Bispecific b7h3 / EGFR-binding protein and conjugate thereof

Abstract

Provided are a bispecific binding protein targeting EGFR and B7H3 and a conjugate thereof, and the use of the bispecific protein and conjugate thereof in the treatment of diseases, particularly in the treatment of tumors.

Description

B7H3 / EGFR bispecific binding protein and its conjugates Technical Field

[0001] This application relates to bispecific binding proteins and conjugates targeting EGFR and B7H3, and the use of said bispecific binding proteins and conjugates in the treatment of diseases. Background Technology

[0002] B7H3 (also known as CD276) is a type I transmembrane protein and an important member of the immune checkpoint inhibitors. B7H3 is constitutively expressed on non-immune cells (including synovial cells, osteoblasts, and endothelial cells) and induced to express on the surface of immune cells (DCs, monocytes, B cells, etc.), participating in the regulation of T-cell immune responses. B7H3 is often associated with poor prognosis in human patients, and inhibiting the B7H3 signaling pathway can mediate effective anti-tumor activity of immune cells. Recent studies have also shown that B7H3 can affect cancer progression beyond its immunomodulatory role. Given the important role of B7H3 in immune checkpoint regulation, several therapeutically targeted B7H3 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.

[0003] EGFR (epidermal growth factor receptor, abbreviated as EGFR, ErbB-1, or HER1) is a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4). The HER family plays an important regulatory role in cellular physiological processes. EGFR is a type I transmembrane glycoprotein, approximately 170 kDa in size. Its main structure includes a ligand-binding extracellular domain, a hydrophobic transmembrane region, and an intracellular region containing tyrosine kinases. It is highly expressed in various tumors such as colorectal cancer, head and neck cancer, and non-small cell lung cancer, and is also expressed to some extent in normal epithelial tissues such as skin and lung. EGFR, which is highly expressed in tumor tissues, homodimerizes or heterodimerizes under the action of ligands such as EGF or TGFα. Dimerization leads to the activation of tyrosine kinases and protein phosphorylation in tumor cells, and the activation of various cell signaling pathways that mediate gene transcription and cell cycle processes. It plays an important role in promoting the survival, proliferation and migration of tumor cells and tumor angiogenesis.

[0004] Bispecific antibodies (BsAbs) are antibodies that can simultaneously target two different antigenic epitopes. Several B7H3 / EGFR bispecific antibodies have been disclosed in this field, such as Hz20G5.26 / Zalu bsAb disclosed in CN202411888761.5. There remains a need to develop high-performance bispecific antibodies targeting both EGFR and B7H3, which will provide patients with more treatment options. Summary of the Invention

[0005] This disclosure provides a bispecific antibody and antibody-drug conjugate targeting EGFR and B7H3, which can specifically recognize and bind to EGFR and B7H3, has high affinity and endocytic activity for cells expressing EGFR and / or B7H3 (e.g., tumor cells), and has a strong killing effect on tumor cells. It has important application value in the treatment of diseases mediated by EGFR and / or B7H3 (e.g., cancer).

[0006] Bispecific antibodies

[0007] In one aspect, this disclosure provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to B7H3.

[0008] 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 EGFR; 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.

[0009] In some embodiments, the bispecific antibody may include one or more first antigen-binding domains and one or more second antigen-binding domains. For example, the bispecific antibody may include two first antigen-binding domains and two second antigen-binding domains.

[0010] I. The first antigen-binding domain that specifically binds to EGFR

[0011] In some embodiments, the first VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 6; and / or, the first VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 7;

[0012] The CDR can be defined by the Chothia, AbM, Kabat, IMGT, MacCallum or AHo numbering system.

[0013] In some implementations, the first VL includes:

[0014] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 10, CDR-L2 containing the sequence shown in SEQ ID NO: 12, and CDR-L3 containing the sequence shown in SEQ ID NO: 14; or,

[0015] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 11, CDR-L2 containing the sequence shown in SEQ ID NO: 13, and CDR-L3 containing the sequence shown in SEQ ID NO: 14.

[0016] In some implementations, the first VH includes:

[0017] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 16, CDR-H2 containing the sequence shown in SEQ ID NO: 20, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0018] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 18, CDR-H2 containing the sequence shown in SEQ ID NO: 22, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0019] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; or,

[0020] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 17, CDR-H2 containing the sequence shown in SEQ ID NO: 21, and CDR-H3 containing the sequence shown in SEQ ID NO: 24.

[0021] In some implementations, the first VL includes:

[0022] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 10, CDR-L2 comprising the sequence shown in SEQ ID NO: 12, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0023] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 11, CDR-L2 comprising the sequence shown in SEQ ID NO: 13, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the IMGT numbering system.

[0024] In some implementations, the first VH includes:

[0025] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 16, CDR-H2 comprising the sequence shown in SEQ ID NO: 20, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Chothia numbering system;

[0026] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 18, CDR-H2 comprising the sequence shown in SEQ ID NO: 22, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the AbM numbering system;

[0027] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Kabat numbering system; or,

[0028] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 17, CDR-H2 comprising the sequence shown in SEQ ID NO: 21, and CDR-H3 comprising the sequence shown in SEQ ID NO: 24; wherein the CDR is defined by the IMGT numbering system.

[0029] In some embodiments, the first VL comprises an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, the first VH comprises an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0030] II. Second antigen-binding domain that specifically binds to B7H3

[0031] In some embodiments, the second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 8 or 67; and / or, the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 9 or 68.

[0032] In some embodiments, the second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 8; and / or, the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 9;

[0033] The CDR can be defined by the Chothia, AbM, Kabat, IMGT, MacCallum or AHo numbering system.

[0034] In some implementations, the second VL includes:

[0035] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 25, CDR-L2 containing the sequence shown in SEQ ID NO: 27, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; or,

[0036] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 26, CDR-L2 containing the sequence shown in SEQ ID NO: 28, and CDR-L3 containing the sequence shown in SEQ ID NO: 29.

[0037] In some implementations, the second VH comprises:

[0038] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 31, CDR-H2 containing the sequence shown in SEQ ID NO: 35, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0039] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 33, CDR-H2 containing the sequence shown in SEQ ID NO: 37, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0040] (iii) CDR-H1 containing the sequence shown in SEQ ID NO: 30, CDR-H2 containing the sequence shown in SEQ ID NO: 34, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0041] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 32, CDR-H2 containing the sequence shown in SEQ ID NO: 36, and CDR-H3 containing the sequence shown in SEQ ID NO: 39.

[0042] In some implementations, the second VL includes:

[0043] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 25, CDR-L2 comprising the sequence shown in SEQ ID NO: 27, and CDR-L3 comprising the sequence shown in SEQ ID NO: 29; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0044] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 26, CDR-L2 comprising the sequence shown in SEQ ID NO: 28, and CDR-L3 comprising the sequence shown in SEQ ID NO: 29; wherein the CDR is defined by the IMGT numbering system.

[0045] In some implementations, the second VH comprises:

[0046] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 31, CDR-H2 comprising the sequence shown in SEQ ID NO: 35, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Chothia numbering system;

[0047] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 33, CDR-H2 comprising the sequence shown in SEQ ID NO: 37, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the AbM numbering system;

[0048] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Kabat numbering system; or,

[0049] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 32, CDR-H2 comprising the sequence shown in SEQ ID NO: 36, and CDR-H3 comprising the sequence shown in SEQ ID NO: 39; wherein the CDR is defined by the IMGT numbering system.

[0050] In some embodiments, the second VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0051] In some embodiments, the second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 67, and / or CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 68;

[0052] The CDR can be defined by the Chothia, AbM, Kabat, IMGT, MacCallum or AHo numbering system.

[0053] In some implementations, the second VL includes:

[0054] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 69, CDR-L2 containing the sequence shown in SEQ ID NO: 71, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; or,

[0055] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 70, CDR-L2 containing the sequence shown in SEQ ID NO: 72, and CDR-L3 containing the sequence shown in SEQ ID NO: 73.

[0056] In some implementations, the second VH comprises:

[0057] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 75, CDR-H2 containing the sequence shown in SEQ ID NO: 79, and CDR-H3 containing the sequence shown in SEQ ID NO: 82;

[0058] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 77, CDR-H2 containing the sequence shown in SEQ ID NO: 81, and CDR-H3 containing the sequence shown in SEQ ID NO: 82;

[0059] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 74, CDR-H2 comprising the sequence shown in SEQ ID NO: 78, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; or,

[0060] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 76, CDR-H2 containing the sequence shown in SEQ ID NO: 80, and CDR-H3 containing the sequence shown in SEQ ID NO: 83.

[0061] In some implementations, the second VL includes:

[0062] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 69, CDR-L2 comprising the sequence shown in SEQ ID NO: 71, and CDR-L3 comprising the sequence shown in SEQ ID NO: 73; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0063] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 70, CDR-L2 comprising the sequence shown in SEQ ID NO: 72, and CDR-L3 comprising the sequence shown in SEQ ID NO: 73; wherein the CDR is defined by the IMGT numbering system.

[0064] In some implementations, the second VH comprises:

[0065] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 75, CDR-H2 comprising the sequence shown in SEQ ID NO: 79, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the Chothia numbering system;

[0066] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 77, CDR-H2 comprising the sequence shown in SEQ ID NO: 81, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the AbM numbering system;

[0067] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 74, CDR-H2 comprising the sequence shown in SEQ ID NO: 78, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the Kabat numbering system; or,

[0068] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 76, CDR-H2 comprising the sequence shown in SEQ ID NO: 80, and CDR-H3 comprising the sequence shown in SEQ ID NO: 83; wherein the CDR is defined by the IMGT numbering system.

[0069] In some embodiments, the second VL comprises an amino acid sequence as shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0070] In some embodiments, the second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 67; and / or, the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 9;

[0071] The CDR can be defined by the Chothia, AbM, Kabat, IMGT, MacCallum or AHo numbering system.

[0072] In some implementations, the second VL includes:

[0073] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 69, CDR-L2 containing the sequence shown in SEQ ID NO: 71, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; or,

[0074] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 70, CDR-L2 containing the sequence shown in SEQ ID NO: 72, and CDR-L3 containing the sequence shown in SEQ ID NO: 73.

[0075] In some implementations, the second VH comprises:

[0076] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 31, CDR-H2 containing the sequence shown in SEQ ID NO: 35, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0077] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 33, CDR-H2 containing the sequence shown in SEQ ID NO: 37, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0078] (iii) CDR-H1 containing the sequence shown in SEQ ID NO: 30, CDR-H2 containing the sequence shown in SEQ ID NO: 34, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0079] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 32, CDR-H2 containing the sequence shown in SEQ ID NO: 36, and CDR-H3 containing the sequence shown in SEQ ID NO: 39.

[0080] In some implementations, the second VL includes:

[0081] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 69, CDR-L2 comprising the sequence shown in SEQ ID NO: 71, and CDR-L3 comprising the sequence shown in SEQ ID NO: 73; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0082] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 70, CDR-L2 comprising the sequence shown in SEQ ID NO: 72, and CDR-L3 comprising the sequence shown in SEQ ID NO: 73; wherein the CDR is defined by the IMGT numbering system.

[0083] In some implementations, the second VH comprises:

[0084] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 31, CDR-H2 comprising the sequence shown in SEQ ID NO: 35, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Chothia numbering system;

[0085] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 33, CDR-H2 comprising the sequence shown in SEQ ID NO: 37, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the AbM numbering system;

[0086] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Kabat numbering system; or,

[0087] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 32, CDR-H2 comprising the sequence shown in SEQ ID NO: 36, and CDR-H3 comprising the sequence shown in SEQ ID NO: 39; wherein the CDR is defined by the IMGT numbering system.

[0088] In some embodiments, the second VL comprises an amino acid sequence as shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0089] In some embodiments, the second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 8; and / or, the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 68;

[0090] The CDR can be defined by the Chothia, AbM, Kabat, IMGT, MacCallum or AHo numbering system.

[0091] In some implementations, the second VL includes:

[0092] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 25, CDR-L2 containing the sequence shown in SEQ ID NO: 27, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; or,

[0093] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 26, CDR-L2 containing the sequence shown in SEQ ID NO: 28, and CDR-L3 containing the sequence shown in SEQ ID NO: 29.

[0094] In some implementations, the second VH comprises:

[0095] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 75, CDR-H2 containing the sequence shown in SEQ ID NO: 79, and CDR-H3 containing the sequence shown in SEQ ID NO: 82;

[0096] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 77, CDR-H2 containing the sequence shown in SEQ ID NO: 81, and CDR-H3 containing the sequence shown in SEQ ID NO: 82;

[0097] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 74, CDR-H2 comprising the sequence shown in SEQ ID NO: 78, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; or,

[0098] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 76, CDR-H2 containing the sequence shown in SEQ ID NO: 80, and CDR-H3 containing the sequence shown in SEQ ID NO: 83.

[0099] In some implementations, the second VL includes:

[0100] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 25, CDR-L2 comprising the sequence shown in SEQ ID NO: 27, and CDR-L3 comprising the sequence shown in SEQ ID NO: 29; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0101] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 26, CDR-L2 comprising the sequence shown in SEQ ID NO: 28, and CDR-L3 comprising the sequence shown in SEQ ID NO: 29; wherein the CDR is defined by the IMGT numbering system.

[0102] In some implementations, the second VH comprises:

[0103] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 75, CDR-H2 comprising the sequence shown in SEQ ID NO: 79, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the Chothia numbering system;

[0104] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 77, CDR-H2 comprising the sequence shown in SEQ ID NO: 81, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the AbM numbering system;

[0105] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 74, CDR-H2 comprising the sequence shown in SEQ ID NO: 78, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the Kabat numbering system; or,

[0106] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 76, CDR-H2 comprising the sequence shown in SEQ ID NO: 80, and CDR-H3 comprising the sequence shown in SEQ ID NO: 83; wherein the CDR is defined by the IMGT numbering system.

[0107] In some embodiments, the second VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0108] III. First antigen-binding domain / Second antigen-binding domain

[0109] In some embodiments of the bispecific antibody disclosed herein:

[0110] (i) the first VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 6, and the first VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7; and

[0111] (ii) The second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 8, and the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 9.

[0112] In some embodiments of the bispecific antibody disclosed herein:

[0113] (i) the first VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 6, and the first VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7; and

[0114] (ii) The second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 67, and the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 68.

[0115] In some embodiments of the bispecific antibody disclosed herein:

[0116] (i) the first VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 6, and the first VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7; and

[0117] (ii) The second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 67, and the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 9.

[0118] In some embodiments of the bispecific antibody disclosed herein:

[0119] (i) the first VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 6, and the first VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7; and

[0120] (ii) The second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 8, and the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 68.

[0121] In some implementations, the CDR is defined by the Chothia, AbM, Kabat, IMGT, MacCallum, or AHo numbering system.

[0122] In some embodiments of the bispecific antibody disclosed herein:

[0123] (a) The first VL includes:

[0124] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 10, CDR-L2 containing the sequence shown in SEQ ID NO: 12, and CDR-L3 containing the sequence shown in SEQ ID NO: 14; or,

[0125] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 11, CDR-L2 containing the sequence shown in SEQ ID NO: 13, and CDR-L3 containing the sequence shown in SEQ ID NO: 14;

[0126] and,

[0127] (b) The first VH includes:

[0128] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 16, CDR-H2 containing the sequence shown in SEQ ID NO: 20, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0129] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 18, CDR-H2 containing the sequence shown in SEQ ID NO: 22, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0130] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; or,

[0131] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 17, CDR-H2 containing the sequence shown in SEQ ID NO: 21, and CDR-H3 containing the sequence shown in SEQ ID NO: 24;

[0132] and

[0133] (c) The second VL includes:

[0134] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 25, CDR-L2 containing the sequence shown in SEQ ID NO: 27, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; or,

[0135] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 26, CDR-L2 containing the sequence shown in SEQ ID NO: 28, and CDR-L3 containing the sequence shown in SEQ ID NO: 29;

[0136] and,

[0137] (d) The second VH includes:

[0138] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 31, CDR-H2 containing the sequence shown in SEQ ID NO: 35, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0139] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 33, CDR-H2 containing the sequence shown in SEQ ID NO: 37, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0140] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; or,

[0141] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 32, CDR-H2 containing the sequence shown in SEQ ID NO: 36, and CDR-H3 containing the sequence shown in SEQ ID NO: 39.

