmol) was dissolved in dichloromethane (30 mL), and TFA (15.3 g, 134 mmol, 10 mL) was added. The reaction mixture was stirred at 25 °C for 6 hours. Water (60 mL) and ethyl acetate (40 mL x 3) were added to the reaction mixture for extraction. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography and then freeze-dried to obtain C-21-2 (2.93 g, 3.63 mmol).

[1032] The preparation method for high performance liquid chromatography is as follows:

[1033] Column: Phenomenex luna C18 (250mm*70mm*10μm)

[1034] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1035] Step 3:

[1036] C-21-2 (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), and then sodium periodate (398.71 mg, 1.86 mmol) and ruthenium trichloride hydrate (15.47 mg, 74.56 μmol) were added to the reaction system. The mixture was stirred at 25 °C for 30 minutes. The reaction system was extracted with water and ethyl acetate and concentrated to obtain C-21-3 (155 mg).

[1037] Step Four:

[1038] IM-6 (27.91 mg, 34.97 μmol), C-21-3 (30 mg, 34.97 μmol), HATU (26.59 mg, 69.93 μmol), and DIPEA (22.60 mg, 174.84 μmol) were added to DMF (3 mL), and the reaction system was reacted at 25 °C for 1 hour. The reaction solution was purified by high performance liquid chromatography and then freeze-dried to obtain C-21 (15 mg).

[1039] The structural characterization data are as follows:

[1040] ESI-MS (m / z): 1591.7 [M+H] + .

[1041] The preparation method for high performance liquid chromatography is as follows:

[1042] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1043] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1044] Example 13: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentoxo-3,18,21,24,27,30,33,36,39-nonoxy-5,8,11,14-tetraazatetraane-4-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-23)

[1045] Step 1:

[1046] C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-oate (2.01 g, 4.04 mmol) were added to DMF (20.0 mL), followed by HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol), and DIEA (2.09 g, 16.2 mmol). The mixture was heated to 60 °C and stirred for 2 hours. After cooling to room temperature, water (10.0 mL) and ethyl acetate (20.0 mL) were added to the reaction mixture. The mixture was separated, and the aqueous phase was extracted twice with ethyl acetate (25.0 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude product C-23-1 (3.00 g), which was used directly in the next step.

[1047] Step Two:

[1048] C-23-1 (3.00 g, 3.53 mmol) was added to dichloromethane (10.0 mL), followed by the addition of TFA (15.4 g, 134 mmol), and the mixture was stirred at 25 °C for 12 hours. The reaction solution was directly concentrated to obtain a crude product, which was then purified by preparative high-performance liquid chromatography and freeze-dried to obtain C-23-2 (1.20 g).

[1049] The preparation method for high performance liquid chromatography is as follows:

[1050] Column: Welch Ultimate C18 (150mm*25mm*5μm)

[1051] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1052] Step 3:

[1053] C-23-2 (500 mg, 0.63 mmol) was added to acetonitrile (10 mL) and water (5 mL), followed by ruthenium trichloride hydrate (13.0 mg, 0.063 mmol) and sodium periodate (1.35 g, 6.29 mmol). The system was reacted at 25 °C for 1 hour, then extracted with water (10 mL) and ethyl acetate (40 mL). The organic phase was concentrated to obtain the crude product, which was purified by column chromatography (MeOH / DCM = 10–20%) and concentrated to obtain C-23-3 (350 mg).

[1054] Step Four:

[1055] IM-6 (20.0 mg, 0.025 mmol) and C-23-3 (21.5 mg, 0.025 mmol) were dissolved in DMF (1 mL), and HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added. The mixture was reacted at room temperature for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain C-23 (17.0 mg).

[1056] The structural characterization data are as follows:

[1057] ESI-MS (m / z): 1592.6 [M+H] + .

[1058] The preparation method for high performance liquid chromatography is as follows:

[1059] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1060] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1061] Example 14: (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(2-(2-(2-(6-(2-((methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamidamido)ethoxy)ethoxy)acetamyl))phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-35)

[1062] Step 1: Synthesis of (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (A-35-3)

[1063] Compound (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetic acid triester (A-35-1, 12.32 g, 31.02 mmol) and 4-hydroxy-3-nitrobenzyl alcohol (compound A-35-2, 5.00 g, 29.56 mmol) were dissolved in acetonitrile (200 mL). Silver oxide (27.40 g, 118.25 mmol) was added with stirring. After nitrogen purging, the mixture was reacted at room temperature in the dark for 12 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:3) to give 12.80 g of the title compound.

[1064] The structural characterization data are as follows:

[1065] ESI-MS (m / z): 503 [M+18] + .

[1066] Step 2: Synthesis of (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (A-35-4)

[1067] Compound A-35-3 (2.20 g, 4.53 mmol) was dissolved in ethyl acetate and tetrahydrofuran (50 mL each), and PtO2 (0.20 g) was added. The reaction system was then purged three times with hydrogen balloons, and the reaction was carried out under a hydrogen atmosphere for 2 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was directly filtered, and the filter cake was washed with ethyl acetate. The filtrate was evaporated under reduced pressure to obtain 2.02 g of the crude title compound, which was directly used in the next step of the reaction.

[1068] The structural characterization data are as follows:

[1069] ESI-MS (m / z): 456.1 [M+1] + .

[1070] Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluorene-9-yl)-3-oxy-2,7,10-trioxa-4-azadodecane-12-amido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (A-35-5)

[1071] Compound A-35-4 (456.00 mg, 1.00 mmol) and [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (385.91 mg, 1.00 mmol) were dissolved in dichloromethane (10 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (495.22 mg, 2.00 mmol) was added with stirring, and the reaction was carried out for 2 hours. The reaction was monitored by high-performance liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (methanol:dichloromethane = 1:20) to give 507.00 mg of the title compound.

[1072] The structural characterization data are as follows:

[1073] ESI-MS (m / z): 823.3 [M+1] + .