[0142] In some embodiments of the bispecific antibody disclosed herein:

[0143] (a) The first VL includes:

[0144] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 10, CDR-L2 containing the sequence shown in SEQ ID NO: 12, and CDR-L3 containing the sequence shown in SEQ ID NO: 14; or,

[0145] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 11, CDR-L2 containing the sequence shown in SEQ ID NO: 13, and CDR-L3 containing the sequence shown in SEQ ID NO: 14;

[0146] and,

[0147] (b) The first VH includes:

[0148] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 16, CDR-H2 containing the sequence shown in SEQ ID NO: 20, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0149] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 18, CDR-H2 containing the sequence shown in SEQ ID NO: 22, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0150] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; or,

[0151] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 17, CDR-H2 containing the sequence shown in SEQ ID NO: 21, and CDR-H3 containing the sequence shown in SEQ ID NO: 24;

[0152] and

[0153] (c) The second VL includes:

[0154] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 69, CDR-L2 containing the sequence shown in SEQ ID NO: 71, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; or,

[0155] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 70, CDR-L2 containing the sequence shown in SEQ ID NO: 72, and CDR-L3 containing the sequence shown in SEQ ID NO: 73;

[0156] and,

[0157] (d) The second VH includes:

[0158] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 75, CDR-H2 containing the sequence shown in SEQ ID NO: 79, and CDR-H3 containing the sequence shown in SEQ ID NO: 82;

[0159] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 77, CDR-H2 containing the sequence shown in SEQ ID NO: 81, and CDR-H3 containing the sequence shown in SEQ ID NO: 82;

[0160] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 74, CDR-H2 comprising the sequence shown in SEQ ID NO: 78, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; or,

[0161] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 76, CDR-H2 containing the sequence shown in SEQ ID NO: 80, and CDR-H3 containing the sequence shown in SEQ ID NO: 83.

[0162] In some embodiments of the bispecific antibody disclosed herein:

[0163] (a) The first VL includes:

[0164] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 10, CDR-L2 containing the sequence shown in SEQ ID NO: 12, and CDR-L3 containing the sequence shown in SEQ ID NO: 14; or,

[0165] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 11, CDR-L2 containing the sequence shown in SEQ ID NO: 13, and CDR-L3 containing the sequence shown in SEQ ID NO: 14;

[0166] and,

[0167] (b) The first VH includes:

[0168] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 16, CDR-H2 containing the sequence shown in SEQ ID NO: 20, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0169] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 18, CDR-H2 containing the sequence shown in SEQ ID NO: 22, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0170] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; or,

[0171] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 17, CDR-H2 containing the sequence shown in SEQ ID NO: 21, and CDR-H3 containing the sequence shown in SEQ ID NO: 24;

[0172] and

[0173] (c) The second VL includes:

[0174] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 69, CDR-L2 containing the sequence shown in SEQ ID NO: 71, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; or,

[0175] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 70, CDR-L2 containing the sequence shown in SEQ ID NO: 72, and CDR-L3 containing the sequence shown in SEQ ID NO: 73;

[0176] and,

[0177] (d) The second VH includes:

[0178] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 31, CDR-H2 containing the sequence shown in SEQ ID NO: 35, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0179] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 33, CDR-H2 containing the sequence shown in SEQ ID NO: 37, and CDR-H3 containing the sequence shown in SEQ ID NO: 38;

[0180] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; or,

[0181] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 32, CDR-H2 containing the sequence shown in SEQ ID NO: 36, and CDR-H3 containing the sequence shown in SEQ ID NO: 39.

[0182] In some embodiments of the bispecific antibody disclosed herein:

[0183] (a) The first VL includes:

[0184] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 10, CDR-L2 containing the sequence shown in SEQ ID NO: 12, and CDR-L3 containing the sequence shown in SEQ ID NO: 14; or,

[0185] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 11, CDR-L2 containing the sequence shown in SEQ ID NO: 13, and CDR-L3 containing the sequence shown in SEQ ID NO: 14;

[0186] and,

[0187] (b) The first VH includes:

[0188] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 16, CDR-H2 containing the sequence shown in SEQ ID NO: 20, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0189] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 18, CDR-H2 containing the sequence shown in SEQ ID NO: 22, and CDR-H3 containing the sequence shown in SEQ ID NO: 23;

[0190] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; or,

[0191] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 17, CDR-H2 containing the sequence shown in SEQ ID NO: 21, and CDR-H3 containing the sequence shown in SEQ ID NO: 24;

[0192] and

[0193] (c) The second VL includes:

[0194] (i) CDR-L1 containing the sequence shown in SEQ ID NO: 25, CDR-L2 containing the sequence shown in SEQ ID NO: 27, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; or,

[0195] (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 26, CDR-L2 containing the sequence shown in SEQ ID NO: 28, and CDR-L3 containing the sequence shown in SEQ ID NO: 29;

[0196] and,

[0197] (d) The second VH includes:

[0198] (i) CDR-H1 containing the sequence shown in SEQ ID NO: 75, CDR-H2 containing the sequence shown in SEQ ID NO: 79, and CDR-H3 containing the sequence shown in SEQ ID NO: 82;

[0199] (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 77, CDR-H2 containing the sequence shown in SEQ ID NO: 81, and CDR-H3 containing the sequence shown in SEQ ID NO: 82;

[0200] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 74, CDR-H2 comprising the sequence shown in SEQ ID NO: 78, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; or,

[0201] (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 76, CDR-H2 containing the sequence shown in SEQ ID NO: 80, and CDR-H3 containing the sequence shown in SEQ ID NO: 83.

[0202] In some embodiments of the bispecific antibody disclosed herein:

[0203] (a) The first VL includes:

[0204] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 10, CDR-L2 comprising the sequence shown in SEQ ID NO: 12, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0205] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 11, CDR-L2 comprising the sequence shown in SEQ ID NO: 13, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the IMGT numbering system;

[0206] and,

[0207] (b) The first VH includes:

[0208] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 16, CDR-H2 comprising the sequence shown in SEQ ID NO: 20, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Chothia numbering system;

[0209] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 18, CDR-H2 comprising the sequence shown in SEQ ID NO: 22, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the AbM numbering system;

[0210] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Kabat numbering system; or,

[0211] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 17, CDR-H2 comprising the sequence shown in SEQ ID NO: 21, and CDR-H3 comprising the sequence shown in SEQ ID NO: 24; wherein the CDR is defined by the IMGT numbering system;

[0212] and

[0213] (c) The second VL includes:

[0214] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 25, CDR-L2 comprising the sequence shown in SEQ ID NO: 27, and CDR-L3 comprising the sequence shown in SEQ ID NO: 29; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0215] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 26, CDR-L2 comprising the sequence shown in SEQ ID NO: 28, and CDR-L3 comprising the sequence shown in SEQ ID NO: 29; wherein the CDR is defined by the IMGT numbering system;

[0216] and,

[0217] (d) The second VH includes:

[0218] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 31, CDR-H2 comprising the sequence shown in SEQ ID NO: 35, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Chothia numbering system;

[0219] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 33, CDR-H2 comprising the sequence shown in SEQ ID NO: 37, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the AbM numbering system;

[0220] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Kabat numbering system; or,

[0221] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 32, CDR-H2 comprising the sequence shown in SEQ ID NO: 36, and CDR-H3 comprising the sequence shown in SEQ ID NO: 39; wherein the CDR is defined by the IMGT numbering system.

[0222] In some embodiments of the bispecific antibody disclosed herein:

[0223] (a) The first VL includes:

[0224] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 10, CDR-L2 comprising the sequence shown in SEQ ID NO: 12, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0225] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 11, CDR-L2 comprising the sequence shown in SEQ ID NO: 13, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the IMGT numbering system;

[0226] and,

[0227] (b) The first VH includes:

[0228] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 16, CDR-H2 comprising the sequence shown in SEQ ID NO: 20, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Chothia numbering system;

[0229] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 18, CDR-H2 comprising the sequence shown in SEQ ID NO: 22, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the AbM numbering system;

[0230] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Kabat numbering system; or,

[0231] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 17, CDR-H2 comprising the sequence shown in SEQ ID NO: 21, and CDR-H3 comprising the sequence shown in SEQ ID NO: 24; wherein the CDR is defined by the IMGT numbering system;

[0232] and

[0233] (c) The second VL includes:

[0234] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 69, CDR-L2 comprising the sequence shown in SEQ ID NO: 71, and CDR-L3 comprising the sequence shown in SEQ ID NO: 73; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0235] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 70, CDR-L2 comprising the sequence shown in SEQ ID NO: 72, and CDR-L3 comprising the sequence shown in SEQ ID NO: 73; wherein the CDR is defined by the IMGT numbering system;

[0236] and,

[0237] (d) The second VH includes:

[0238] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 75, CDR-H2 comprising the sequence shown in SEQ ID NO: 79, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the Chothia numbering system;

[0239] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 77, CDR-H2 comprising the sequence shown in SEQ ID NO: 81, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the AbM numbering system;

[0240] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 74, CDR-H2 comprising the sequence shown in SEQ ID NO: 78, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the Kabat numbering system; or,

[0241] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 76, CDR-H2 comprising the sequence shown in SEQ ID NO: 80, and CDR-H3 comprising the sequence shown in SEQ ID NO: 83; wherein the CDR is defined by the IMGT numbering system.

[0242] In some embodiments of the bispecific antibody disclosed herein:

[0243] (a) The first VL includes:

[0244] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 10, CDR-L2 comprising the sequence shown in SEQ ID NO: 12, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0245] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 11, CDR-L2 comprising the sequence shown in SEQ ID NO: 13, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the IMGT numbering system;

[0246] and,

[0247] (b) The first VH includes:

[0248] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 16, CDR-H2 comprising the sequence shown in SEQ ID NO: 20, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Chothia numbering system;

[0249] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 18, CDR-H2 comprising the sequence shown in SEQ ID NO: 22, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the AbM numbering system;

[0250] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Kabat numbering system; or,

[0251] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 17, CDR-H2 comprising the sequence shown in SEQ ID NO: 21, and CDR-H3 comprising the sequence shown in SEQ ID NO: 24; wherein the CDR is defined by the IMGT numbering system;

[0252] and

[0253] (c) The second VL includes:

[0254] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 69, CDR-L2 comprising the sequence shown in SEQ ID NO: 71, and CDR-L3 comprising the sequence shown in SEQ ID NO: 73; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0255] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 70, CDR-L2 comprising the sequence shown in SEQ ID NO: 72, and CDR-L3 comprising the sequence shown in SEQ ID NO: 73; wherein the CDR is defined by the IMGT numbering system;

[0256] and,

[0257] (d) The second VH includes:

[0258] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 31, CDR-H2 comprising the sequence shown in SEQ ID NO: 35, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Chothia numbering system;

[0259] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 33, CDR-H2 comprising the sequence shown in SEQ ID NO: 37, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the AbM numbering system;

[0260] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Kabat numbering system; or,

[0261] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 32, CDR-H2 comprising the sequence shown in SEQ ID NO: 36, and CDR-H3 comprising the sequence shown in SEQ ID NO: 39; wherein the CDR is defined by the IMGT numbering system.

[0262] In some embodiments of the bispecific antibody disclosed herein:

[0263] (a) The first VL includes:

[0264] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 10, CDR-L2 comprising the sequence shown in SEQ ID NO: 12, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0265] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 11, CDR-L2 comprising the sequence shown in SEQ ID NO: 13, and CDR-L3 comprising the sequence shown in SEQ ID NO: 14; wherein the CDR is defined by the IMGT numbering system;

[0266] and,

[0267] (b) The first VH includes:

[0268] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 16, CDR-H2 comprising the sequence shown in SEQ ID NO: 20, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Chothia numbering system;

[0269] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 18, CDR-H2 comprising the sequence shown in SEQ ID NO: 22, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the AbM numbering system;

[0270] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Kabat numbering system; or,

[0271] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 17, CDR-H2 comprising the sequence shown in SEQ ID NO: 21, and CDR-H3 comprising the sequence shown in SEQ ID NO: 24; wherein the CDR is defined by the IMGT numbering system;

[0272] and

[0273] (c) The second VL includes:

[0274] (i) CDR-L1 comprising the sequence shown in SEQ ID NO: 25, CDR-L2 comprising the sequence shown in SEQ ID NO: 27, and CDR-L3 comprising the sequence shown in SEQ ID NO: 29; wherein the CDR is defined by the Chothia, AbM, or Kabat numbering system; or,

[0275] (ii) CDR-L1 comprising the sequence shown in SEQ ID NO: 26, CDR-L2 comprising the sequence shown in SEQ ID NO: 28, and CDR-L3 comprising the sequence shown in SEQ ID NO: 29; wherein the CDRs are defined by the IMGT numbering system; and,

[0276] (d) The second VH includes:

[0277] (i) CDR-H1 comprising the sequence shown in SEQ ID NO: 75, CDR-H2 comprising the sequence shown in SEQ ID NO: 79, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the Chothia numbering system;

[0278] (ii) CDR-H1 comprising the sequence shown in SEQ ID NO: 77, CDR-H2 comprising the sequence shown in SEQ ID NO: 81, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the AbM numbering system;

[0279] (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 74, CDR-H2 comprising the sequence shown in SEQ ID NO: 78, and CDR-H3 comprising the sequence shown in SEQ ID NO: 82; wherein the CDR is defined by the Kabat numbering system; or,

[0280] (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 76, CDR-H2 comprising the sequence shown in SEQ ID NO: 80, and CDR-H3 comprising the sequence shown in SEQ ID NO: 83; wherein the CDR is defined by the IMGT numbering system.

[0281] In some embodiments of the bispecific antibody disclosed herein:

[0282] (i) the first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0283] (ii) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0284] In some embodiments of the bispecific antibody disclosed herein:

[0285] (i) the first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0286] (ii) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0287] In some embodiments of the bispecific antibody disclosed herein:

[0288] (i) the first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0289] (ii) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0290] In some embodiments of the bispecific antibody disclosed herein:

[0291] (i) the first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0292] (ii) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0293] In some embodiments of the bispecific antibody disclosed herein:

[0294] (i) The first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0295] (ii) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and the second VH comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0296] In some embodiments of the bispecific antibody disclosed herein:

[0297] (i) The first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0298] (ii) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and the second VH comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0299] In some embodiments of the bispecific antibody disclosed herein:

[0300] (i) The first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0301] (ii) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and the second VH comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0302] In some embodiments of the bispecific antibody disclosed herein:

[0303] (i) The first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0304] (ii) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and the second VH comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0305] IV. Structure

[0306] The first and second antigen-binding domains of the bispecific antibodies disclosed herein may include structural forms of various antigen-binding fragments.

[0307] IgG-scFv

[0308] 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.

[0309] In some implementations, the scFv is connected, with or without a connector, to the C-terminus or N-terminus of the heavy or light chain of the full-length antibody.

[0310] In some embodiments, one scFv is connected to the N-terminus or C-terminus of one heavy chain of the full-length antibody, with or without a connector. In some embodiments, two scFvs are optionally connected to the N-terminus and C-terminus of one heavy chain of the full-length antibody, respectively, with or without a connector. In some embodiments, one scFv is connected to the N-terminus of one heavy chain of the full-length antibody, with or without a connector, and another scFv is connected to the C-terminus of another heavy chain of the full-length antibody, with or without a connector. In some embodiments, two scFvs are connected to the N-termins of two heavy chains of the full-length antibody, with or without a connector. In some embodiments, two scFvs are connected to the C-termins of two heavy chains of the full-length antibody, with or without a connector.

[0311] In some embodiments, the first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), the second antigen-binding domain is an scFv, and the bispecific antibody comprises:

[0312] (i) a peptide chain IA, comprising the VL of the first antigen-binding domain and a light chain constant region (CL); and

[0313] (ii) A peptide chain IB comprising the VH of the first antigen-binding domain, the CH1 region of the heavy chain, the Fc domain monomer, and the second antigen-binding domain.

[0314] In some embodiments, the second antigen-binding domain is connected to the N-terminus of the VH of the first antigen-binding domain, either via a connector or not; or, the second antigen-binding domain is connected to the C-terminus of the Fc domain monomer, either via a connector or not.

[0315] In some embodiments, the first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), the second antigen-binding domain is an scFv, and the bispecific antibody comprises:

[0316] (i) a peptide chain IA, which includes, from the N-terminus to the C-terminus, the VL of the first antigen-binding domain and the light chain constant region (CL); and

[0317] (ii) A peptide chain IB, which includes, from the N-terminus to the C-terminus, the VH of the first antigen-binding domain, the CH1 region of the heavy chain, the Fc domain monomer, and the second antigen-binding domain.

[0318] In some embodiments, the first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), the second antigen-binding domain is an scFv, and the bispecific antibody comprises:

[0319] (i) a peptide chain IA, which includes, from the N-terminus to the C-terminus, the VL of the first antigen-binding domain and the light chain constant region (CL); and

[0320] (ii) A peptide chain IB, which includes, from the N-terminus to the C-terminus, the second antigen-binding domain, the VH of the first antigen-binding domain, the heavy chain CH1 region, and the Fc domain monomer.

[0321] In some embodiments, the bispecific antibody comprises two identical or different peptide chains IA and two identical or different peptide chains IB, wherein the two peptide chains IB form a dimer through their respective Fc domain monomers.

[0322] In some embodiments, the bispecific antibody comprises two distinct peptide chains IA, wherein the VL and / or light chain constant region (CL) of the first antigen-binding domain are different.

[0323] In some embodiments, the bispecific antibody comprises different peptide chains IB, wherein the VH, heavy chain CH1 region, Fc domain monomer of the first antigen-binding domain and / or the second antigen-binding domain are different.

[0324] In some embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example having a structure as shown in (GGGGS)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 40, 41 or 44-51); or the peptide linkers each independently contain an amino acid sequence shown in any one of SEQ ID NO: 40, 41 or 44-52.

[0325] In some embodiments, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker containing one or more glycine (G) and / or serine (S), for example, having a structure as shown in (GGGGS)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 40, 41 or 44-51); or each peptide linker independently contains an amino acid sequence shown in any one of SEQ ID NO: 40, 41 or 44-52.

[0326] In some embodiments, the CL is a kappa light chain constant region, for example, the CL has a sequence as shown in SEQ ID NO: 42, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0327] In some embodiments, 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.

[0328] In some embodiments, the Fc domain monomer has a sequence as shown in SEQ ID NO: 60, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0329] In some embodiments, the Fc domain monomers further include, independently, modifications capable of altering effector function. Preferably, the modifications altering effector function include the following mutations with EU numbers: L234A / L235A / G237A; more preferably, the Fc domain monomers have a sequence as shown in SEQ ID NO: 53 or 61, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0330] In some embodiments, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), the first antigen-binding domain is an scFv, and the bispecific antibody comprises:

[0331] (i) peptide chain IV-A, which includes the VL of the second antigen-binding domain and the light chain constant region (CL); and

[0332] (ii) peptide chain IV-B, comprising the VH of the second antigen-binding domain, the heavy chain CH1 region, the Fc domain monomer, and the first antigen-binding domain.

[0333] In some embodiments, the first antigen-binding domain is connected to the N-terminus of the VH of the second antigen-binding domain, either via a connector or not; or, the first antigen-binding domain is connected to the C-terminus of the Fc domain monomer, either via a connector or not.

[0334] In some embodiments, the bispecific antibody comprises:

[0335] (i) peptide chain IV-A, which includes the VL of the second antigen-binding domain and the light chain constant region (CL) from the N-terminus to the C-terminus; and

[0336] (ii) Peptide chain IV-B, which includes, from the N-terminus to the C-terminus, the VH of the second antigen-binding domain, the heavy chain CH1 region, the Fc domain monomer, and the first antigen-binding domain.

[0337] In some embodiments, the bispecific antibody comprises:

[0338] (i) peptide chain IV-A, which includes the VL of the second antigen-binding domain and the light chain constant region (CL) from the N-terminus to the C-terminus; and

[0339] (ii) Peptide chain IV-B, which includes, from the N-terminus to the C-terminus, the first antigen-binding domain, the VH of the second antigen-binding domain, the heavy chain CH1 region, and the Fc domain monomer.

[0340] In some embodiments, the bispecific antibody comprises:

[0341] (i) peptide chain IV-A, which includes the VL of the second antigen-binding domain and the light chain constant region (CL) from the N-terminus to the C-terminus; and

[0342] (ii) Peptide chain IV-B, which includes, from the N-terminus to the C-terminus, the VH of the second antigen-binding domain, the heavy chain CH1 region, the Fc domain monomer, and the first antigen-binding domain.

[0343] In some embodiments, the bispecific antibody comprises two identical or different peptide chains IV-A and two identical or different peptide chains IV-B, wherein the two peptide chains IV-B form a dimer through their respective Fc domain monomers.

[0344] In some embodiments, the bispecific antibody comprises two distinct peptide chains IV-A, wherein the VL and / or light chain constant region (CL) of the second antigen-binding domain are different.

[0345] In some embodiments, the bispecific antibody comprises different peptide chains IV-B, wherein the VH, heavy chain CH1 region, Fc domain monomer of the second antigen-binding domain and / or the first antigen-binding domain are different.

[0346] In some embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example having a structure as shown in (GGGGS)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 40, 41 or 44-51); or the peptide linkers each independently contain an amino acid sequence shown in any one of SEQ ID NO: 40, 41 or 44-52.

[0347] In some embodiments, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker containing one or more glycine (G) and / or serine (S), for example, having a structure as shown in (GGGGS)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 40, 41 or 44-51); or each peptide linker independently contains an amino acid sequence shown in any one of SEQ ID NO: 40, 41 or 44-52.

[0348] In some embodiments, the CL is a kappa light chain constant region, for example, the CL has a sequence as shown in SEQ ID NO: 42, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0349] In some embodiments, 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.

[0350] In some embodiments, the Fc domain monomer has a sequence as shown in SEQ ID NO: 60, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0351] In some embodiments, the Fc domain monomers further include, independently, modifications capable of altering effector function. Preferably, the modifications altering effector function include the following mutations with EU numbers: L234A / L235A / G237A; more preferably, the Fc domain monomers have a sequence as shown in SEQ ID NO: 53 or 61, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0352] DVD-IgG

[0353] 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 includes a heavy chain variable region VH' and a light chain variable region VL'.

[0354] In some embodiments, the VH' is connected to the N-terminus of the heavy chain of the full-length antibody via or without a connector; and the VL' is connected to the N-terminus of the light chain of the full-length antibody via or without a connector.

[0355] In some embodiments, the VH' is connected to the N-terminus of one of the heavy chains of the full-length antibody via or without a connector; and the VL' is connected to the N-terminus of one of the light chains of the full-length antibody via or without a connector.

[0356] In some embodiments, the first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0357] (i) peptide chain II-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); and

[0358] (ii) Peptide chain II-B, comprising VH of the second antigen-binding domain, VH of the first antigen-binding domain, heavy chain CH1 region, and Fc domain monomer.

[0359] In some embodiments, the VL of the second antigen-binding domain is connected to the N-terminus of the VL of the first antigen-binding domain, with or without a connector; and / or

[0360] The VH of the second antigen-binding domain is connected to the N-terminus of the VH of the first antigen-binding domain, either through a connector or not.

[0361] In some embodiments, the bispecific antibody comprises:

[0362] (i) peptide chain II-A, which includes, from the N-terminus to the C-terminus, a VL of the second antigen-binding domain, a VL of the first antigen-binding domain, and a light chain constant region (CL); and

[0363] (ii) Peptide chain II-B, which includes, from the N-terminus to the C-terminus, the VH of the second antigen-binding domain, the VH of the first antigen-binding domain, the heavy chain CH1 region, and the Fc domain monomer.

[0364] In some embodiments, the bispecific antibody comprises two identical or different peptide chains II-A and two identical or different peptide chains II-B, wherein the two peptide chains II-B form a dimer through their respective Fc domain monomers.

[0365] In some embodiments, the bispecific antibody comprises two distinct peptide chains II-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.

[0366] In some embodiments, the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises:

[0367] (i) peptide chain III-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); and

[0368] (ii) Peptide chain III-B, comprising the VH of the first antigen-binding domain, the VH of the second antigen-binding domain, the heavy chain CH1 region, and the Fc domain monomer.

[0369] In some embodiments, the VL of the first antigen-binding domain is connected to the N-terminus of the VL of the second antigen-binding domain, with or without a connector; and / or

[0370] The VH of the first antigen-binding domain is connected to the N-terminus of the VH of the second antigen-binding domain, either through a connector or not.

[0371] In some embodiments, the bispecific antibody comprises:

[0372] (i) peptide chain III-A, which includes, from the N-terminus to the C-terminus, a VL of the first antigen-binding domain, a VL of the second antigen-binding domain, and a light chain constant region (CL); and

[0373] (ii) Peptide chain III-B, which includes, from the N-terminus to the C-terminus, the VH of the first antigen-binding domain, the VH of the second antigen-binding domain, the heavy chain CH1 region, and the Fc domain monomer.

[0374] In some embodiments, the bispecific antibody comprises two distinct peptide chains III-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.

[0375] In some embodiments, the bispecific antibody comprises different peptide chains III-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.

[0376] In some embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example having a structure as shown in (GGGGS)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 40, 41 or 44-51); or the peptide linkers each independently contain an amino acid sequence shown in any one of SEQ ID NO: 40, 41 or 44-52.

[0377] In some embodiments, the scFv has a structure as shown in [VH]-[L]-[VL] or [VL]-[L]-[VH], where [L] is a peptide linker containing one or more glycine (G) and / or serine (S), for example, having a structure as shown in (GGGGS)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 40, 41 or 44-51); or each peptide linker independently contains an amino acid sequence shown in any one of SEQ ID NO: 40, 41 or 44-52.

[0378] In some embodiments, the CL is a kappa light chain constant region, for example, the CL has a sequence as shown in SEQ ID NO: 42, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0379] In some embodiments, 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.

[0380] In some embodiments, the Fc domain monomer has a sequence as shown in SEQ ID NO: 60, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0381] In some embodiments, the Fc domain monomers further include, independently, modifications capable of altering effector function. Preferably, the modifications altering effector function include the following mutations with EU numbers: L234A / L235A / G237A; more preferably, the Fc domain monomers have a sequence as shown in SEQ ID NO: 53 or 61, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0382] In some embodiments, the Fc domain monomer contains modifications of one or more amino acids that promote dimerization of the Fc domain monomer.

[0383] 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.

[0384] In some embodiments, the bispecific antibody comprises:

[0385] (1) A peptide chain IA comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or a peptide chain IB comprising the amino acid sequence shown in SEQ ID NO: 2 or 5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0386] (2) Peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0387] (3) A peptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 62 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or a peptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 63 or 64 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or

[0388] (4) A peptide chain III-A comprising the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or a peptide chain III-B comprising the amino acid sequence shown in SEQ ID NO: 66 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0389] In some embodiments, the bispecific antibody comprises:

[0390] (1) A peptide chain IA comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and a peptide chain IB comprising the amino acid sequence shown in SEQ ID NO: 2 or 5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0391] (2) Peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0392] (3) Peptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 62 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and peptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 63 or 64 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or

[0393] (4) Peptide chain III-A comprising the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and peptide chain III-B comprising the amino acid sequence shown in SEQ ID NO: 66 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0394] In certain embodiments of the bispecific antibodies disclosed herein, the heavy chain constant region of the bispecific antibody 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 may contain a glutamic acid or glutamine residue, or the glutamic acid or glutamine residue may be cyclized to pyroglutamic acid.

[0395] In some embodiments, the N-terminal amino acid of the variable region of the bispecific antibody may be cyclized to pyroglutamic acid. Therefore, in the compositions containing bispecific antibodies provided in this disclosure, each bispecific antibody 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 the N-terminal glutamine or glutamate cyclized to a pyroglutamate salt.

[0396] Therefore, in some embodiments, this disclosure further provides compositions comprising the bispecific antibodies described herein, wherein the bispecific antibody in the composition may comprise (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 and / or light 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 and / or light 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 and / or light 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 and / or light chain.

[0397] Preparation of bispecific antibodies

[0398] The bispecific antibodies disclosed herein can be prepared by various methods known in the art, such as recombinant genetic engineering techniques. For example, the bispecific antibodies of this disclosure can be generated by co-expressing multiple polynucleotides encoding the respective polypeptide chains of the bispecific antibody. The polypeptide chains generated by co-expression can be linked via, for example, disulfide bonds or other means to form a functional bispecific antibody. For example, the light chain portion of the Fab fragment can be encoded by separate polynucleotides with a portion of the heavy chain portion of the Fab fragment in the bispecific antibody (which may further contain an Fc domain monomer and optionally other antigen-binding domains). When co-expressed, the polypeptide containing the heavy chain portion of the Fab fragment links with the polypeptide containing the light chain portion of the Fab fragment to form the Fab fragment. As another example, a portion of one of the two Fc domain monomers in the bispecific antibody provided herein (which may further contain an antigen-binding domain) can be encoded by separate polynucleotides with a portion of the other of the two Fc domain monomers (which may further contain an antigen-binding domain). When co-expressed, the two Fc domain monomers link to form an Fc domain.

[0399] On the other hand, this disclosure provides isolated nucleic acid molecules or groups of nucleic acid molecules comprising a nucleotide sequence encoding a bispecific antibody of this disclosure or at least one peptide chain thereof. According to codon degeneracy in the art, in some embodiments, the nucleotide sequence can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence is codon-optimized.

[0400] In some embodiments, the isolated nucleic acid molecule comprises nucleotide sequences encoding peptide chains of the bispecific antibody disclosed herein, and the nucleotide sequences encoding the peptide chains are present on the same or different isolated nucleic acid molecules.

[0401] On the other hand, this disclosure provides vectors (e.g., expression vectors) that contain nucleic acid molecules encoding the isolated samples described above.

[0402] In some embodiments, the vector contains nucleotide sequences encoding each peptide chain of the bispecific antibody disclosed herein, and the nucleotide sequences encoding each peptide chain are present on the same or different vectors.

[0403] For example, the vectors disclosed herein comprise: a first vector comprising a nucleotide sequence encoding peptide chain IA, and a second vector comprising a nucleotide sequence encoding peptide chain IB.

[0404] For example, the vectors disclosed herein include: a third vector containing a nucleotide sequence encoding peptide chain II-A, and a fourth vector containing a nucleotide sequence encoding peptide chain II-B.

[0405] For example, the vectors disclosed herein include: a fifth vector containing a nucleotide sequence encoding peptide III-A, and a sixth vector containing a nucleotide sequence encoding peptide III-B.

[0406] For example, the vectors disclosed herein include: a seventh vector containing a nucleotide sequence encoding peptide IV-A, and an eighth vector containing a nucleotide sequence encoding peptide IV-B.

[0407] On the other hand, this disclosure provides host cells that contain the nucleic acid molecules or vectors described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., *E. coli* cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cells of this disclosure are mammalian cells, such as CHO (e.g., CHO-EBNA, CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0408] On the other hand, this disclosure provides a method for preparing the bispecific antibody described herein, comprising culturing host cells as described above under conditions that allow protein expression, and recovering the bispecific antibody from the cultured host cell culture.

[0409] Derived antibodies

[0410] The bispecific antibodies of this disclosure can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, antibody derivatization (e.g., labeling) does not adversely affect its binding to EGFR and B7H3 (particularly human EGFR and human B7H3). Therefore, the bispecific antibodies of this disclosure are also intended to include such derivatized forms. For example, the bispecific antibodies of this disclosure can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody, a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., avidin or a multihistidine tag) capable of mediating the binding of the bispecific antibody to another molecule.

[0411] In some embodiments, the bispecific antibody is conjugated to at least one label. For example, the label is selected from enzymes, fluorescent dyes, radioisotopes, biotin, and colloidal gold.

[0412] Antibody-drug conjugates

[0413] In one aspect, this disclosure provides an antibody-drug conjugate comprising a bispecific antibody against EGFR and B7H3 provided herein, and at least one therapeutic agent. For example, the therapeutic agent is a cytotoxic agent.

[0414] In some embodiments, the antibody-drug conjugate has the formula Ab'-[MLED]. x The structure shown, wherein:

[0415] Ab' is a bispecific antibody against EGFR and B7H3 disclosed herein;

[0416] M is the linker site that is connected to the bispecific antibody;

[0417] L is a structural segment that connects the joint portions M and E;

[0418] E is a structural segment connecting L and D;

[0419] D is the cytotoxic agent or a fragment thereof; and

[0420] x is any integer selected from 1 to 10.

[0421] I. ADC connector

[0422] The ADC provided herein includes a linker-MLE- for linking a cytotoxic drug fragment D to the antibody. In one aspect, this disclosure provides a linker unit having the following structure:

[0423] -MLE-

[0424] Wherein: M is the linker site where the linker unit connects to the bispecific antibody or its antigen-binding fragment;

[0425] L is the structural segment connecting the joint portions M and E;

[0426] E is a structural segment that connects L and the toxin molecule.

[0427] 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.

[0428] 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.

[0429] 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.

[0430] In some implementations, M is selected from

[0431] In some implementation schemes, M is

[0432] In some implementations, M is selected from

[0433] In some implementations, M is selected from

[0434] 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: 54)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 55)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 56)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 57)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 58))),

[0435] 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.

[0436] 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: 54)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 55)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 56)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 57)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 58)).

[0437] 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, Glt-Glu-Gly, Glt-Glt-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: 54)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 55)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 56)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 57)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 58)), Where s is selected from integers from 1 to 20.

[0438] In some implementations, L is selected from one or more of the following structures:

[0439] In some implementations, L is selected from the following structures:

[0440] In some implementations, L is selected from the following structures:

[0441] In some implementations, L is selected from the following structures:

[0442] In some implementations, L is selected from the following structures:

[0443] In some implementations, L is selected from the following structures:

[0444] In some implementations, E is a single bond or selected from the following structures:

[0445] -NHCH2-, -NHCH2-O-CH2-CO-, -CO-O-CH2-CO-,

[0446] In some implementations, E is a single bond, -NHCH2-, -NHCH2-O-CH2-CO-,

[0447] In some implementations, E is -NHCH2- or

[0448] In some implementations, E is -NHCH2- or a single bond.