[1074] Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluorene-9-yl)-3-oxy-2,7,10-trioxa-4-azadodecane-12-amido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (A-35-6)

[1075] Compound A-35-5 (507.00 mg, 616.18 μmol) and diisopropylethylamine (238.91 mg, 1.85 mmol) were dissolved in dichloromethane (20 mL). Then, p-nitrophenyl chloroformate (372.60 mg, 1.85 mmol) was dissolved in dichloromethane (1 mL) and slowly added dropwise to the reaction solution. After the addition was complete, the reaction was allowed to proceed at room temperature for 15 h. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (methanol:dichloromethane = 1:20) to obtain 496.00 mg of the title compound.

[1076] The structural characterization data are as follows:

[1077] ESI-MS (m / z): 988.5 [M+1] + .

[1078] Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluorene-9-yl)-3-oxy-2,7,10-trioxa-4-azadodecane-12-amido)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (A-35-7)

[1079] Compound A-35-6 (165.51 mg, 0.17 mmol), (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (compound 2-2, 40.00 mg, 0.084 mmol) and 1-hydroxybenzotriazole (33.96 mg, 0.25 mmol) were dissolved in DMF (4 mL), and diisopropylethylamine (32.48 mg, 0.25 mmol) was added dropwise. The mixture was stirred for 12 h, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with saturated brine and dried. After concentration under reduced pressure, 100.00 mg of the crude title compound was obtained and proceeded directly to the next reaction.

[1080] The structural characterization data are as follows:

[1081] ESI-MS (m / z): 1326.2 [M+1] + .

[1082] Step Six: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-35-8)

[1083] Compound A-35-7 (100.00 mg, 0.08 mmol) was dissolved in MeOH (5 mL), 1 drop of dichloromethane was added, and 1 mL of an aqueous solution of lithium hydroxide monohydrate (15.82 mg, 0.377 mmol) was added. The mixture was stirred for 2 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The pH of the reaction solution was adjusted to 4 by adding 3N hydrochloric acid aqueous solution. After concentration under reduced pressure, the solution was purified by preparative high performance liquid chromatography (under the following conditions). The preparative solution was freeze-dried to obtain 27.00 mg of the title compound.

[1084] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1085] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1086] The structural characterization data are as follows:

[1087] ESI-MS (m / z): 964.2 [M+1] + .

[1088] Step Seven: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(2-(2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamidamido)ethoxy)ethoxy)acetamyl))phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-35)

[1089] Compound A-35-8 (27.00 mg, 0.03 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (A-35-09), 11.26 mg, 0.03 mmol) were dissolved in DMF (1 mL). Diisopropylethylamine (3.62 mg, 0.03 mmol) was added dropwise with stirring. The reaction was carried out at room temperature for 4 hours, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The reaction solution was purified by preparative HPLC (under the conditions described below), and the preparative solution was freeze-dried to give 11.70 mg of the title compound.

[1090] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1091] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1092] The structural characterization data are as follows:

[1093] ESI-MS (m / z): 1214.4 [M+1] + .

[1094] Example 15: N-((10S,19S)-10-benzyl-1-(((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino) -30-(2-(methylsulfonyl)pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontane-29-yn-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctacosane-38-amide or N-((10S,19S)-10-benzyl-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-30-(2 -(methylsulfonyl)pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontane-29-yne-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctacosane-38-amide (A-36-A)

[1095] Step 1: N 6 Synthesis of -(((9H-fluorene-9-yl)methoxy)carbonyl)-N2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanoctane-38-acyl)-D-lysine(2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanoctane-38-amido)-6-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)hexanoic acid (A-36-03)

[1096] The hydrochloride salt of compound A-36-02 (389.68 mg, 962.45 μmol) was dissolved in dichloromethane (8 mL), and DIPEA (518.28 mg, 4.01 mmol, 713.88 μL) and compound A-36-01 (550.00 mg, 802.04 μmol) were added. The reaction mixture was reacted at 25 °C for 1.5 h. The pH of the reaction solution was adjusted to neutral with dilute hydrochloric acid, and the solvent was dried under reduced pressure. The concentrate was purified by preparative high-performance liquid chromatography to give the title compound A-17-03 (450.00 mg).

[1097] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1098] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1099] ESI-MS (m / z): 939.3 [M+H] + .

[1100] Step 2: Synthesis of (40S,49S)-40-(4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)butyl)-49-benzyl-38,41,44,47,50,53-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,56-tetraoxo-39,42,45,48,51,54-hexaazaoctacosane-58-acid (A-36-04)

[1101] Compound A-36-03 (50.00 mg, 118.09 μmol) was dissolved in DMF (2.5 mL), and HATU (49.39 mg, 129.89 μmol), compound A-07-2 (133.07 mg, 141.70 μmol), and DIPEA (45.78 mg, 354.26 μmol, 63.06 μL) were added. The mixture was reacted at 25 °C for 1 hour. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high-performance liquid chromatography to give the title compound A-36-04 (40.00 mg).

[1102] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1103] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1104] ESI-MS (m / z): 1344.4 [M+H] + .

[1105] Step 3: (9H-fluorene-9-ylmethyl)((40S)-40-(((10S)-10-benzyl-1-(((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b Synthesis of quinoline-1-yl)amino)-1,6,9,12,15-pentoxy-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxa-39-aza-44-yl)carbamate (A-36-05)

[1106] Compound A-36-04 (29.86 mg, 59.50 μmol) was dissolved in DMF (3 mL), and HATU (27.15 mg, 71.40 μmol), compound 1-5-A (80.00 mg, 59.50 μmol), and DIPEA (38.45 mg, 297.51 μmol, 52.96 μL) were added. The mixture was reacted at 25 °C for 1 hour. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high-performance liquid chromatography to give the title compound A-36-05 (50.00 mg).

[1107] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1108] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1109] ESI-MS (m / z): 1781.6 [M+H] + .