[0449] In some implementations, E is -NHCH2- or -NHCH2-O-CH2-CO-.

[0450] In some implementations, E is

[0451] In some implementations, the connection unit (-MLE-) is selected from the following structures:

[0452] In some implementations, the connection unit has the following structure:

[0453] In another aspect, this disclosure provides compounds comprising the aforementioned connecting units.

[0454] In some embodiments, the compound is selected from:

[0455] 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.

[0456] ADC drug-linker

[0457] 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-.

[0458] 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.

[0459] 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.

[0460] 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.

[0461] 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.

[0462] In some implementations, M is selected from

[0463] In some implementations, M is

[0464] In some implementations, M is selected from

[0465] In some implementations, M is selected from

[0466] 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-Lts, 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: 54)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 55)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 56)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 57)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 58))),

[0467] 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.

[0468] 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: 54)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 55)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 56)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 57)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 58)).

[0469] 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: 54)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 55)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 56)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 57)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 58)), Where s is selected from integers from 1 to 20.

[0470] In some implementations, L is selected from one or more of the following structures:

[0471] In some implementations, L is selected from the following structures:

[0472] In some implementations, L is selected from the following structures:

[0473] In some implementations, L is selected from the following structures:

[0474] In some implementations, L is selected from the following structures:

[0475] In some implementations, L is selected from the following structures:

[0476] In some implementations, E is a single bond or selected from the following structures:

[0477] -NHCH2-, -NHCH2-O-CH2-CO-, -CO-O-CH2-CO-,

[0478] In some implementations, E is a single bond, -NHCH2-, -NHCH2-O-CH2-CO-,

[0479] In some implementations, E is -NHCH2- or

[0480] In some implementations, E is -NHCH2- or a single bond.

[0481] In some implementations, E is -NHCH2- or -NHCH2-O-CH2-CO-.

[0482] In some implementations, E is

[0483] In some implementation schemes, Selected from the following structures:

[0484] In some implementation schemes, Selected from the following structures:

[0485] In some embodiments, the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

[0486] In some embodiments, the microtubule inhibitor is an olistatin or maytansine compound.

[0487] In some embodiments, the olistatin compound is selected from the following:

[0488] In some embodiments, the DNA intercalating agent is pyrrolobenzodiazepine. (PBD)

[0489] In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0490] 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.

[0491] In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analogue thereof.

[0492] 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.

[0493] Among them, R1 and R2 are each independently selected from C. 1-6 Alkyl and halogen;

[0494] R3 is selected from H, -CO-CH2OH, and

[0495] R4 and R5 are each independently selected from H, halogen, hydroxyl, methyl, and amino; or R4 and R5 are connected to form a 5-6 membered oxygen-containing heterocycle;

[0496] R6 is selected from hydrogen, C 2-6 alkenyl or -C 1-4 Alkylene-NR a R b ;

[0497] 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, -CO-C 1-6 Alkyl and -C 1-4 Alkylene-NR d R e Or, R a and R b The atoms connected to it may optionally be substituted with R f 5-6 membered nitrogen-containing heterocycles; among which, R d R e and R f Each time it appears, it is independently selected from H and C. 1-6 alkyl;

[0498] Alternatively, R6 and R7, together with the carbon atoms they are attached to, form a 5-6 membered ring.

[0499] In some implementation schemes, R d and R e H is used each time it appears. In some implementations, R... f It is methyl in every occurrence.

[0500] In some embodiments, the cytotoxic agent is selected from compounds of Formula I, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of Formula I.

[0501] Among them, R1 is selected from C 1-6 Alkyl groups (e.g., methyl) and halogens such as F or Cl,

[0502] R2 is a halogen, such as F or Cl.

[0503] R3 is selected from H, -CO-CH2OH, and

[0504] In some embodiments, the cytotoxic agent is selected from compounds of Formula II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of Formula II.

[0505] in,

[0506] R4 and R5 are each independently selected from H, hydroxyl, methyl, and amino (-NH2); or R4 and R5 are connected to form a 5-6 membered oxygen-containing heterocycle (e.g., dioxanepentyl ring or dioxanehexyl ring);

[0507] R6 is selected from hydrogen, C 2-6 alkenyl or -C 1-4 Alkylene-NR a R b For example, R6 can be selected from hydrogen, allyl (i.e., 2-propenyl) or -CH2N(CH3)2;

[0508] 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, -CO-C 1-6 Alkyl and -C 1-4 Alkylene-NR d R e Or, R a and R b The atoms connected to it may optionally be substituted with R f5-6 membered nitrogen-containing heterocycles; among which, R d R e and R f Each time it appears, it is independently selected from H and C. 1-6 Alkyl groups; for example, R7 can be selected from methyl, ethyl, -CH2CH2NH (isopropyl), -CH2CH2NH (isopropyl), -CH2CH2N (isopropyl)(SO2CH3), -CH2CH2N (isopropyl)(COCH3), -CH2CH2Si(CH3)3, -CH2CH2Si(CH3)2 (tert-butyl), -C=NO-tert-butyl, -C=NO-CH2CH2NH2,

[0509] Alternatively, R6 and R7, together with the carbon atoms they are attached to, form a 5-6 membered ring.

[0510] In some embodiments, the cytotoxic agent is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds:

[0511] The fragment of the cytotoxic drug obtained after the cytotoxic drug is linked to the linker is D in the above general formula.

[0512] In some embodiments, the cytotoxic agent is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds:

[0513] The fragment of the cytotoxic drug obtained after the cytotoxic drug is linked to the linker is D in the above general formula.

[0514] D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amine group on the cytotoxic drug.

[0515] Connection between connector and drug (load)

[0516] 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.

[0517] 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.

[0518] ADC Examples

[0519] 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, ADC D-01 to ADC D-03, and ADC E-01 to ADC E-02, wherein the structure of the ADC is as follows:

[0520] Wherein, Ab-(S-) is the bispecific antibody provided in this disclosure, namely Ab';

[0521] In some implementations, This indicates the specific linkage between the thiol group of the cysteine ​​residue in the bispecific antibody or its antigen-binding fragment and the pyrimidin or succinimide group in the antibody-drug conjugate.

[0522] In some embodiments, 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 the pyrimidin or succinimide group in the antibody-drug conjugate through an addition reaction or substitution reaction.

[0523] In a specific implementation plan, Ab' can be selected from bispecific antibodies BsAb01, BsAb02, BsAb03, BsAb04, BsAb05, or BsAb06.

[0524] In some implementations, BsAb01, BsAb02, BsAb03, BsAb04, BsAb05, or BsAb06 may be obtained according to Embodiment 2 of this document.

[0525] 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.

[0526] In some embodiments, the N-terminal amino acid of the bispecific antibody in 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.

[0527] Therefore, in some embodiments, this disclosure further provides compositions comprising the antibody-drug conjugates described herein, wherein the primary 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 and / or light 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 and / or light 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 and / or light 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 and / or light chain.

[0528] In some embodiments, the DAR value (drug-antibody conjugate ratio) of the composition is 1 to 10, for example: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10.

[0529] In some embodiments, the DAR value of the composition is 3 to 9, or 4 to 8, for example, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 to 5.5, 3.5 to 6.0, 3.5 to 6.5, 3.5 to 7.0, 3.5 to 7.5, 3.5 to 8.0, 4.0 to 4.5, 4.0 to 5.0, 4.0 to 5.5, 4.0 to 6.0, 4.0 to 6.5, 4.0 to 7.0, 4.0 to 7.5, 4.0 to 8.0, 4.5 to 5.0, 4.5 to 5.5, 4.5 to 6.0, 4.5 to 6.5, 4.5 to 7.0, 4.5 to 7.5, 4.5 to 8.0, 5.0 to 5.5, 5.0 to 6.0, 5.0 to 6.5, 5.0 to 7.0, 5.0 to 7.5, 5.0 to 8.0, 5.5 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 6.0 to 7.5, 6.0 to 8.5, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.5, 7.0 to 7.5, 7.0 to 9.0 or 7.5 to 9.0.

[0530] In some embodiments, the DAR of the ADC compositions described herein is about 6.0 to 9.0, preferably about 6.0-8.0, for example about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19, about 6.2, about 6.21, about 6.22, about 6.23, about 6.24, about 6.25, about 6.26, about 6.27, about 6.28, about 6.29, about 6.3, about 6.31, about 6.32, about 6.33, about 6. .34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.4, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.5, about 6.51, about 6.52, about 6.53, about 6.54, about 6. 55, approximately 6.56, approximately 6.57, approximately 6.58, approximately 6.59, approximately 6.6, approximately 6.61, approximately 6.62, approximately 6.63, approximately 6.64, approximately 6.65, approximately 6.66, approximately 6.67, approximately 6.68, approximately 6.69, approximately 6.7, approximately 6.71, approximately 6.72, approximately 6.73, approximately 6.74, approximately 6.75, approximately 6.7 6, approximately 6.77, approximately 6.78, approximately 6.79, approximately 6.8, approximately 6.81, approximately 6.82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.9, approximately 6.91, approximately 6.92, approximately 6.93, approximately 6.94, approximately 6.95, approximately 6.96, approximately 6.97 Approximately 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, 7.1, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18 Approximately 7.19, 7.2, 7.21, 7.22, 7.23, 7.24, 7.25, 7.26, 7.27, 7.28, 7.29, 7.3, 7.31, 7.32, 7.33, 7.34, 7.35, 7.36, 7.37, 7.38, 7.39, approximately 7.4, approximately 7.41, approximately 7.42, approximately 7.43, approximately 7.44, approximately 7.45, approximately 7.46, approximately 7.47, approximately 7.48, approximately 7.49, approximately 7.5, approximately 7.51, approximately 7.52, approximately 7.53, approximately 7.54, approximately 7.55, approximately 7.56, approximately 7.57, approximately 7.58, approximately 7.59, approximately 7.6, approximately 7.61, approximately 7.62, approximately 7.63, approximately 7.64, approximately 7.65, approximately 7.66, approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.7, approximately 7.71, approximately 7.72, approximately 7.73, approximately 7.74, approximately 7.75, approximately 7.76, approximately 7.77, approximately 7.78, approximately 7.79, approximately 7.8, approximately 7.81, approximately 7.82, approximately 7.83, approximately 7.84, approximately 7.85, approximately 7.86, approximately 7.87, approximately 7.88, approximately 7.89, approximately 7.9, approximately 7.91, approximately 7.92, approximately 7.93, approximately 7.94, approximately 7.95, approximately 7.96, approximately 7.97, approximately 7.98, approximately 7.99, approximately 8.0, approximately 8.01, approximately 8.02, approximately 8.03, approximately 8.04, approximately 8.05, approximately 8.06, approximately 8.07, approximately 8.08, approximately 8.09, approximately 8.1, approximately 8.11, approximately 8.12, approximately 8.13, approximately 8.14, approximately 8.15, approximately 8.16, approximately 8.17, approximately 8.18, approximately 8.19, approximately 8.2, approximately 8.21, approximately 8.22, approximately 8.23, approximately 8.24, approximately 8.25, approximately 8.26, approximately 8.27, approximately 8.28, approximately 8.29, approximately 8.3, approximately 8.31, approximately 8.32, approximately 8.33, approximately 8.34, approximately 8.35, approximately 8.36, approximately 8.37, approximately 8.38, approximately 8.39, approximately 8.4, approximately 8.41, approximately 8.42, approximately 8.43, approximately 8.44, approximately 8.45, approximately 8.46, approximately 8.47, approximately 8.48, approximately 8.49, approximately 8.5, approximately 8.51, approximately 8.52, approximately 8.53, approximately 8.54, approximately 8.55, approximately 8.56, approximately 8.57, approximately 8.58, approximately 8.59, approximately 8.6, approximately 8.61, approximately 8.62, approximately 8.63, approximately 8.64, approximately 8.65, Approximately 8.66, 8.67, 8.68, 8.69, 8.7, 8.71, 8.72, 8.73, 8.74, 8.75, 8.76, 8.77, 8.78, 8.79, 8.8, 8.81, 8.82, 8.83, 8.84, 8.85, 8.86, 8.87, 8.88, 8.89, 8.9, 8.91, 8.92, 8.93, 8.94, 8.95, 8.96, 8.97, 8.98, 8.99, 9.0.

[0531] In some embodiments, the DAR value of the composition is approximately 8.

[0532] Couplet

[0533] In another aspect, this disclosure provides a method for preparing the antibody-drug conjugate (ADC) described herein by combining the adapter-load described herein with the antibody described herein.

[0534] In some implementations, the antibody described herein binds to the adapter-load described herein by binding to lysine residues in the antibody.

[0535] 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.

[0536] 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.

[0537] 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 a further embodiment, the interchain disulfide bond in the antibody is located between the heavy chain and the light chain.

[0538] 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).

[0539] 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.

[0540] 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.

[0541] In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0542] Pharmaceutical compositions or combinations

[0543] In another aspect, this disclosure provides a pharmaceutical composition comprising one or more antibody-drug conjugates or compositions as described in any of the preceding claims, and a pharmaceutically acceptable pharmaceutical carrier and / or excipient.

[0544] 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.

[0545] In some embodiments, the ADC or composition in the pharmaceutical compositions of this disclosure is sufficient (e.g., in a subject) to exert a tumor-suppressive effect. For example, the tumor-suppressive effect of the ADC or composition in the pharmaceutical compositions of this disclosure is superior to the tumor-suppressive effect of a monospecific anti-EGFR antibody and / or a monospecific anti-B7H3 antibody, wherein the amino acid sequence of the CDR of the monospecific anti-EGFR 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-EGFR antibody is identical to the amino acid sequence of the CDR of the second antigen-binding domain.

[0546] In some embodiments, the antibody-drug conjugates described herein are typically formulated in unit injection form with a pharmaceutically acceptable parenteral carrier for parenteral use, such as intravenous bolus, intravenous injection, intratumoral injection, etc. 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.

[0547] Therapeutic applications of ADC

[0548] 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 the prevention and / or treatment and / or as adjunctive therapy for diseases associated with EGFR and / or B7H3 in subjects, and / or for the inhibition of EGFR and / or B7H3 activity in vitro or in subjects; optionally, the disease associated with EGFR and / or B7H3 is a tumor; optionally, the tumor is B7H3 and / or EGFR positive.

[0549] 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, 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, pancreatic cancer, colorectal cancer, or melanoma; more preferably, the tumor is non-small cell lung cancer.

[0550] 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.

[0551] In another aspect, this disclosure provides a method for inhibiting the activity of EGFR and / or B7H3 in cells, comprising contacting the cells with the antibody-drug conjugate or pharmaceutical composition described herein. The cells are cells expressing EGFR and / or B7H3, such as tumor cells.

[0552] In another aspect, this disclosure provides a method for preventing and / or treating and / or acting as an adjunct therapy for diseases associated with EGFR 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.

[0553] In some embodiments, the EGFR and / or B7H3-related disease is a tumor. In some embodiments, the tumor is B7H3 and / or EGFR positive.

[0554] 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, 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, pancreatic cancer, colorectal cancer, or melanoma; more preferably, the tumor is non-small cell lung cancer.

[0555] On the other hand, this disclosure provides the use of the antibody-drug conjugates, compositions, pharmaceutical compositions, or combinations thereof described herein in the prevention and / or treatment and / or as adjuvant therapy for diseases associated with EGFR and / or B7H3, and / or, in vitro or in subjects, the use of inhibiting the activity of EGFR and / or B7H3; optionally, the disease associated with EGFR and / or B7H3 is a tumor; optionally, the tumor is B7H3 and / or EGFR positive.

[0556] 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, 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, pancreatic cancer, colorectal cancer, or melanoma; more preferably, the tumor is non-small cell lung cancer.

[0557] 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.

[0558] In some implementations, the second therapy may be administered simultaneously, alone, or sequentially with the methods described above.

[0559] 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 EGFR and / or B7H3.

[0560] In some embodiments, the EGFR and / or B7H3-related disease is a tumor. In some embodiments, the tumor is B7H3 and / or EGFR positive.

[0561] 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, 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, pancreatic cancer, colorectal cancer, or melanoma; more preferably, the tumor is non-small cell lung cancer.

[0562] In some embodiments, the prevention, treatment, and / or adjunctive therapy of EGFR and / or B7H3-related diseases 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.

[0563] 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.

[0564] Furthermore, the ADC disclosed herein can be present in the pharmaceutical composition in unit dose form for convenient administration.

[0565] 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, groin, bladder, 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.

[0566] 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.

[0567] In this disclosure, the subjects may be mammals, such as humans.

[0568] Reagent test kit

[0569] 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.