[1110] Step 4: N-((10S,19S)-23-amino-10-benzyl-1-(((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentazatritane-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctadecane-38-amide N-(( Synthesis of 10S,19S)-23-amino-10-benzyl-1-(((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolezido[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaza-230alkyl-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxa-octacosane-38-amide (A-36-06)

[1111] Compound A-36-05 (20.00 mg, 11.22 μmol) was dissolved in DMF (2.5 mL) and diethylamine (0.5 mL) and reacted at 25 °C for 2 hours. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to give the formate of the title compound A-36-06 (10.00 mg).

[1112] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1113] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1114] ESI-MS (m / z): 1559.7 [M+H]+.

[1115] Step 5: N-((10S,19S)-10-benzyl-1-(((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-3 0-(2-(methylsulfonyl)pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontane-29-yn-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctacosane-38-amide or N-( (10S,19S)--10-benzyl-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-30-(2-(methyl) Synthesis of (A-36-A) pyrimidin-5-yl)-1,6,9,12,15,18,25-heptaoxo-3-oxa-5,8,11,14,17,24-hexaazatriacontane-29-yne-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctacosane-38-amide

[1116] The formate of compound A-36-06 (7.00 mg, 4.49 μmol) and compound A-07-1 (3.28 mg, 8.97 μmol) were dissolved in DMF (1 mL), and DIPEA (1.74 mg, 13.46 μmol, 2.40 μL) was added. The mixture was reacted at 25 °C for 2 hours. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to give the title compound A-36-A (5.60 mg).

[1117] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1118] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1119] ESI-MS (m / z): 1809.8 [M+H] + .

[1120] Example 16: 4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-((methanesulfonyl)pyrimidin-5-yl)hexyl-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butamido)butamido)-5-ureidopentanoyl)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate (A-37)

[1121] Step 1: Synthesis of 4-(4-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-4-oxobutyl)piperidine-1-carboxylic acid tert-butyl ester

[1122] At 25°C, compounds Val-Cit-PABC (A-37-1, 1 g, 2.64 mmol), 4-(N-Boc-4-piperidinyl)butyric acid (929.65 mg, 3.43 mmol), and EEDQ (977.55 mg, 3.95 mmol) were dissolved in a mixture of methanol (20 mL) and dichlo...

Claims

An antibody-drug conjugate containing the structure shown below: Ab-[M-L-E-D] x in: Ab is a bispecific antibody or its antigen-binding fragment containing a first antigen-binding domain that specifically binds to PDL1 and a second antigen-binding domain that specifically binds to B7H3. M is the linker site that is linked to the bispecific antibody or its antigen-binding fragment; L is a structural segment connecting the joint portions M and E; E is a structural segment connecting L and D; D is a cytotoxic drug or a fragment thereof; and x is any integer from 1 to 10. The antibody-drug conjugate of claim 1, wherein, The first antigen-binding domain includes a first light chain variable region (VL) and a first heavy chain variable region (VH), wherein the first VL and the first VH together form a domain capable of specifically binding PDL1; the second antigen-binding domain includes a second VL and a second VH, wherein the second VL and the second VH together form a domain capable of specifically binding B7H3. The antibody-drug conjugate according to claim 1 or 2, wherein, The first VL includes LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO:61; and / or, the first VH includes HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:60; The CDRs are defined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system. The antibody-drug conjugate of any one of claims 1-3, wherein: The first VL includes: (i) LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system; (ii) LCDR1 comprising the sequence shown in SEQ ID NO:32, LCDR2 comprising the sequence shown in SEQ ID NO:35, and LCDR3 comprising the sequence shown in SEQ ID NO:38; wherein the CDRs are defined by the Contact numbering system; or, (iii) LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the IMGT numbering system; And / or, The first VH includes: (i) HCDR1 containing the sequence shown in SEQ ID NO:18, HCDR2 containing the sequence shown in SEQ ID NO:23, and HCDR3 containing the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system; (ii) HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system; (iii) HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system; (iv) HCDR1 comprising the sequence shown in SEQ ID NO:21, HCDR2 comprising the sequence shown in SEQ ID NO:26, and HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or, (v) HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system. The antibody-drug conjugate of any one of claims 1-4, wherein, The first VL contains an amino acid sequence as shown in SEQ ID NO:61, and / or the first VH contains an amino acid sequence as shown in SEQ ID NO:

60. The antibody-drug conjugate of any one of claims 1-5, wherein, The second VL includes LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO:63; and / or, the second VH includes HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:62; The CDRs are defined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system. The antibody-drug conjugate of any one of claims 1-6, wherein: The second VL includes: (i) LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system. (ii) LCDR1 comprising the sequence shown in SEQ ID NO:53, LCDR2 comprising the sequence shown in SEQ ID NO:56, and LCDR3 comprising the sequence shown in SEQ ID NO:59; wherein the CDRs are defined by the Contact numbering system; or, (iii) LCDR1 comprising the sequence shown in SEQ ID NO:54, LCDR2 comprising the sequence shown in SEQ ID NO:57, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the IMGT numbering system; And / or, The second VH includes: (i) HCDR1 containing the sequence shown in SEQ ID NO:39, HCDR2 containing the sequence shown in SEQ ID NO:44, and HCDR3 containing the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system; (ii) HCDR1 comprising the sequence shown in SEQ ID NO:40, HCDR2 comprising the sequence shown in SEQ ID NO:45, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system; (iii) HCDR1 comprising the sequence shown in SEQ ID NO:41, HCDR2 comprising the sequence shown in SEQ ID NO:46, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system; (iv) HCDR1 comprising the sequence shown in SEQ ID NO:42, HCDR2 comprising the sequence shown in SEQ ID NO:47, and HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or, (v) HCDR1 comprising the sequence shown in SEQ ID NO:43, HCDR2 comprising the sequence shown in SEQ ID NO:48, and HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system. The antibody-drug conjugate of any one of claims 1-7, wherein, The second VL contains an amino acid sequence as shown in SEQ ID NO:63, and / or the second VH contains an amino acid sequence as shown in SEQ ID NO:

62. The antibody-drug conjugate of any one of claims 1-8, wherein, (i) the first VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:61, and the first VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:60; and (ii) The second VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:63, and the second VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:

62. The CDRs are defined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system. The antibody-drug conjugate of any one of claims 1-9, wherein, (a) The first VL includes: (i) LCDR1 comprising the sequence shown in SEQ ID NO:31, LCDR2 comprising the sequence shown in SEQ ID NO:34, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system; (ii) LCDR1 comprising the sequence shown in SEQ ID NO:32, LCDR2 comprising the sequence shown in SEQ ID NO:35, and LCDR3 comprising the sequence shown in SEQ ID NO:38; wherein the CDRs are defined by the Contact numbering system; or, (iii) LCDR1 comprising the sequence shown in SEQ ID NO:33, LCDR2 comprising the sequence shown in SEQ ID NO:36, and LCDR3 comprising the sequence shown in SEQ ID NO:37; wherein the CDRs are defined by the IMGT numbering system; and, (b) The first VH includes: (i) HCDR1 containing the sequence shown in SEQ ID NO:18, HCDR2 containing the sequence shown in SEQ ID NO:23, and HCDR3 containing the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Chothia numbering system; (ii) HCDR1 comprising the sequence shown in SEQ ID NO:19, HCDR2 comprising the sequence shown in SEQ ID NO:24, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the AbM numbering system; (iii) HCDR1 comprising the sequence shown in SEQ ID NO:20, HCDR2 comprising the sequence shown in SEQ ID NO:25, and HCDR3 comprising the sequence shown in SEQ ID NO:28; wherein the CDRs are defined by the Kabat numbering system; (iv) HCDR1 comprising the sequence shown in SEQ ID NO:21, HCDR2 comprising the sequence shown in SEQ ID NO:26, and HCDR3 comprising the sequence shown in SEQ ID NO:29; wherein the CDRs are defined by the Contact numbering system; or, (v) HCDR1 comprising the sequence shown in SEQ ID NO:22, HCDR2 comprising the sequence shown in SEQ ID NO:27, and HCDR3 comprising the sequence shown in SEQ ID NO:30; wherein the CDRs are defined by the IMGT numbering system; and (c) The second VL includes: (i) LCDR1 comprising the sequence shown in SEQ ID NO:52, LCDR2 comprising the sequence shown in SEQ ID NO:55, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the Chothia, AbM, or Kabat numbering system. (ii) LCDR1 comprising the sequence shown in SEQ ID NO:53, LCDR2 comprising the sequence shown in SEQ ID NO:56, and LCDR3 comprising the sequence shown in SEQ ID NO:59; wherein the CDRs are defined by the Contact numbering system; or, (iii) LCDR1 comprising the sequence shown in SEQ ID NO:54, LCDR2 comprising the sequence shown in SEQ ID NO:57, and LCDR3 comprising the sequence shown in SEQ ID NO:58; wherein the CDRs are defined by the IMGT numbering system; and, (d) The second VH includes: (i) HCDR1 containing the sequence shown in SEQ ID NO:39, HCDR2 containing the sequence shown in SEQ ID NO:44, and HCDR3 containing the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Chothia numbering system; (ii) HCDR1 comprising the sequence shown in SEQ ID NO:40, HCDR2 comprising the sequence shown in SEQ ID NO:45, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the AbM numbering system; (iii) HCDR1 comprising the sequence shown in SEQ ID NO:41, HCDR2 comprising the sequence shown in SEQ ID NO:46, and HCDR3 comprising the sequence shown in SEQ ID NO:49; wherein the CDRs are defined by the Kabat numbering system; (iv) HCDR1 comprising the sequence shown in SEQ ID NO:42, HCDR2 comprising the sequence shown in SEQ ID NO:47, and HCDR3 comprising the sequence shown in SEQ ID NO:50; wherein the CDRs are defined by the Contact numbering system; or, (v) HCDR1 comprising the sequence shown in SEQ ID NO:43, HCDR2 comprising the sequence shown in SEQ ID NO:48, and HCDR3 comprising the sequence shown in SEQ ID NO:51; wherein the CDRs are defined by the IMGT numbering system. The antibody-drug conjugate of any one of claims 1-10, wherein, (i) the first VL contains the amino acid sequence shown in SEQ ID NO:61, and / or the first VH contains the amino acid sequence shown in SEQ ID NO:60; and (ii) The second VL contains an amino acid sequence as shown in SEQ ID NO:63, and / or the second VH contains an amino acid sequence as shown in SEQ ID NO:

62. The antibody-drug conjugate according to any one of claims 1-11, wherein, One of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the other is an scFv; Preferably, the scFv is optionally linked to the C-terminus or N-terminus of the heavy chain of the full-length antibody via a peptide linker. The antibody-drug conjugate of claim 12, wherein, The first antigen-binding domain is scFv, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises: (i) A peptide chain VA, comprising a VL of the second antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; (ii) A peptide chain VB comprising a VH region of the second antigen-binding domain, a heavy chain CH1 region, an Fc domain monomer, and the first antigen-binding domain; preferably, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1; preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3; preferably, the first antigen-binding domain is connected to the C-terminus of the Fc domain monomer via a peptide linker; preferably, the peptide linker comprises an amino acid sequence such as SEQ ID NO: 66 or 68; Preferably, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68; Preferably, the scFv may or may not contain a disulfide bond between VH and VL; Preferably, the bispecific antibody comprises two identical or different peptide chains VA and two identical or different peptide chains VB, wherein the two peptide chains VB form a dimer through their respective Fc domain monomers. The antibody-drug conjugate of claim 13, wherein, Each of the Fc domain monomers further includes, independently, modifications that can alter the function of the effector. Preferably, the modification that alters the effector function includes the following mutations in EU number: L234A / L235A / G237A; Preferably, the Fc domain monomer comprises a sequence selected from the following sequence: SEQ ID NO: 74 or 75. The antibody-drug conjugate of claim 12, wherein, The first antigen-binding domain is scFv, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises: (i) peptide chain III-A, which includes the VL of the second antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; (ii) Peptide chain III-B, comprising the first antigen-binding domain, and a VH region of the second antigen-binding domain, a heavy chain CH1 region, and an Fc domain monomer; preferably, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1; preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3; preferably, the first antigen-binding domain is connected to the N-terminus of the VH region of the second antigen-binding domain via a peptide linker; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:67; Preferably, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68; Preferably, the scFv may or may not contain a disulfide bond between VH and VL; Preferably, the bispecific antibody comprises two identical or different peptide chains III-A and two identical or different peptide chains III-B, wherein the two peptide chains III-B form a dimer through their respective Fc domain monomers. The antibody-drug conjugate of claim 15, wherein, Each of the Fc domain monomers further includes, independently, modifications that can alter the function of the effector. Preferably, the modification that alters the effector function includes the following mutations in EU number: L234A / L235A / G237A; Preferably, the Fc domain monomer contains the sequence shown in SEQ ID NO:

74. The antibody-drug conjugate of claim 12, wherein, The first antigen-binding domain is scFv, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises: (i) peptide chain IV-A, which includes the VL of the second antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; (ii) Peptide chain IV-B, comprising the VH of the second antigen-binding domain, the CH1 region of the heavy chain, and a first Fc domain monomer (or a second Fc domain monomer); preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3. (iii) A peptide chain IV-C comprising the first antigen-binding domain, a VH region of the second antigen-binding domain, a heavy chain CH1 region, and a monomer of the second Fc domain (or a monomer of the first Fc domain); preferably, the first antigen-binding domain is connected to the N-terminus or C-terminus of the VH region of the second antigen-binding domain via a peptide linker; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO: 67 or 68. Preferably, the first and second Fc domain monomers are Fc domain monomers of IgG, such as Fc domain monomers of IgG1; preferably, the Fc domain monomers comprise a hinge region, CH2, and CH3. Preferably, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68; Preferably, the scFv may or may not contain a disulfide bond between VH and VL; Preferably, the bispecific antibody comprises two identical or different peptide chains IV-A, one peptide chain IV-B, and one peptide chain IV-C, wherein peptide chains IV-B and IV-C form a dimer through their respective Fc domain monomers. The antibody-drug conjugate of claim 17, wherein, The first and second Fc domain monomers each independently contain one or more amino acid modifications that promote dimerization of the first and second Fc domain monomers. 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 "knob-into-hole" modification; Preferably, the "knob" modification is the EU-numbered T366W / S354C mutation; Preferably, the "hole" is modified to be the EU numbered T366S / L368A / Y407V / Y349C mutation. The antibody-drug conjugate of claim 18, wherein, The first and second Fc domain monomers of the Fc domain further independently contain modifications that can change the function of the effector. Preferably, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / P329A; Preferably, (i) The first Fc domain monomer contains the sequence shown in SEQ ID NO: 72, and the second Fc domain monomer contains the sequence shown in SEQ ID NO: 73; or (ii) The first Fc domain monomer contains the sequence shown in SEQ ID NO: 73, and the second Fc domain monomer contains the sequence shown in SEQ ID NO:

72. The antibody-drug conjugate of any one of claims 1-11, wherein, One of the first antigen-binding domain and the second antigen-binding domain is Fab, and the other is scFv or Fab; and the bispecific antibody 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 peptide linkers. The antibody-drug conjugate of claim 20, wherein, The first antigen-binding domain is scFv, the second antigen-binding domain is Fab, and the bispecific antibody comprises: (i) A peptide chain IA comprising a first antigen-binding domain and a first Fc domain monomer; preferably, the first Fc domain monomer comprises a hinge region, CH2 and CH3; preferably, the first antigen-binding domain is connected to the N-terminus of the first Fc domain monomer via a peptide linker; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:

64. (ii) a peptide chain IB comprising a second antigen-binding domain VH, a heavy chain CH1 region, and a second Fc domain monomer; preferably, the second Fc domain monomer comprises a hinge region, CH2, and CH3; and (iii) Peptide chain IC, which includes a second protobinding domain VL and a light chain constant region (CL). Preferably, the second Fc domain monomer forms a dimer with the first Fc domain monomer; Preferably, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker, such as a flexible peptide containing (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68; Preferably, the scFv may or may not contain a disulfide bond between VH and VL; Preferably, the bispecific antibody comprises a peptide chain IA, a peptide chain IB, and a peptide chain IC, wherein peptide chains IA and IB form a dimer through their respective Fc domain monomers. The antibody-drug conjugate of claim 20, 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)CrossMabFab: In Fab, the antibody light chain CL-VL and antibody heavy chain CH1-VH are interchanged; (b) CrossMabVH-VL: In the Fab, the VL and VH of the antibody were interchanged; or (c)CrossMabCH1-CL: In Fab, the CH and CL of the antibody are interchanged. The antibody-drug conjugate of claim 22, wherein, The bispecific antibody comprises: (i) Peptide chain II-A, which includes the VL of the first antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; (ii) peptide chain II-B, which includes the VH of the first antigen-binding domain, the CH1 region of the heavy chain, and the monomer of the first Fc domain. (iii) Peptide chain II-C, comprising the VH of the second antigen-binding domain, the light chain constant region (CL), and the second Fc domain monomer; (iv) Peptide chain II-D, 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; Preferably, the bispecific antibody comprises a peptide chain II-A, a peptide chain II-B, a peptide chain II-C, and a peptide chain II-D, wherein peptide chains II-B and II-C form a dimer through their respective Fc domain monomers. The antibody-drug conjugate according to any one of claims 20-23, wherein, The first and second Fc domain monomers each independently contain one or more amino acid modifications that promote dimerization of the first and second Fc domain monomers. 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 "knob-into-hole" modification; Preferably, the "knob" modification is the EU-numbered T366W / S354C mutation; Preferably, the "hole" is modified to be the EU numbered T366S / L368A / Y407V / Y349C mutation. The antibody-drug conjugate of claim 24, wherein, The first and second Fc domain monomers of the Fc domain further independently contain modifications that can change the function of the effector. Preferably, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / P329A; Preferably, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:72 and 73, or SEQ ID NO:73 and 72. The antibody-drug conjugate of any one of claims 1-11, wherein, One of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the other is VH / VL; Preferably, the VH is attached to the N-terminus of the heavy chain of the full-length antibody via a peptide linker; and the VL is attached to the N-terminus of the light chain of the full-length antibody via a peptide linker. Preferably, the VH is connected to the N-terminus of one of the heavy chains of the full-length antibody via a peptide linker; and the VL is connected to the N-terminus of one of the light chains of the full-length antibody via a peptide linker. The antibody-drug conjugate of claim 26, wherein, The first antigen-binding domain is VH / VL, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises: (i) Peptide chain VI-A, comprising a VL of the first antigen-binding domain, a VL of the second antigen-binding domain, and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; preferably, the VL of the first antigen-binding domain is connected to the N-terminus of the VL of the second antigen-binding domain via a peptide linker. (ii) Peptide chain VI-B, comprising a VH of the first antigen-binding domain, a VH of the second antigen-binding domain, a heavy chain CH1 region, and an Fc domain monomer; preferably, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1; preferably, the Fc domain monomer comprises a hinge region, CH2, and CH3; preferably, the VH of the first antigen-binding domain is connected to the N-terminus of the VH of the second antigen-binding domain via a peptide linker; Preferably, each of the peptide linkers is 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, 4, 5, 6, 7, 8, 9 or 10. Preferably, each peptide linker independently comprises an amino acid sequence as shown in SEQ ID NO:68; Preferably, the antibody-drug conjugate comprises two identical or different peptide chains VI-A and two identical or different peptide chains VI-B, wherein the two peptide chains VI-B form a dimer through their respective Fc domain monomers. The antibody-drug conjugate of claim 27, wherein, The Fc domain monomer contains modifications that can have altered effector functions; Preferably, the modification of the altered effector function includes the following mutations with EU numbers: L234A / L235A / G237A. Preferably, the Fc domain monomer contains the sequence shown in SEQ ID NO:

74. The antibody-drug conjugate of claim 26, wherein, The first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), the second antigen-binding domain is VH / VL, and the bispecific antibody comprises: (i) Peptide chain VII-A, comprising a VL of the second antigen-binding domain, a VL of the first antigen-binding domain, and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; preferably, the VL of the second antigen-binding domain is connected to the N-terminus of the VL of the first antigen-binding domain via a peptide linker. (ii) Peptide chain VII-B, comprising VH of the second antigen-binding domain, VH of the first antigen-binding domain, heavy chain CH1 region, and monomer of the second Fc domain; preferably, VH of the second antigen-binding domain is connected to the N-terminus of VH of the first antigen-binding domain via a peptide linker. (iii) A peptide chain VII-C, comprising the VL of the second antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; and (iv) Peptide chain VII-D, comprising the VH of the second antigen-binding domain, the CH1 region of the heavy chain, and a monomer of the first Fc domain; Preferably, the first and second Fc domain monomers are Fc domain monomers of IgG, such as Fc domain monomers of IgG1; preferably, the Fc domain monomers comprise a hinge region, CH2, and CH3. Preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:68; Preferably, the antibody-drug conjugate comprises a peptide chain VII-A, a peptide chain VII-B, a peptide chain VII-C, and a peptide chain VII-D, wherein peptide chains VII-B and VII-D form a dimer through their respective Fc domain monomers. The antibody-drug conjugate of claim 29, wherein, The first and second Fc domain monomers each independently contain one or more amino acid modifications that promote dimerization of the first and second Fc domain monomers. 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 "knob-into-hole" modification; Preferably, the "knob" modification is the EU-numbered T366W / S354C mutation; Preferably, the "hole" is modified to be the EU numbered T366S / L368A / Y407V / Y349C mutation. The antibody-drug conjugate of claim 30, wherein, The first and second Fc domain monomers of the Fc domain further independently contain modifications that can change the function of the effector. Preferably, the modification that alters the effector function includes the following mutations with EU numbers: L234A / L235A / P329A; Preferably, the first and second Fc domain monomers respectively comprise sequences selected from: SEQ ID NO:72 and 73, or SEQ ID NO:73 and 72. The antibody-drug conjugate of any one of claims 1-31, wherein, The bispecific antibody or its antigen-binding fragment comprises: (1) A peptide chain VA comprising the amino acid sequence shown in SEQ ID NO:2, and / or a peptide chain VB comprising the amino acid sequence shown in SEQ ID NO:5; (2) peptide chain III-A comprising the amino acid sequence shown in SEQ ID NO:2, and / or peptide chain III-B comprising the amino acid sequence shown in SEQ ID NO:4; (3) A peptide chain IV-A containing the amino acid sequence shown in SEQ ID NO:2, a peptide chain IV-B containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain IV-C containing the amino acid sequence shown in SEQ ID NO:

12. (4) A peptide chain IV-A containing the amino acid sequence shown in SEQ ID NO:2, a peptide chain IV-B containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain IV-C containing the amino acid sequence shown in SEQ ID NO:

15. (5) A peptide chain IA containing the amino acid sequence shown in SEQ ID NO:3, a peptide chain IB containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain IC containing the amino acid sequence shown in SEQ ID NO:

2. (6) Peptide chain II-A containing the amino acid sequence shown in SEQ ID NO:9, peptide chain II-B containing the amino acid sequence shown in SEQ ID NO:8, peptide chain II-C containing the amino acid sequence shown in SEQ ID NO:10, and / or peptide chain II-D containing the amino acid sequence shown in SEQ ID NO:

11. (7) peptide chain VI-A comprising the amino acid sequence shown in SEQ ID NO:7, and / or peptide chain VI-B comprising the amino acid sequence shown in SEQ ID NO:6; or (8) A peptide chain VII-A containing the amino acid sequence shown in SEQ ID NO:14, a peptide chain VII-B containing the amino acid sequence shown in SEQ ID NO:13, a peptide chain VII-C containing the amino acid sequence shown in SEQ ID NO:1, and / or a peptide chain VII-D containing the amino acid sequence shown in SEQ ID NO:

2. The antibody-drug conjugate according to any one of claims 1-32, wherein, M includes Wherein, ring A is a 5-6 membered aliphatic heterocycle or a 5-20 membered aromatic ring system, wherein the aliphatic heterocycle and aromatic ring system are optionally selected independently by one or more groups selected from oxygen (=O), halogen, cyano, amino, carboxyl, mercapto, and C. 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group. The antibody-drug conjugate according to any one of claims 1-32, wherein, M includes Wherein ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by one or more 6-membered heteroaromatic rings connected to a benzene ring via single bonds, or a polycyclic ring formed by multiple 6-membered heteroaromatic rings connected via single bonds, wherein the aliphatic heterocycle is optionally surrounded by one or more elements selected from oxygen (=O), halogens, and C. 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group. The antibody-drug conjugate according to any one of claims 1-32, wherein, M includes Where ring A is selected from M1 is selected from single bond, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl or amino group. The antibody-drug conjugate according to any one of claims 1-32, wherein, M is selected from The antibody-drug conjugate according to any one of claims 1-32, wherein, M is The antibody-drug conjugate according to any one of claims 1-32, wherein, M is selected from The antibody-drug conjugate according to any one of claims 1-32, wherein, M is selected from The antibody-drug conjugate according to any one of claims 1-39, wherein, L can be selected from one or more of the following structures: C 1-6 Alkyl group, -N(R')-, carbonyl group, -O-, selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) s Natural or non-natural amino acids and their analogues containing 1, 2, 3 or 4 amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG(SEQ)) ID NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81))), Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20. The antibody-drug conjugate of claim 40, wherein, The short peptide is selected from Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-G lu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG (SEQ ID NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81)). The antibody-drug conjugate according to any one of claims 1-39, wherein, The L is selected from a structure that includes one or more of the following: C 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly(GGFG(SEQ ID NO:77)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:78)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:79)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:80)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:81)), Where s is selected from integers from 1 to 20. The antibody-drug conjugate according to any one of claims 1-39, wherein, The L is selected from one or more of the following structures: The antibody-drug conjugate according to any one of claims 1-39, wherein, The L is selected from the following structures: The antibody-drug conjugate according to any one of claims 1-39, wherein, The L is selected from the following structures: The antibody-drug conjugate according to any one of claims 1-39, wherein, The L is selected from the following structures: The antibody-drug conjugate according to any one of claims 1-39, wherein, The L is selected from the following structures: The antibody-drug conjugate according to any one of claims 1-39, wherein, The L is selected from the following structures: The antibody-drug conjugate according to any one of claims 1-48, wherein, E is a single bond or is selected from the following structures: -NHCH2-, -NHCH2-O-CH2-CO-, -CO-O-CH2-CO-, The antibody-drug conjugate according to any one of claims 1-48, wherein, E represents a single bond, -NHCH2-, -NHCH2-O-CH2-CO-, The antibody-drug conjugate according to any one of claims 1-48, wherein, E is -NHCH2- or The antibody-drug conjugate according to any one of claims 1-48, wherein, E can be -NHCH2- or a single bond. The antibody-drug conjugate according to any one of claims 1-48, wherein, E is either -NHCH2- or -NHCH2-O-CH2-CO-. The antibody-drug conjugate according to any one of claims 1-48, wherein, E is The antibody-drug conjugate according to any one of claims 1-54, wherein, Selected from the following structures: The antibody-drug conjugate according to any one of claims 1-54, wherein, Selected from the following structures: The antibody-drug conjugate according to any one of claims 1-56, wherein, The cytotoxic drugs are selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors. The antibody-drug conjugate of claim 57, wherein, The microtubule inhibitor is an olistatin or maytansine compound. The antibody-drug conjugate of claim 58, wherein, The olistatin compounds are selected from the following: The antibody-drug conjugate of claim 57, wherein, The DNA intercalating agent is pyrrolobenzodiazepine. (PBD) The antibody-drug conjugate of claim 57, wherein, The DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor. The antibody-drug conjugate of claim 61, wherein, The topoisomerase I inhibitor is selected from camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, rubotecan, and pharmaceutically acceptable salts, esters, or analogs thereof; the topoisomerase II inhibitor is selected from doxorubicin, PNU-159682, docalimcin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, and pharmaceutically acceptable salts, esters, or analogs thereof. The antibody-drug conjugate of claim 57, wherein, The RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analogue thereof. The antibody-drug conjugate according to any one of claims 1-56, wherein, The cytotoxic drug is selected from compounds of Formula I and II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of Formula I and II. Among them, R1 and R2 are each independently selected from C. 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; R4 and R5 are each independently selected from H, halogens, and hydroxyl groups; or R4 and R5 are linked to form a 5-6 membered oxygen-containing heterocycle; R6 is selected from hydrogen or -C. 1-4 Alkylene-NR a R b ; R7 is selected from C 1-6 Alkyl, -C 1-4 Alkylene-NR a R b -C 1-4 Alkylene-SiR a R b R c -SiR a R b R c -C 1-4 Alkylene = N-OR a ;where R a R b and R c Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, -SO2-C 1-6 Alkyl and -CO-C 1-6 Alkyl; wherein R is optionally present a and R b It forms 5-6 member nitrogen-containing heterocycles with the connected atoms. The antibody-drug conjugate according to any one of claims 1-56, wherein, The cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of the compounds: The cytotoxic drug fragment obtained after being linked to the linker is D in the general formula of claim 1. The antibody-drug conjugate according to any one of claims 1-56, wherein, The cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of the compounds: The fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula of claim 1. The antibody-drug conjugate of claim 65 or 66, wherein, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amine group on the cytotoxic drug. The antibody-drug conjugate according to any one of claims 1-67, wherein, The antibody-drug conjugate is selected from: Wherein, Ab or HA is a bispecific antibody or its antigen-binding fragment as defined in any one of claims 1-32; This indicates the specific linkage between the thiol group of the cysteine ​​residue in the bispecific antibody or its antigen-binding fragment and M in the antibody-drug conjugate; and x represents the amount of drug load. The antibody-drug conjugate of claim 68, wherein, The antibody-drug conjugate is obtained by forming a thioether bond between the thiol group in the bispecific antibody or its antigen-binding fragment and M in the antibody-drug conjugate through an addition reaction or a substitution reaction. The antibody-drug conjugate of claim 68 or 69, wherein, The Ab or HA is a bispecific antibody selected from bsAb01, bsAb02, bsAb03, bsAb04, bsAb05, bsAb06, bsAb07, and bsAb08, preferably bsAb01, bsAb02, bsAb03, and bsAb04. Antibody-drug conjugates selected from bsAb01-A-10, bsAb02-A-10, bsAb03-A-10, and bsAb04-A-10. A composition comprising one or more antibody-drug conjugates as described in any one of claims 1-71. The composition of claim 72, wherein, The composition has a DAR (drug-antibody conjugate ratio) of 1-10. The composition of claim 72, wherein, The DAR value of the composition is 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. The composition of claim 72, wherein, The composition has a DAR value of 3 to 9. The composition of claim 72, wherein, The composition has a DAR value of 4 to 8. The composition of claim 72, wherein, The DAR values ​​of the composition are 3.0–3.5, 3.0–4.0, 3.0–4.5, 3.0–5.0, 3.0–5.5, 3.0–6.0, 3.5–4.0, 3.5–4.5, 3.5–5.0, 3.5–5.5, 3.5–6.0, 3.5–6.5, 3.5–7.0, 3.5–7.5, 3.5–8.0, 4.0–4.5, 4.0–5.0, 4.0–5.5, 4.0–6.0, 4.0–6.5, 4.0–7.0, 4.0–7.5, 4.0–8.0, 4.5–5.0, and 4.5–5.