[0570] 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 EGFR and / or B7H3 antigens as a control. In another particular embodiment, the kit described herein also includes a control antibody that does not react with the EGFR or B7H3 antigen. In yet another particular embodiment, the kit described herein includes one or more elements for detecting the binding of an antibody to the EGFR 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 a first antibody may be conjugated to a detectable substrate). In a particular embodiment, the kits provided herein may include recombinantly generated or chemically synthesized EGFR 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 EGFR 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.

[0571] 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.

[0572] Beneficial effects of the invention

[0573] This disclosure provides bispecific antibodies and antibody-drug conjugates targeting EGFR and B7H3, which possess excellent cell affinity, endocytic activity, in vitro tumor cell killing ability, and in vivo tumor suppression effect. Detailed Implementation

[0574] The present disclosure is further illustrated below through a description of specific embodiments, but this is not intended to limit the scope of the disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of the present disclosure without departing from its basic ideas and scope.

[0575] Terminology Definition

[0576] 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.

[0577] 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 K (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.

[0578] 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.

[0579] 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.

[0580] 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 31, pp. 422-439, Springer-Verlag, Berlin. The definitions in the CDR system (2001), AHo numbering system (Honegger and Plückthun, A., J. Mol. Biol. 309: 657-670 (2001)), or IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003) are readily apparent to those skilled in the art. 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).

[0581] 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.

[0582] 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, VH 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.

[0583] 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.

[0584] 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.

[0585] 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.

[0586] 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.

[0587] 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.

[0588] 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.

[0589] 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: 44) or a variant thereof, for example, the amino acid sequence (GGGGS)4 (SEQ ID NO: 41), 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.

[0590] 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).

[0591] 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.

[0592] 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 one half of the bispecific antibody, enabling the light chain to bind correctly 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: CrossMabFab, referring to the crossover or exchange of all VH-CH1 and VL-CL domain positions in one half of the bispecific antibody; CrossMabVH-VL, referring to the crossover or exchange of only the VH and VL domain positions in one half of the bispecific antibody; and CrossMabCH1-CL, referring to the crossover or exchange of the CH1 and CL domain positions within the Fab region of one half of the 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, 10811187-1191 (2011); Kleinet et al., MAbs 8(6): 1010-1020 (2016); and Klein et al., MAbs 4(6): 653-663 (2012).

[0593] 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.

[0594] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) or half-maximal effective concentration (EC50) of the interaction. 50 )express.

[0595] 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.

[0596] 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.

[0597] 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).

[0598] 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.

[0599] 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.

[0600] The twenty common amino acids involved in this disclosure are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (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.

[0601] 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).

[0602] 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 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10. In additional embodiments, for compositions of the present disclosure containing an ADC, the DAR of the composition is 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 to 5.5, 3.5 to 6.0, 3.5 to 6.5, 3.5 to 7.0, 3.5 to 7.5, 3.5 to 8.0, 4.0 to 4.5, 4.0 to 5.0, 4.0 to 5.5, 4.0 to 6.0, 4.0 to 6.5, 4.0 to 7.0, 4.0 to 7.5, 4.0 to 8.0, or 4.5 to 5. Integers or decimals from 0, 4.5 to 5.5, 4.5 to 6.0, 4.5 to 6.5, 4.5 to 7.0, 4.5 to 7.5, 4.5 to 8.0, 5.0 to 5.5, 5.0 to 6.0, 5.0 to 6.5, 5.0 to 7.0, 5.0 to 7.5, 5.0 to 8.0, 5.5 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 5.5 to 7.5, 5.5 to 8.0, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.5, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.5, 7.0 to 7.5, 7.0 to 9.0, or 7.5 to 9.0. 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.

[0603] 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).

[0604] 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.

[0605] 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.

[0606] 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.

[0607] 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.

[0608] 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).

[0609] 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.

[0610] 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.

[0611] 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 sites 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.

[0612] As used herein, terms such as "first" and "second" are used only to distinguish technical features and do not represent order, quantity, priority, or other limitations. The actual number of relevant features may be adjusted according to the technical solution and is not limited by the above description. For example, in some embodiments of the bispecific antibody disclosed herein, the bispecific antibody may include two first antigen-binding domains and two second antigen-binding domains.

[0613] 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 1-3 Alkyl 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.

[0614] "Alkenyl" is intended to include hydrocarbon chains with a straight or branched configuration, having a specified number of carbon atoms and one or more, preferably one to two, carbon-carbon double bonds, which can be present at any stable point along the chain. For example, "C 2-6 "Alkenyl" is intended to include C2, C3, C4, C5, and C6 alkenyl groups. Examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, and 4-methyl-3-pentenyl.

[0615] 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.

[0616] 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" 5-8Examples 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.

[0617] 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.

[0618] 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.

[0619] 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.

[0620] 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.

[0621] As used herein, the terms “about” or “approximately” when used in conjunction with numerical variables generally mean that the value of the variable is within the experimental error range (e.g., within the 95% confidence interval of the mean) or within ±10%.

[0622] abbreviations

[0623] Sequence information

[0624] Descriptions of the sequences involved in this disclosure are provided in the table below.

[0625] Example

[0626] The present disclosure is further illustrated below through a description of specific embodiments, but this is not intended to limit the scope of the disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of the present disclosure without departing from its basic ideas and scope.

[0627] Example 1: Preparation of payload linker

[0628] The structures of the compounds described in the following examples were determined by NMR ( 1 It can be determined by ¹H NMR or mass spectrometry (MS).

[0629] Nuclear magnetic resonance (NMR) 1 The H NMR measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0630] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0631] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0632] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0633] Example 1.1 Preparation of 1N-((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]indolazine[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-14)

[0634] Step 1:

[0635] 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), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain 700 mg of compound IM-5.

[0636] The preparation method for high performance liquid chromatography purification is as follows:

[0637] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0638] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0639] Step Two:

[0640] 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.

[0641] The preparation method for high performance liquid chromatography purification is as follows:

[0642] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0643] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0644] Step 3:

[0645] 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 mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 50.56 mg of compound A-14.

[0646] Its structural characterization data are as follows:

[0647] MS m / z (ESI): 1002.4 [M+H]+

[0648] The preparation method for high performance liquid chromatography purification is as follows:

[0649] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0650] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0651] 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).

[0652] Example 1.2: 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-hydroxyacetamyl)-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)

[0653] Step 1: Preparation of ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3′,4′:6,7] indo[1,2-b]quinoline-1-yl)amino)-2-oxoethyl acetate (B-03-1)

[0654] (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).

[0655] Its structural characterization data are as follows:

[0656] ESI-MS (m / z): 552.2 [M+1] + .

[0657] 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)

[0658] 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).

[0659] Its structural characterization data are as follows:

[0660] ESI-MS (m / z): 1352.8 [M+1] + .

[0661] 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)

[0662] 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 ethyl acetate (200 mg, 0.122 mmol) of 0,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indazosin[1,2-b]quinoline-1-yl)amino)-2-oxoethyl acetate was dissolved in THF (3 mL) and MeOH (3 mL). While stirring, 1 mL of an aqueous solution of sodium carbonate (25.88 mg, 0.224 mmol) was added dropwise. After the addition was complete, stirring was continued for 1 hour. The reaction mixture was neutralized by adding dilute hydrochloric acid dropwise, and the solution was concentrated under reduced pressure before proceeding to the next step.

[0663] Its structural characterization data are as follows:

[0664] ESI-MS (m / z): 1596.7 [M+1] + .

[0665] 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-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 (B-03-4)

[0666] 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 the addition of 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).

[0667] Its structural characterization data are as follows:

[0668] ESI-MS (m / z): 1323.6 [M+1] + .

[0669] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylamido)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)

[0670] 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 stirring was continued 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).

[0671] The structural characterization data are as follows:

[0672] ESI-MS (m / z): 1628.7 [M+1] + .

[0673] The preparative high performance liquid chromatography method is as follows:

[0674] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0675] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0676] Example 1.3: 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)

[0677] 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.

[0678] The separation and purification method for A-05 is as follows:

[0679] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0680] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0681] The structural characterization data for A-05 are as follows:

[0682] MS m / z (ESI): 1107.3 [M+H]+

[0683] 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).

[0684] Example 1.4: 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)

[0685] 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.

[0686] The separation and purification method for A-07 is as follows:

[0687] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0688] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0689] The structural characterization data for A-07 are as follows:

[0690] ESI-MS (m / z): 1111.3 [M+H] + .

[0691] Example 1.5: N-((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26-heptaoxin-2,14-dioxin-5,8,11,16,19,22,25-heptaazaheptan-27-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl (D-03)

[0692] Step 1: Synthesis of (9H-fluorene-9-yl)methyl((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptylheptanamide-27-yl)carbamate

[0693] Weigh out compound IM-6 (50.0 mg, 0.077 mmol) and (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropane)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-N,3-dimethyl-2-((S)-3-methyl 2-(methylamino)butyrylamino)butyramide (MMAE, 55.60 mg, 0.077 mmol) was dissolved in DMF (1 mL), and then HATU (32.37 mg, 85.18 μmol) and DIPEA (20.02 mg, 154.88 μmol) were added. After the addition was complete, the reaction was carried out at room temperature for 1 h. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 35.0 mg of the title compound.

[0694] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0695] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid)

[0696] The structural characterization data are as follows:

[0697] ESI-MS (m / z): 1345.1 [M+H] + .

[0698] Step 2: Synthesis of (S)-2-((2S,13S)-19-amino-13-benzyl-2-isopropyl-3-methyl-4,9,12,15,18-pentazo-6-oxa-3,8,11,14,17-heptaneazine)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-N,3-dimethylbutyramide

[0699] Compound D-03-1 (20.00 mg, 0.015 mmol) was dissolved in dichloromethane (2 mL), and then diethylamine (1 mL) was added. After the addition was complete, the reaction was carried out at room temperature for 1 h. The reaction was detected by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain 20.0 mg of crude product.

[0700] The structural characterization data are as follows:

[0701] ESI-MS (m / z): 1123.1 [M+H] + .

[0702] Step 3: Synthesis of N-((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptaazaheptan-27-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl

[0703] Compound D-03-2 (20.00 mg, 0.015 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid (17.00 mg, 0.015 mmol) were dissolved in DMF (1 mL), and then HATU (6.33 mg, 16.65 μmol) and DIPEA (3.91 mg, 30.27 μmol) were added. After the addition was complete, the reaction was carried out at room temperature for 1 h. The reaction was detected by liquid chromatography-mass spectrometry (LC-MS), and the reaction solution was purified by preparative high performance liquid chromatography (under the following conditions) to give 3.52 mg of the title compound.

[0704] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0705] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0706] The structural characterization data are as follows:

[0707] ESI-MS (m / z): 1374.1 [M+H] + .

[0708] Example 1.6: 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)

[0709] Step 1:

[0710] 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.

[0711] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0712] Mobile phase A: Acetonitrile; Mobile phase B: Water

[0713] Step Two:

[0714] 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.

[0715] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0716] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0717] The structural characterization data are as follows: ESI-MS (m / z): 1631.7 [M+H] + 816.0 [M / 2+H] + .

[0718] Example 1.7: 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)

[0719] Step 1:

[0720] 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.

[0721] Step Two:

[0722] 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-diazapentadecanoamide (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.

[0723] Step 3:

[0724] 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.

[0725] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0726] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0727] Step Four:

[0728] 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.

[0729] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0730] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0731] Step 5:

[0732] B-02-4 (50 mg, 26.52 μmol) was dissolved in dichloromethane (1 mL) at 25 °C, 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.

[0733] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0734] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0735] The structural characterization data of B-02 are as follows:

[0736] ESI-MS (m / z): 1613.6 [M+H] + .

[0737] Example 1.8: 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)

[0738] Step 1:

[0739] The starting material C-07-1 (4.80 g, 16.33 mmol), tributyltin (2-methylsulfamylpyrimidin-4-yl) (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.

[0740] Step Two:

[0741] 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.

[0742] Step 3:

[0743] 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 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 40 mg of compound C-07-3.

[0744] The preparation method for high performance liquid chromatography is as follows:

[0745] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0746] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0747] Step Four:

[0748] 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.

[0749] Step 5:

[0750] 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.

[0751] Step Six:

[0752] 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.

[0753] Step Seven:

[0754] 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.

[0755] The preparation method for high performance liquid chromatography is as follows:

[0756] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0757] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0758] Step 8:

[0759] 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.

[0760] The structural characterization data are as follows:

[0761] ESI-MS (m / z): 1342.4 [M+H] + .

[0762] The preparation method for high performance liquid chromatography is as follows:

[0763] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0764] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0765] Example 1.9: 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]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-5-yl)benzamide (C-10)

[0766] Step 1:

[0767] 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.

[0768] Step Two:

[0769] 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.

[0770] Step 3:

[0771] 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.

[0772] The preparation method for high performance liquid chromatography is as follows:

[0773] Chromatographic column: Phenomenex Luna C18 200*40mm*10um.

[0774] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% hydrochloric acid)

[0775] Mobile phase: [water(HCl)-ACN]; B%: 13%-43%, 10 min).

[0776] Step Four:

[0777] 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 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 51 mg of compound C-10-4.

[0778] The preparation method for high performance liquid chromatography is as follows:

[0779] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0780] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0781] Step 5:

[0782] 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.

[0783] Step Six:

[0784] 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 DMF (3 mL) 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.

[0785] The structural characterization data are as follows:

[0786] ESI-MS (m / z): 1342.5 [M+H]+.

[0787] The preparation method for high performance liquid chromatography is as follows:

[0788] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0789] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0790] Example 1.10: 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]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-5-yl)benzamide (C-17)

[0791] Step 1:

[0792] 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.

[0793] Step Two:

[0794] 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).

[0795] The preparation method for high performance liquid chromatography is as follows:

[0796] Column: Phenomenex luna C18 (250mm*70mm*10μm)

[0797] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0798] Step 3:

[0799] 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).

[0800] Step Four:

[0801] 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 sequentially added 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).

[0802] The structural characterization data are as follows:

[0803] ESI-MS (m / z): 1371.4 [M+H] + .

[0804] The preparation method for high performance liquid chromatography is as follows:

[0805] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0806] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0807] Example 1.11: 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)

[0808] Step 1:

[0809] 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, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was then slurried with petroleum ether and filtered again. The filter cake was dried under vacuum to obtain C-19-2 (5.65 g).

[0810] Step Two:

[0811] 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).

[0812] Step 3:

[0813] C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 12-amino-4,7,10-trioxadodecanoate (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.

[0814] Step Four:

[0815] 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).

[0816] The preparation method for high performance liquid chromatography is as follows:

[0817] Column: Phenomenex luna C18 (150mm*25mm*10μm)

[0818] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0819] Step 5:

[0820] 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).

[0821] Step Six:

[0822] 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 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).

[0823] The structural characterization data are as follows:

[0824] ESI-MS (m / z): 1372.4 [M+H] + .

[0825] The preparation method for high performance liquid chromatography is as follows:

[0826] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

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

[0828] Example 1.12: 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)

[0829] Step 1:

[0830] 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.

[0831] Step Two:

[0832] 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).

[0833] The preparation method for high performance liquid chromatography is as follows:

[0834] Column: Phenomenex luna C18 (250mm*70mm*10μm)

[0835] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0836] Step 3:

[0837] 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).

[0838] Step Four:

[0839] 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).

[0840] The structural characterization data are as follows:

[0841] ESI-MS (m / z): 1591.7 [M+H] + .

[0842] The preparation method for high performance liquid chromatography is as follows:

[0843] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0844] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0845] Example 1.13: 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)

[0846] Step 1:

[0847] 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.

[0848] Step Two:

[0849] 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).

[0850] The preparation method for high performance liquid chromatography is as follows:

[0851] Column: Welch Ultimate C18 (150mm*25mm*5μm)

[0852] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0853] Step 3:

[0854] 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).

[0855] Step Four:

[0856] 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).

[0857] The structural characterization data are as follows:

[0858] ESI-MS (m / z): 1592.6 [M+H] + .

[0859] The preparation method for high performance liquid chromatography is as follows:

[0860] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0861] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0862] Example 1.14: (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)

[0863] 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)

[0864] 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 reaction was carried out 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.

[0865] The structural characterization data are as follows:

[0866] ESI-MS (m / z): 503 [M+18] + .

[0867] 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)

[0868] 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.

[0869] The structural characterization data are as follows:

[0870] ESI-MS (m / z): 456.1 [M+1] + .

[0871] 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)

[0872] 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.

[0873] The structural characterization data are as follows:

[0874] ESI-MS (m / z): 823.3 [M+1] + .

[0875] 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)

[0876] 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.

[0877] The structural characterization data are as follows:

[0878] ESI-MS (m / z): 988.5 [M+1] + .

[0879] 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)

[0880] 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.

[0881] The structural characterization data are as follows:

[0882] ESI-MS (m / z): 1326.2 [M+1] + .

[0883] Step Six: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-(2-aminoethoxy)ethoxy)acetamyl)-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)

[0884] 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.

[0885] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0886] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0887] The structural characterization data are as follows:

[0888] ESI-MS (m / z): 964.2 [M+1] + .

[0889] 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)

[0890] 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.

[0891] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0892] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0893] The structural characterization data are as follows:

[0894] ESI-MS (m / z): 1214.4 [M+1] + .

[0895] Example 1.15: 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-yne-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-yyn-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctacosane-38-amide (A-36-A)

[0896] 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-dodecoxaoctacosane-38-acyl)-D-lysine(2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctacosane-38-amido)-6-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)hexanoic acid (A-36-03)

[0897] 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).

[0898] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0899] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0900] ESI-MS (m / z): 939.3 [M+H] + .

[0901] 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-tridecyloxo-39,42,45,48,51,54-hexaazaoctacosane-58-acid (A-36-04)

[0902] 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).

[0903] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0904] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0905] ESI-MS (m / z): 1344.4 [M+H] + .

[0906] 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)

[0907] 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).

[0908] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0909] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0910] ESI-MS (m / z): 1781.6 [M+H] + .

[0911] 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]indolazino[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)

[0912] 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).

[0913] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0914] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0915] ESI-MS (m / z): 1559.7 [M+H]+.

[0916] 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

[0917] 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).

[0918] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0919] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0920] ESI-MS (m / z): 1809.8 [M+H] + .

[0921] Example 1.16: 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)

[0922] 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

[0923] 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 dichloromethane (20 mL), and the mixture was heated to 45°C for 2 h. The reaction solution was evaporated to dryness, and methyl tert-butyl ether (50 mL) was added and stirred for 30 min. A turbid precipitate was formed, and after filtration, 1.3 g of the title compound was obtained.

[0924] The structural characterization data are as follows:

[0925] ESI-MS (m / z): 633.2 [M+H] + .

[0926] Step 2: Synthesis of (S)N-(4-(hydroxymethyl)phenyl)-2-((S)-3-methyl-2-(4-(piperidin-4-yl)butamido)butamido)-5-ureidopentamido

[0927] Compound A-37-2 (1.5 g, 2.37 mmol) was dissolved in dichloromethane (3.00 mL) at 25 °C, and trifluoroacetic acid (1.5 mL) was added in one batch. The reaction was carried out at 25 °C for 2 h. The reaction was monitored by liquid chromatography-mass spectrometry to ensure the reaction was complete. The solvent in the reaction solution was evaporated under reduced pressure. The crude product was dissolved in methanol (3.00 mL), and potassium carbonate (1.64 g, 11.85 mmol) was added. The mixture was stirred for 30 min. The reaction solution was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain 500 mg of the title compound.

[0928] The structural characterization data are as follows:

[0929] ESI-MS (m / z): 533.3 [M+H] + .

[0930] Step 3: Synthesis of (S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butamido)-3-methylbutamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide

[0931] At 25 °C, 26-azido-3,6,9,12,15,18,21,24-octaoxahexadecyl 4-methylbenzenesulfonate (668.72 mg, 1.13 mmol) and compound A-37-3 (1 g, 1.88 mmol) were dissolved in N,N-dimethylformamide (5 mL), and potassium carbonate (518.89 mg, 3.75 mmol) was added in one batch. The mixture was heated to 80 °C and reacted for 2 h. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to give 600 mg of the title compound.

[0932] The structural characterization data are as follows:

[0933] ESI-MS (m / z): 954.5 [M+H] + .

[0934] Step 4: Synthesis of 4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butamido)-3-methylbutamido)-5-ureidopentamido)benzyl(4-nitrophenyl)carbamate

[0935] At 25°C, compound A-37-4 (733 mg, 0.77 mmol) was dissolved in N,N-dimethylformamide (2 mL), DIPEA (396.40 mg, 3.07 mmol) was added, and a solution of di(p-nitrobenzene) carbonate (701.10 mg, 2.30 mmol) in N,N-dimethylformamide (1 mL) was added dropwise. The mixture was stirred at 25°C for 16 h. The reaction was monitored by liquid chromatography-mass spectrometry (LC-MS). The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was freeze-dried to obtain 400 mg of the title compound.

[0936] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0937] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0938] The structural characterization data are as follows:

[0939] ESI-MS (m / z): 1119.5 [M+H] + .

[0940] Step 5: Synthesis of 4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butamido)-3-methylbutamido)-5-ureidopentamido)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

[0941] At 25°C, belotecone (20 mg, 0.046 mmol) and compound A-37-5 (46.47 mg, 0.042 mmol) were dissolved in N,N-dimethylformamide (2 mL), and HOBT (9.35 mg, 69.20 μmol) and DIPEA (11.90 mg, 0.092 mmol) were added. The reaction system was stirred at 25°C for 1 h. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was freeze-dried to obtain 41 mg of the title compound.

[0942] Column: SunFire Prep C18 ODS 19mm×150mm×5.0μm

[0943] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0944] The structural characterization data are as follows:

[0945] ESI-MS (m / z): 1413.5 [(M+H)] + .

[0946] Step Six: Synthesis of 4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-((methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynylamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butamido)butamido)-5-ureidopentanoylamino)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3′,4′:6,7]indolazin[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate

[0947] At 25 °C, 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynylamide (B-03-5, 12.96 mg, 0.042 mmol) and compound A-37-6 (40 mg, 0.028 mmol) were dissolved in dimethyl sulfoxide (1 mL) and water (0.25 mL). After adding cuprous bromide (8.20 mg, 0.057 mmol), the reaction system was reacted at 25 °C for 11 minutes. The reaction was monitored by liquid chromatography-mass spectrometry (LC-MS). The reaction solution was purified by high performance liquid chromatography (under the following conditions). The prepared solution was freeze-dried to obtain 29 mg of the title compound.

[0948] Column: SunFire Prep C18 ODS 19mm×150mm×5.0μm

[0949] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0950] The structural characterization data are as follows:

[0951] ESI-MS (m / z): 1718.8 [(M+H)] + .

[0952] Example 1.17: (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxapentano[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-((18-(2-(methanesulfonyl)pyrimidin-5-yl)-13-oxo-3,6,9-trioxa-12-azaoctadecyl-17-yn-1-yl)carbamoyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-38)

[0953] Step 1: Synthesis of tert-butyl (1-(5-formyl-2-hydroxyphenyl)-1-oxo-5,8,11-trioxo-2-azatridecane-13-yl)carbamate

[0954] 5-Formyl-2-hydroxybenzoic acid (A-38-2, 625.04 mg, 3.76 mmol) was dissolved in dichloromethane (20 mL), followed by the addition of 1 drop of DMF and then 2 mL of thionyl chloride. The mixture was heated under reflux for 2 hours. After concentration under reduced pressure, the solution was dissolved in dichloromethane (5 mL) to obtain an acyl chloride intermediate solution for later use. Tert-Butoxycarbonyltriethylene glycolamine (A-38-1, 1 g, 3.42 mmol) was dissolved in dichloromethane (20 mL). After cooling to 0 °C, the above acyl chloride solution and diisopropylethylamine (36.47 mg, 0.282 mmol) were added dropwise. The reaction system was slowly heated to room temperature and reacted for 12 hours. Subsequently, the solution was concentrated under reduced pressure and purified (under the following conditions) to obtain A-38-3 (271 mg).

[0955] ESI-MS (m / z): 441.2 [M+1] + .

[0956] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0957] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0958] Step 2: Synthesis of (2R,3R,4S,5S,6S)-2-(2-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecane-16-yl)carbamoyl)-4-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate)

[0959] A-38-3 (270 mg, 0.613 mmol) and (2R,3R,5S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (A-38-4, 267.80 mg, 0.674 mmol) were dissolved in acetonitrile (30 mL), and silver oxide (568.18 mg, 2.45 mmol) and 4A molecular sieve powder (1 g) were added. The mixture was stirred under nitrogen protection for 16 hours. The filtrate after filtration was concentrated under reduced pressure to obtain 460 mg of crude A-38-5, which was directly used for the next step of the reaction.

[0960] ESI-MS (m / z): 757.4 [M+1] + .

[0961] Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(2-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecane-16-yl)carbamoyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate

[0962] A-38-5 (460 mg, 0.608 mmol) was dissolved in dichloromethane (5 mL) and isopropanol (5 mL). Silica gel powder (1 g) and sodium borohydride (11.50 mg, 0.304 mmol) were added with stirring. The mixture was reacted for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to prepare purified A-38-6 (343 mg) (under the conditions below).

[0963] ESI-MS (m / z): 759.3 [M+1] + .

[0964] Column: Waters XBridge Prep C18OBD (8μm*45mm*450mm)

[0965] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0966] Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(2-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecane-16-yl)carbamoyl)-4-(((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate)

[0967] A-38-6 (343 mg, 0.452 mmol) was dissolved in dry dichloromethane (25 mL), and diisopropylethylamine (175.27 mg, 1.36 mmol) was added. While stirring, p-nitrophenyl chloroformate (273.35 mg, 1.36 mmol, dissolved in 25 mL dichloromethane) was added dropwise. The reaction was carried out at room temperature for 12 hours. The reaction system was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: 6% methanol / dichloromethane) to obtain compound A-38-7 (342 mg).

[0968] Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(2-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecane-16-yl)carbamoyl)-4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxapentano[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-triethyl acetate)

[0969] A-38-7 (53.93 mg, 0.058 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 (A-38-8, 20 mg, 0.039 mmol) and HOBt (15.77 mg, 0.117 mmol) were dissolved in DMF (1 mL), and diisopropylethylamine (15.09 mg, 0.117 mmol) was added dropwise. The mixture was stirred for 12 hours, and 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. The solution was concentrated under reduced pressure to obtain 49 mg of crude A-38-9, which was then directly used in the next step of the reaction.

[0970] ESI-MS (m / z): 1262.5 [M+1] + .

[0971] Step Six: Synthesis of (2S,3S,4S,5R,6S)-6-(2-((2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamoyl)-4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxapentano[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

[0972] A-38-9 (49 mg, 0.039 mmol) was dissolved in methanol (2 mL), and lithium hydroxide aqueous solution (13.03 mg, 0.311 mmol, dissolved in 0.5 mL water) was added dropwise. The mixture was stirred for 1 hour, concentrated under reduced pressure, and then trifluoroacetic acid (2 mL) was added dropwise. The mixture was stirred for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry until complete. The reaction mixture was concentrated under reduced pressure and then purified (under the conditions below) to obtain compound A-38-10 (23 mg).

[0973] ESI-MS (m / z): 1022.0 [M+1] + .

[0974] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0975] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0976] Step 7: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxapentano[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-((18-(2-(methanesulfonyl)pyrimidin-5-yl)-13-oxo-3,6,9-trioxa-12-azaoctadecyl-17-yn-1-yl)carbamoyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid)

[0977] A-38-10 (23 mg, 0.020 mmol) and (2,5-dioxopyrrolidone-1-yl)-6-(2-methylsulfonylpyrimidin-5-yl)hexanoic acid-5-ynyl ester (IM-3, 8.14 mg, 0.022 mmol) were dissolved in DMF (1 mL), and diisopropylethylamine (7.85 mg, 0.061 mmol) was added dropwise. The mixture was stirred for 2 hours, and TLC monitoring showed that the reaction was essentially complete. The reaction system was purified (under the following conditions) to obtain compound A-38 (5.10 mg).

[0978] ESI-MS (m / z): 1272.3 [M+1] + .

[0979] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0980] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0981] Example 1.18: (2S,3S,4S,5R,6S)-6-(4-((((((((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyran[3′,4′:6,7]indolazine[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-((methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamidamido)ethoxy)acetamyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-39)

[0982] Step 1: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10-trioxo-4-azadodecanoamide)-4-((((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolazine[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triethyl acetate)

[0983] A-35-6 (185.85 mg, 0.188 mmol), eczema (50 mg, 0.094 mmol), and HOBt (38.13 mg, 0.282 mmol) were dissolved in DMF (4 mL), and diisopropylethylamine (36.47 mg, 0.282 mmol) was added dropwise. The mixture was stirred for 12 hours. The reaction mixture was monitored by LCMS to ensure complete reaction of the starting materials. The reaction mixture was extracted with water and ethyl acetate. The organic phase was washed with saturated brine, dried, and concentrated under reduced pressure to obtain 120 mg of a yellow oily crude product, which was directly used for the next reaction.

[0984] ESI-MS (m / z): 1285.4 [M+1] +

[0985] Step 2: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-aminoethoxy)ethoxy)acetamyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyran[3′,4′:6,7]indolazine[1,2-b]quinoline-1-yl)carbamoyl)oxy)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0986] A-39-1 (120 mg, 0.093 mmol) was dissolved in methanol (5 mL) and dichloromethane (approximately 50 μL), and then an aqueous solution of lithium hydroxide (31.37 mg, 0.747 mmol, dissolved in 1 mL of water) was added dropwise. The mixture was stirred for 2 hours. The reaction mixture was monitored by LCMS to ensure complete reaction of the starting materials. The pH of the reaction mixture was adjusted to 4 by adding 3N hydrochloric acid solution dropwise. After concentration under reduced pressure, 32 mg of a white solid, A-39-2, was obtained.

[0987] ESI-MS (m / z): 922.3 [M+1] +

[0988] Step 3: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((((((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyran[3′,4′:6,7]indolazine[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)ethoxy)acetamyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid)

[0989] A-39-2 (30 mg, 0.033 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (IM-3, 13.08 mg, 0.036 mmol) were dissolved in DMF (1 mL), and diisopropylethylamine (4.21 mg, 0.033 mmol) was added dropwise with stirring. The reaction was carried out at room temperature for 4 hours. The reaction mixture was monitored by LCMS to ensure complete reaction of the starting materials. The reaction system was purified to obtain 18.54 mg of A-39 as a white solid. ESI-MS (m / z): 1171.8 [M+1] +

[0990] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0991] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0992] Example 1.19: (2S,3S,4S,5R,6S)-6-(4-(((((((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-((methanesulfonyl)pyrimidin-5-yl)hex-5-acetamyl)ethoxy)acetamyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid and (2S ,3S,4S,5R,6S)-6-(4-(((((((((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-((methanesulfonyl)pyrimidin-5-yl)hex-5-acetamyl)ethoxy)acetamyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-40-A and A-40-B)

[0993] Step 1: Synthesis of (2S,3S,4S,5R,6S)-2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10-trioxo-4-azadodecanoamide)-4-(((((((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triethyl acetate

[0994] Starting materials A-35-6 (303 mg, 0.307 mmol), 1-4 (100 mg, 0.205 mmol), and HOBt (83 mg, 0.614 mmol) were dissolved in DMF (5 mL), and diisopropylethylamine (79 mg, 0.614 mmol) was added dropwise. The mixture was stirred for 12 hours. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with saturated brine, dried, and concentrated under reduced pressure to obtain 260 mg of crude A-40-1, which was then directly used in the next reaction.

[0995] ESI-MS (m / z): 1300.7 [M+1] + .

[0996] Step 2: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-aminoethoxy)ethoxy)acetamyl)-4-(((((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0997] A-40-1 (260 mg, 0.200 mmol) was dissolved in methanol (5 mL), then two drops of dichloromethane were added to dissolve it completely. Lithium hydroxide aqueous solution (67 mg, 1.60 mmol, dissolved in 0.5 mL water) was then added dropwise, and the mixture was stirred for 2 hours. LC-MS was used to monitor the reaction until complete. 3N hydrochloric acid aqueous solution was added to the reaction system to adjust the pH to 4, and the mixture was concentrated under reduced pressure to prepare purified A-40-2 (63 mg) (under the conditions below).

[0998] ESI-MS (m / z): 938.3 [M+1] + .

[0999] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1000] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1001] Step 3: (2S,3S,4S,5R,6S)-6-(4-(((((((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetamyl)ethoxy)acetamyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) Synthesis of (2S,3S,4S,5R,6S)-6-(4-(((((((((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydroxybenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetamyl)ethoxy)acetamyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid)

[1002] A-40-2 (63 mg, 0.067 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (IM-3, 27 mg, 0.074 mmol) were dissolved in DMF (1 mL), and diisopropylethylamine (9 mg, 0.067 mmol) was added dropwise with stirring. The reaction was carried out at room temperature for 4 hours. The reaction mixture was monitored by LCMS to ensure complete reaction. The reaction solution was prepared, separated, and purified (under the following conditions) to obtain A-40-A 10.14 mg and A-40-B 14.60 mg.

[1003] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1004] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1005] The retention time of peak A-40-A was 8.90 min. ESI-MS (m / z): 1188.6 [M+1]+ .

[1006] The retention time of peak A-40-B is 9.10 min. ESI-MS (m / z): 1188.6 [M+1] + .