0. 5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0~5.5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.

0. A pharmaceutical composition comprising one or more antibody-drug conjugates as described in any one of claims 1-71 or compositions as described in any one of claims 72-77, and a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition of claim 78, wherein, The pharmaceutical composition further comprises one or more additional pharmaceutically active agents selected from: TROP2 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, B7H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof. The drug combination comprises a first therapeutic agent and a second therapeutic agent, wherein, The first therapeutic agent and the second therapeutic agent are administered simultaneously or sequentially. The first therapeutic agent is selected from one or more antibody-drug conjugates according to any one of claims 1-71 or compositions according to any one of claims 72-77. The second therapeutic agent is selected from: TROP2 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, B7H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof. Use in the preparation of a medicament of any one of claims 1-71, any one of claims 72-77, any one of claims 78 or 79, or any one of claims 80, wherein, The drug is used in subjects for the prevention and / or treatment of diseases associated with PDL1 and / or B7H3 and / or as an adjuvant for the treatment of such diseases, and / or for inhibiting the activity of PDL1 and / or B7H3 in vitro or in subjects; optionally, the disease associated with PDL1 and / or B7H3 is a tumor; optionally, the tumor is B7H3 and / or PDL1 positive. The use as described in claim 81, wherein, The tumor is lung cancer, stomach cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Preferably, the tumor is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma. A method for inhibiting the activity of PDL1 and / or B7H3 in cells, the method comprising contacting the cells with an antibody-drug conjugate according to any one of claims 1-71, a composition according to any one of claims 72-77, a pharmaceutical composition according to claim 78 or 79, or a pharmaceutical combination according to claim 80, wherein the cells are cells expressing PDL1 and / or B7H3. A method for use in subjects to prevent and / or treat and / or as an adjunct to the treatment of diseases associated with PDL1 and / or B7H3, the method comprising administering to a subject in need an effective amount of an antibody-drug conjugate of any one of claims 1-71, a composition of any one of claims 72-77, a pharmaceutical composition of claim 78 or 79, or a pharmaceutical combination of claim 80, optionally, wherein the disease associated with PDL1 and / or B7H3 is a tumor; optionally, the tumor is B7H3 and / or PDL1 positive. The method of claim 84, wherein, The tumor is lung cancer, stomach cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Preferably, the tumor is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma. The method of claim 84 or 85, wherein the method further comprises administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy may be administered simultaneously, separately, or sequentially. The antibody-drug conjugate of any one of claims 1-71, the composition of any one of claims 72-77, the pharmaceutical composition of claim 78 or 79, or the pharmaceutical combination of claim 80, for use in subjects for the prevention and / or treatment and / or as an adjunct to the treatment of diseases associated with PDL1 and / or B7H3, wherein... The disease associated with PDL1 and / or B7H3 is a tumor; optionally, the tumor is B7H3 and / or PDL1 positive. The antibody-drug conjugate, composition, pharmaceutical composition, or pharmaceutical combination of claim 87, wherein, The tumor is lung cancer, stomach cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Preferably, the tumor is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma. The antibody-drug conjugate, composition, pharmaceutical composition, or combination of drugs according to claim 87 or 88, wherein the prevention and / or treatment and / or adjuvant treatment of diseases associated with PDL1 and / or B7H3 in the subject further comprises administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy may be administered simultaneously, separately, or sequentially.

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