[1007] Example 1.20: Synthesis of (2S,3S,4S,5R,6S)-6-(4-(((((1S,9S)-4-chloro-9-ethyl-9-hydroxy-5-fluoro-10,13-dioxo-2,3,9,10,13,15-octahydroxybenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetamyl)ethoxy)acetamyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-41)

[1008] Step 1: (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10-trioxo-4-azadodecanoamide)-4-(((((1S,9S)-4-chloro-9-ethyl-9-hydroxy-5-fluoro-10,13-dioxo-2,3,9,10,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate

[1009] The starting material A-35-6 (56.6 mg, 0.057 mmol), compound 1-11 (30.0 mg, 0.048 mmol) and HOBt (12.9 mg, 0.095 mmol) were dissolved in DMF (1 mL), and diisopropylethylamine (18.5 mg, 0.143 mmol) was added dropwise. The mixture was stirred at 25 °C for 4 hours. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 29.0 mg of the title compound.

[1010] ESI-MS (m / z): 1304.3 [M+1]+.

[1011] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1012] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1013] Step 2: (2S,3S,4S,5R,6S)-6-(2-(2-(2-aminoethoxy)ethoxy)acetamyl)-4-(((((1S,9S)-4-chloro-9-ethyl-9-hydroxy-5-fluoro-10,13-dioxo-2,3,9,10,13,15-octahydroxybenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[1014] Compound A-35-1 (29.0 mg, 0.022 mmol) was dissolved in methanol (1 mL) / tetrahydrofuran (1 mL), and lithium hydroxide aqueous solution (7.7 mg, 0.184 mmol, dissolved in 0.5 mL of water) was added dropwise. The mixture was stirred at 25 °C for 2 hours. Water (5 mL) was added to the reaction system, and the pH was adjusted to 2-3 with 3N hydrochloric acid. Impurities were extracted with ethyl acetate (5 mL), and the aqueous phase was lyophilized to obtain the crude product. The crude product was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 5.0 mg of the title compound.

[1015] ESI-MS (m / z): 942.2 [M+1]+.

[1016] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1017] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1018] Step 3: (2S,3S,4S,5R,6S)-6-(4-(((((1S,9S)-4-chloro-9-ethyl-9-hydroxy-5-fluoro-10,13-dioxo-2,3,9,10,13,15-octahydroxybenzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)-2-(2-(2-(6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetamyl)ethoxy)acetamyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid)

[1019] Compound A-35-2 (5.0 mg, 0.0053 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (IM-3, 2.3 mg, 0.0064 mmol) were dissolved in DMF (0.5 mL), and diisopropylethylamine (2.0 mg, 0.016 mmol) was added dropwise. The reaction was carried out at 25 °C for 4 hours. The reaction solution was directly purified by preparative high performance liquid chromatography (under the following conditions) to obtain 0.90 mg of the title compound.

[1020] ESI-MS (m / z): 1192.0 [M+1]+.

[1021] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1022] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1023] Example 1.21: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecane-5-acetylamido)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-(isopropylamino)ethyl)-3,14-dioxy-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl) carbonate (B-08)

[1024] Step 1: Synthesis of (S)-(2-(4-ethyl-4-hydroxy-3,14-dioxy-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate tert-butyl

[1025] Compound 2-1 (500.00 mg, 1.15 mmol) was placed in 15 mL of dry dichloromethane at 20 °C, and tert-butyl carbonate (276.90 mg, 1.27 mmol) and DIPEA (447.21 mg, 3.46 mmol) were added. The reaction mixture was stirred at 20 °C for 16 h. The reaction was detected by liquid chromatography-mass spectrometry. The reaction mixture was purified by silica gel column chromatography (mobile phase: dichloromethane / methanol = 50 / 1) to give 400 mg of the title compound.

[1026] The structural characterization data are as follows:

[1027] ESI-MS (m / z): 534.0 [M+H] + .

[1028] Step 2: Synthesis of tert-butyl (2-((S)-4-(((4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl)oxy)carbonyl)-4-ethyl-3,14-dioxy-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate

[1029] Compound B-08-1 (30.00 mg, 0.056 mmol) was added to dry dichloromethane (1 mL) at 0 °C under nitrogen protection. DMAP (54.95 mg, 0.450 mmol) was added, followed by the dropwise addition of triphosgene (16.68 mg, 0.056 mmol) in dichloromethane (1 mL). The reaction mixture was kept at 0 °C for 30 min. The reaction mixture was then purged with nitrogen under reduced pressure, and (S)-2-(32-azido-5-)- was added dropwise at 0 °C. A 20 mL solution of dry dichloromethane was prepared with 117.39 mg (0.111 mmol) of oxo-3,9,12,15,18,21,24,27,30-nonoxa-6-aza-triapentadecanoamide (N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexamethyleneamide). The reaction solution was stirred at 20 °C for 60 min, and the reaction was detected by liquid chromatography-mass spectrometry. The concentrated reaction solution was used directly in the next step of the reaction.

[1030] The structural characterization data are as follows:

[1031] ESI-MS (m / z): 1620.2 [M+H] + .

[1032] Step 3: Synthesis of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(isopropylamino)ethyl)-3,14-dioxy-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl) carbonate (B-08-3)

[1033] Compound B-08-2 (80.00 mg, 0.049 mmol) was placed in acetonitrile (1.0 mL) and trifluoroacetic acid (0.4 mL) was added. The reaction solution was kept at 20 °C for 1 h. The reaction was detected by liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was freeze-dried to obtain 41.0 mg of trifluoroacetate of the title compound.

[1034] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1035] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1036] The structural characterization data are as follows:

[1037] ESI-MS (m / z): 1248.2 [M+H] + .

[1038] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecane-5-acetylamido)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-(isopropylamino)ethyl)-3,14-dioxy-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl) carbonate

[1039] Compound B-08-3 (40.00 mg, 0.032 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynylamide (B-03-5, 14.69 mg, 0.048 mmol) were added to a mixed solution of DMSO (0.5 mL) and H2O (0.1 mL) at 25 °C, and cuprous bromide (9.20 mg, 0.064 mmol) was added. The reaction was carried out at 20 °C for 2 h under nitrogen protection. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was freeze-dried to obtain 20.0 mg of trifluoroacetate of the title compound.

[1040] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1041] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1042] The structural characterization data are as follows:

[1043] ESI-MS (m / z): 1552.6 [M+H] + .

[1044] Example 1.22: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatriapentadecanoamide)benzyl((1S,9S)-1-(dimethylamino)-9-ethyl-5-fluoro-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazine[1,2-b]quinoline-9-yl) carbonate (B-09)

[1045] Step 1: Synthesis of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((1S,9S)-1-(dimethylamino)-9-ethyl-5-fluoro-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

[1046] Compounds 1-2 (20.00 mg, 0.040 mmol) were added to dry dichloromethane (1 mL) at 0 °C under nitrogen protection. DMAP (39.10 mg, 0.320 mmol) was added, followed by the dropwise addition of triphosgene (11.87 mg, 0.040 mmol) in dichloromethane (1 mL). The reaction mixture was kept at 0 °C for 30 min. The reaction mixture was then purged with nitrogen under reduced pressure, and (S)-2-(3,2-azido-5-oxo-3,9,12,15)- was added dropwise at 0 °C. A dry dichloromethane (20 mL) solution of 18,21,24,27,30-nonoxa-6-aza-triapentadecanoamide)N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexamethyleneamide (84.66 mg, 0.080 mmol) was prepared. The reaction solution was stirred at 20 °C for 1 h, and the reaction was detected by liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was freeze-dried to obtain 40.0 mg of the title compound.

[1047] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1048] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1049] The structural characterization data are as follows:

[1050] ESI-MS (m / z): 1550.2 [M+H] + .

[1051] Step 2: (1S,9S)-1-(dimethylamino)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-9-yl(4-((S)-2-(4-((4-methoxyphenyl)diphenyl) Synthesis of (methyl)amino)butyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecyl-5-acetylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatriapentadecanoamide)benzyl)carbonate

[1052] Compound B-09-1 (30.00 mg, 0.019 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynylamide (B-03-5, 8.87 mg, 0.029 mmol) were added to a mixed solution of DMSO (0.5 mL) and H2O (0.1 mL) at 25 °C, and cuprous bromide (5.55 mg, 0.039 mmol) was added. The reaction was carried out at 20 °C for 1 h under nitrogen protection. The reaction was detected by liquid chromatography-mass spectrometry (LC-MS). The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was freeze-dried to obtain 25.0 mg of the title compound.

[1053] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1054] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1055] The structural characterization data are as follows:

[1056] ESI-MS (m / z): 1855.2 [M+H] + .

[1057] Step 3: Synthesis of 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-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((1S,9S)-1-(dimethylamino)-9-ethyl-5-fluoro-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

[1058] Compound B-09-2 (15.00 mg, 0.017 mmol) was placed in acetonitrile (0.5 mL) at 25 °C, and trifluoroacetic acid (0.2 mL) was added. The reaction solution was kept at 20 °C for 0.5 h, and the reaction was detected by liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was freeze-dried to obtain 9.0 mg of trifluoroacetate of the title compound.

[1059] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1060] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1061] The structural characterization data are as follows:

[1062] ESI-MS (m / z): 1583.1 [M+H] + .

[1063] Example 1.23: 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)-5-ureidopentamido)benzyl((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (D-01)

[1064] Step 1: Synthesis of 4-((S)-2-((S)-2-(((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutylamino)-5-ureidopentamido)benzyl((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[1065] At 25℃, (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohep-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyramide ( MMAE (10 mg, 0.014 mmol) and compound D-01-1 (12.82 mg, 0.017 mmol) were dissolved in DMF (1.0 mL), HOBt (2.82 mg, 0.021 mmol) and DIPEA (3.60 mg, 0.028 mmol) were added, and the reaction was maintained at 25 °C for 1 h. The reaction was detected by liquid chromatography-mass spectrometry. The reaction solution was used directly for the next step of the reaction without any treatment.

[1066] The structural characterization data are as follows:

[1067] ESI-MS (m / z): 1345.2 [M+H] + .

[1068] Step 2: Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutylamino)-5-ureidopentamido)benzyl((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl(methyl)carbamate

[1069] Diethylamine (0.1 mL) was added to the reaction solution of compound D-01-2 at 25 °C, and the reaction was maintained at 25 °C for 1 h. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product, which was directly used in the next step of the reaction.

[1070] The structural characterization data are as follows:

[1071] ESI-MS (m / z): 1123.2 [M+H] + .

[1072] Step 3: Synthesis of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)-5-ureidopentamido)benzyl((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[1073] At 25°C, crude D-01-3 and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid (7.16 mg, 0.027 mmol) were dissolved in DMF (0.5 mL), and HATU (10.15 mg, 0.027 mmol) and DIPEA (3.45 mg, 0.027 mmol) were added. The reaction was maintained at 25°C for 1 h. The reaction was monitored by liquid chromatography-mass spectrometry. The reaction solution was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 5.0 mg of the title compound.

[1074] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1075] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1076] The structural characterization data are as follows:

[1077] ESI-MS (m / z): 1373.2 [M+H] + .

[1078] Example 1.24: Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)-3-((4S,7S,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)thiazolidin-4-carboxamide (E-01)

[1079] Step 1: Preparation of (9H-fluorene-9-yl)methyl((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropane-2-yl)carbamate (E-01-1)

[1080] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (70 mg, 127.13 μmol) was dissolved in THF (10 mL). After purging with nitrogen three times, lithium hydroxide monohydrate (32.01 mg, 762.80 μmol) was added under ice bath conditions. After stirring at room temperature for 10 minutes, (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)methyl ester (97.23 mg, 254.27 μmol) was added. After the addition was complete, the reaction was stirred at 22 °C for 5 hours. Add 20 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract three times with dichloromethane (10 mL x 3), wash with 10 mL of brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product of the title compound. After purification by preparative high performance liquid chromatography, freeze-dry to obtain the title compound (70 mg, 80.19 μmol).

[1081] Its structural characterization data are as follows:

[1082] MS m / z (ESI): 873.3 [M+H]+

[1083] Its preparation method is as follows:

[1084] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1085] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1086] Step 2: Preparation of (S)-3-(((S)-2-aminopropamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (E-01-2)

[1087] (9H-fluorene-9-yl)methyl((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropane-2-yl)carbamate (40 mg, 45.82 μmol) was dissolved in DMF (2 mL), and diethylamine (16.76 mg, 229.11 μmol) was added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (34 mg, 44.46 μmol).

[1088] Its structural characterization data are as follows:

[1089] MS m / z (ESI): 651.2 [M+H]+

[1090] Its preparation method is as follows:

[1091] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1092] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1093] Step 3: Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)-3-((4S,7S,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)thiazolidin-4-carboxamide (E-01)

[1094] (S)-3-(((S)-2-aminopropamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (40 mg, 52 mg) (31 μmol) and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (25.76 mg, 62.77 μmol) were dissolved in DMF (2 mL), and DIPEA (27.04 mg, 209.22 μmol) and HATU (39.75 mg, 104.61 μmol) were added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (41 mg, 38.52 μmol).

[1095] Its structural characterization data are as follows:

[1096] MS m / z (ESI): 1043.3 [M+H]+

[1097] Its preparation method is as follows:

[1098] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1099] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1100] Example 2: Preparation of Antibodies

[1101] 2.1 Construction and expression of recombinant anti-EGFR and anti-B7H3 bispecific antibodies

[1102] The bispecific antibodies BsAb01, BsAb02, BsAb03, BsAb04, BsAb05, and BsAb06 disclosed herein all employ a 2+2 symmetrical structure, containing two identical polypeptide chains 1 and two identical polypeptide chains 2, and maintain affinity for EGFR and B7H3, exhibiting strong killing activity against tumor cells. The structures and amino acid sequences of the bispecific antibodies are shown in Table 1.

[1103] Table 1: Structure and sequence of bispecific antibodies

[1104] The above sequence underwent codon optimization and DNA synthesis. The two peptide chains of the bispecific antibody were cloned into the pKLGS1 expression vector from Kelun Biotech (containing two expression cassettes. The first expression cassette has a mouse CMV promoter and sv40 polyA as the polyA; the second expression cassette has a mouse CMV promoter and TK polyA as the polyA). The expression plasmid was transfected into CHO-K1 cells (obtained from the American Type Culture Collection (ATCC)). TM After a period of expression (using Manasas, VA), the supernatant was harvested, and bispecific antibodies were captured using Protein A (MabSelect SuRe LX, GE) affinity chromatography. These were then purified using cation exchange chromatography (Eshmuno CPX, Merck) to obtain the bispecific antibodies BsAb01, BsAb02, BsAb03, BsAb04, BsAb05, and BsAb06.

[1105] 2.2 Construction and expression of recombinant anti-EGFR and anti-B7H3 bispecific antibodies and hIgG1-M antibody

[1106] The recombinant anti-EGFR and anti-B7H3 bispecific antibody Hz20G5.26 / Zalu bsAb sequence was derived from patent CN202411888761.5 (sequence numbers SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 in that patent). hIgG1-M is an anti-chicken lysozyme antibody; its variable region sequence is derived from patent CA2309763A1, with the heavy chain variable region fused to the mutated human IgG1 heavy chain constant region (SEQ ID NO: 43), and the light chain variable region fused to the human kappa light chain constant region (SEQ ID NO: 42). The expression plasmids were transfected into CHO-K1 cells, and the transfected cells were cultured in cell culture medium under suitable conditions to express the antibodies. After a period of expression, the supernatant was harvested, and the control antibody was captured using Protein A (MabSelect SuRe LX, GE) affinity chromatography. Then, cationic ( Purification was performed by CPX (Merck) chromatography to obtain the control double antibody Hz20G5.26 / Zalu bsAb and hIgG1-M.

[1107] Example 3: Conjugation of compounds containing cellular bioactive molecules and linkers with antibodies

[1108] 3.1 Preparation of ADCs conjugated with bispecific antibodies BsAb01, BsAb02, BsAb03, Hz20G5.26 / Zalu bsAb, and hIgG1-M to A-14

[1109] Bispecific antibodies BsAb01, BsAb02, BsAb03, Hz20G5.26 / Zalu bsAb, and control group hIgG1-M solution were diluted with 20 mM PB + 0.1 M EDTA at pH 7.6. The pH was then adjusted to 7.6 with 1 M Na2HPO4 solution. A 10 mM TCEP-HCl solution (pH 7.6) at a molar ratio of 5.5:1 to the antibody was added and mixed thoroughly. The solution was incubated at room temperature for 2 hours to obtain reduced antibody solutions. 10 mM A-14 dimethyl sulfoxide solution was added to each reduced antibody solution at a molar ratio of A-14 to antibody of 10:1. All resulting solutions were incubated at room temperature for 2 hours and analyzed using NAP. TM The buffer solution was replaced with 20 mM histidine buffer at pH 6.0 using a gel column (Cytiva) to obtain the following ADCs: BsAb01-A-14, BsAb02-A-14, BsAb03-A-14, Hz20G5.26 / Zalu bsAb-A-14, and hIgG1-MA-14. Mass spectrometry determined the DAR values ​​of BsAb01-A-14, BsAb02-A-14, BsAb03-A-14, Hz20G5.26 / Zalu bsAb-A-14, and hIgG1-MA-14 to be approximately 8.

[1110] 3.2 Preparation of ADCs (antibodies) of bispecific antibodies BsAb04, BsAb05, BsAb06, and hIgG1-M conjugated with E-01

[1111] Bispecific antibodies BsAb04, BsAb05, BsAb06, and the control group hIgG1-M solution were diluted with 20 mM PB + 0.1 M EDTA at pH 7.6. The pH was then adjusted to 7.6 with 1 M Na2HPO4 solution. A 10 mM TCEP-HCl solution (pH 7.6) at a molar ratio of 5.5:1 to the antibody was added and mixed thoroughly. The solution was incubated at room temperature for 2 hours to obtain the reduced antibody solution. 10 mM A-14 dimethyl sulfoxide solution was added to each reduced antibody solution at a molar ratio of E-01 to antibody of 10:1. All resulting solutions were incubated at room temperature for 2 hours and analyzed using NAP. TM The buffer solution was replaced with 20 mM histidine buffer at pH 6.0 using a gel column (Cytiva) to obtain ADC BsAb04-E-01, ADC BsAb05-E-01, ADC BsAb06-E-01, and ADC hIgG1-ME-01. Mass spectrometry determined the DAR values ​​of ADC BsAb04-E-01, ADC BsAb05-E-01, ADC BsAb06-E-01, and ADC hIgG1-ME-01 to be approximately 8.

[1112] 3.3 Preparation of ADC molecule Hz20G5.26 / Zalu bsAb-Exatecan

[1113] Following the method in Example 2 of patent CN202411888761.5, Hz20G5.26 / Zalu bsAb was coupled into an ADC Hz20G5.26 / Zalu bsAb-Exatecan, and the DAR value measured by mass spectrometry was approximately 4.

[1114] Example 4: Detection of antibody-drug conjugate activity

[1115] 4.1 Cell affinity assay of anti-human B7H3 / EGFR bispecific antibody and its conjugate

[1116] The cell affinity of the anti-human B7H3 / EGFR bispecific antibody and its conjugates was detected using a flow cytometer (Beckman, model Cytoflex). The cell lines used included PC-9 (Kangyuan Bochuang) human non-small cell lung cancer cells with high EGFR and high B7H3 expression, NCI-H1975 (Kangyuan Bochuang) human non-small cell lung cancer cells with moderate EGFR and moderate B7H3 expression, NCI-H358 (Nanjing Kebai) human non-small cell lung cancer cells with high EGFR and high B7H3 expression, A375 (Nanjing Kebai) human melanoma cells with very low EGFR and low B7H3 expression, BxPc-3 (Nanjing Kebai) human pancreatic cancer cells with high EGFR and high B7H3 expression, PC9-B7H3 KO (Kelun Biotech) human non-small cell lung cancer cells with high EGFR and low B7H3 expression (B7H3 / EGFR knockdown) human non-small cell lung cancer cells PC9-B7H3 / EGFR low (Kelun Biotech) human non-small cell lung cancer cells with low EGFR and low B7H3 expression (B7H3 / EGFR knockdown). The detection methods are as follows:

[1117] Adherent cells were digested with Trypsin-EDTA (0.25%) solution (Shanghai Yuanpei), and digestion was terminated with complete culture medium. After centrifugation (500g, 3min), the supernatant was discarded, and the cell density was adjusted to 2.0 × 10⁶ cells / year with RPMI 1640 (containing 1% FBS). 6 Add 50 μl of cell suspension per well to a 96-well conical plate, resulting in a cell count of 1 × 10⁶ cells / ml. 5Cells / well. Dilute the antibody and its conjugate with RPMI 1640 (containing 1% FBS) to a maximum concentration of 200 nM. Perform 11 serial dilutions (3-fold) and add 50 μl / well of the diluted antibody and its conjugate to each well of a 96-well conical plate containing cells, resulting in a final concentration of 100 nM. Incubate at 4°C for 30 min. After primary antibody incubation, wash cells twice with RPMI 1640 (containing 1% FBS). Flow cytometry secondary antibody anti-human IgG-PE (Jackson ImmunoResearch, 109-115-170) was diluted 1:400 with RPMI 1640 (containing 1% FBS) and used as a pre-treatment. Resuspend cells in 50 μl / well and incubate at 4°C in the dark for 30 min. After secondary antibody incubation, wash cells twice with RPMI 1640 (containing 1% FBS) and add 200 μl / well of RPMI 1640. Cells were resuspended in 1640 (containing 1% FBS) and then analyzed by flow cytometry.

[1118] Data processing: Export Median PE and import the data into analysis software to perform four-parameter fitting of affinity curves and calculate EC. 50 .

[1119] The tumor cell affinity results of anti-human B7H3 / EGFR bispecific antibodies and their conjugates are shown in Tables 2-1 to 2-3. As long as EGFR or B7H3 is expressed, the bispecific antibodies can effectively bind to it. The tested bispecific antibodies BsAb01, BsAb02, BsAb03, and their corresponding conjugates all exhibited high tumor cell affinity (ECG). 50 All were less than 1 nM, and all were superior to the control antibody Hz20G5.26 / Zalu bsAb and the isotoxin conjugate Hz20G5.26 / Zalu bsAb-A-14. The tested bispecific antibodies BsAb04, BsAb05, BsAb06 and their corresponding conjugates all exhibited high tumor cell affinity (ECG). 50 All are less than 1 nM), and all are superior to the control ADC Hz20G5.26 / Zalu bsAb-Exatecan.

[1120] Table 2-1: Cell affinity against human B7H3 / EGFR BsAb

[1121] Table 2-2: Cell Affinity of Anti-Human B7H3 / EGFR BsAb Conjugates

[1122] Table 2-3: Cell affinity of anti-human B7H3 / EGFR BsAb and its conjugates

[1123] 4.2 Detection of cellular endocytic activity of anti-human B7H3 / EGFR bispecific antibodies and their conjugates

[1124] The endocytic activity of the anti-human B7H3 / EGFR bispecific antibody and its conjugates was detected using flow cytometry (Beckman, Cytoflex). Cells used included human non-small cell lung cancer (NSCLC) cells NCI-H1975 (Kangyuan Bochuang) expressing both EGFR and B7H3, NCI-H358 (Nanjing Kebai) expressing high levels of B7H3 and B7H3, A375 (Nanjing Kebai) expressing very low levels of EGFR and B7H3, PC9-B7H3EGFR KO (Kelun Biotech) expressing very high levels of B7H3 but no EGFR (overexpression of B7H3 and EGFR knockout), and PC9-B7H3 / EGFR low (Kelun Biotech) expressing low levels of both EGFR and B7H3 (B7H3 / EGFR knockdown). The detection methods are as follows:

[1125] Adherent cells were digested with Trypsin-EDTA (0.25%) (Shanghai Yuanpei) solution and counted. Digestion was terminated with complete culture medium. After centrifugation (500g, 3min), the supernatant was discarded, and the cell density was adjusted to 1×10⁶ cells / year with complete culture medium. 5 Add 100 μl of cell suspension to each well of a 96-well plate, resulting in a cell count of 1 × 10⁶ cells / ml. 4 Cells were cultured overnight at 37°C with 5% CO2. Bispecific antibodies and their conjugates were prepared using complete culture medium, with a maximum primary antibody concentration of 40 nM and a maximum Phrodo-deepred (Colombe) concentration of 120 nM. Equal volumes were mixed and incubated at 37°C for 30 min to form an antibody-Phrodo mixture. The mixture was then serially diluted 3-fold to eight concentration points using complete culture medium. The cell plate was removed, and 50 μl / well of old culture medium was aspirated. The diluted antibody-Phrodo mixture solution (maximum final primary antibody concentration of 10 nM and maximum final Phrodo-deepred concentration of 30 nM) was added to each well. The plate was incubated at 37°C with 5% CO2 for 24 h. After incubation, the culture medium was aspirated, and the cells were washed once with 200 μl / well of PBS. Cells were digested with 30 μl / well of Trypsin-EDTA (0.25%) (Shanghai Yuanpei), and digestion was terminated with 170 μl / well of PBS containing 10% FBS. After thoroughly resuspending the cells with a pipette, flow cytometry was performed (APC voltage 300V).

[1126] Data processing: The Median APC was exported and the data were imported into analysis software to calculate AUC, and the endocytic activity of each antibody and conjugate was compared. Anti-human B7H3 / EGFR bispecific antibodies and conjugates showed good endocytic activity against various tumor cells (as shown in Tables 3-1 and 3-2). The strength of endocytic activity varied with the expression levels of EGFR and B7H3 and cell type. In various cell types, the bispecific antibodies BsAb01, BsAb02, BsAb03, and their antibody-drug conjugates obtained by conjugating them with A-14 showed stronger endocytic activity than the control antibody Hz20G5.26 / Zalu bsAb and its isotoxin conjugate Hz20G5.26 / Zalu bsAb-A-14.

[1127] Table 3-1: Anti-human B7H3 / EGFR BsAb endocytosis activity

[1128] Table 3-2: Internalization activity against human B7H3 / EGFR BsAb and its conjugates

[1129] 4.3 In vitro cell killing assay of anti-human B7H3 / EGFR bispecific antibody-drug conjugate

[1130] Adherent tumor cells were digested with Trypsin-EDTA (0.25%) solution (Shanghai Yuanpei), and digestion was terminated with complete culture medium. After centrifugation (500g, 3min), the supernatant was discarded, and the cells were resuspended in complete culture medium and counted. 100 μl of cell suspension was added to each well of a 96-well plate (Corning, catalog number 3903). The cell addition amounts for different tumor cell types were as follows: 1000 cells / well of HCC827 (Nanjing Kebai) human non-small cell lung cancer cells with EGFR high expression and B7H3 expression; 5000 cells / well of NCI-H358 (Nanjing Kebai) human non-small cell lung cancer cells with EGFR high expression and B7H3 expression; 500 cells / well of PC9-B7H3 (Kelunbotai) human non-small cell lung cancer cells with B7H3 extremely high expression and EGFR high expression; 500 cells / well of PC9-B7H3 KO (Kelunbotai) human non-small cell lung cancer cells with EGFR high expression and no B7H3 expression; and 500 cells / well of PC9-B7H3 EGFR KO (Kelunbotai) human non-small cell lung cancer cells with B7H3 extremely high expression and no EGFR expression. The 96-well plates were placed in an evaporation-proof aluminum container containing an appropriate amount of PBS and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[1131] The antibody-drug conjugate (ADC) was diluted with complete culture medium to a maximum final concentration of 2500 nM, and serially diluted 3-fold to 11 concentration points. 100 μl of the diluted ADC was added to each well of a 96-well plate, resulting in a maximum final concentration of 1250 nM, and the plates were incubated for 5 days under the same conditions. CCL2.0 (Novazia, catalog number DD1101-03) and the 96-well plates were brought to room temperature beforehand for equilibration. 20 μl of CCL2.0 was added to each well of the 96-well plate, and the plates were shaken at 400 rpm for 15 min. The Luminescence signal was then measured using a microplate reader.

[1132] Data processing: Import the data into analysis software to perform four-parameter fitting of the kill curve and calculate the IC. 50 As shown in Table 4, the tested bispecific antibody-drug conjugates exhibited significantly superior killing activity against all tumor cells compared to hIgG1-MA-14, indicating that the killing activity was target-mediated. Furthermore, the killing activity of the anti-human B7H3 / EGFR bispecific antibody-drug conjugate was superior to the control isotoxin conjugate Hz20G5.26 / Zalu bsAb-A-14 in different cell types.

[1133] Table 4: In vitro cell killing effect of bispecific antibody-drug conjugates

[1134] 4.4 Payload in vitro cell killing detection

[1135] Adherent tumor cells were digested with Trypsin-EDTA (0.25%) solution (Shanghai Yuanpei), and digestion was terminated with complete culture medium. After centrifugation (1000 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in complete culture medium and counted. 90 μl of cell suspension was added to each well of a 96-well plate (Corning, catalog number 3903). The cell addition amounts were as follows for different tumor cell types: 3000 PC-9 (Nanjing Kebai) human non-small cell lung cancer cells with high EGFR and high B7H3 expression (Nanjing Kebai); 5000 BxPC-3 (Nanjing Kebai) human pancreatic cancer cells with high EGFR and low B7H3 expression (Nanjing Kebai); and 3000 A375 (Nanjing Kebai) human melanoma cells with high B7H3 and low EGFR expression (Nanjing Kebai). The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 72 h.

[1136] Small molecule compounds 2-19 and Exatecan were diluted with complete culture medium to a maximum final concentration of 10. 4nM, serially diluted 10-fold to 8 concentration points, add 10 μl / well of the diluted small molecule compound to a 96-well plate, and continue incubation for 3 days under the above conditions. Bring CCL2.0 (Novazia, catalog number DD1101-03) and the 96-well plate to room temperature beforehand to equilibrate. Add 50 μl / well of CCL2.0 to the 96-well plate, shake at 650 rpm for 10 min, and detect the Luminescence signal value using a microplate reader.

[1137] Data processing: Import the data into analysis software to perform four-parameter fitting of the kill curve and calculate the IC. 50 As shown in Table 5, the tested small molecules all exhibited significant killing activity against various tumor cells. The payloads 2-19 of BsAb04-E-01, BsAb05-E-01, and BsAb06-E-01 showed weaker killing activity in all three tumor cell types compared to the Exatecan payload of Hz20G5.26 / Zalu bsAb-Exatecan. Specifically, the toxicity of 2-19 against PC-9 cells was approximately 50% of that of Exatecan; the toxicity of 2-19 against BxPC-3 cells was approximately 37% of that of Exatecan; and the toxicity of 2-19 against A375 cells was approximately 18% of that of Exatecan. It is reasonable to expect that, at the same toxin dosage, the safety profiles of the ADCs BsAb04-E-01, BsAb05-E-01, and BsAb06-E-01 are superior to the control group Hz20G5.26 / Zalu bsAb-Exatecan.

[1138] Table 5: Payload in vitro cell killing

[1139] 4.5 In vivo efficacy detection of different antibody-drug conjugates in the PC-9 model - 1

[1140] PC-9 human non-small cell lung cancer cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) with high EGFR and B7H3 expression were cultured in vitro as a monolayer in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were digested twice weekly with trypsin-EDTA and passaged. When the cells reached the exponential growth phase, the medium was removed for mycoplasma detection, and the cells were collected. 5 × 10⁵ cells were subcutaneously inoculated into the right scapula of each mouse. 6 PC-9 cells were suspended in 0.1 ml of serum-free culture medium containing 50% Matrigel. The cells were allowed to grow to an average tumor volume of 100–250 mm. 3Mice with irregular tumor volumes or those that were too small or too large were excluded. The remaining mice were randomly divided into groups of 6 or 7 mice each, based on tumor volume and body weight. Each group received a single intravenous (iv) tail vein injection on the day of grouping. Dosage settings: BsAb01-A-14, BsAb02-A-14, and BsAb03-A-14 were administered at doses of 3 mg / kg and 10 mg / kg, respectively. hIgG1-MA-14 and Hz20G5.26 / Zalu bsAb-A-14 were also administered at the same molar toxin doses as the 3 mg / kg and 10 mg / kg doses of BsAb01-A-14, BsAb02-A-14, and BsAb03-A-14, respectively. After grouping, tumors were measured twice weekly using calipers, and tumor volume was calculated using the following formula: V = 0.5a × b 2 Where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily. Tumor growth inhibition rate (TGI) (%) was calculated using the formula: TGI (%) (tumor volume) = [1 - (T...] Vt -T V0 ) / (C Vt -C V0 []×100%; When tumor regression occurs, TGI(%) (tumor volume) = 100% - (T Vt -T V0 ) / T V 0×100%. T V0 T represents the average tumor volume in the test drug group during the administration of the drug to the test drug group. Vt C represents the average tumor volume in the test drug group on day t after drug administration; V0 C represents the average tumor volume in the solvent group during the grouped drug administration. Vt The mean tumor volume of the solvent group is t days after drug administration.

[1141] The results showed that a single intravenous administration of different conjugate drugs significantly enhanced the efficacy against human non-small cell lung cancer PC-9 cell subcutaneous xenografts in mice. At the same molar dose of toxin, the anti-human B7H3 / EGFR bispecific antibody conjugates BsAb01-A-14, BsAb02-A-14, and BsAb03-A-14 exhibited superior tumor-inhibiting activity compared to the control conjugate Hz20G5.26 / Zalu bsAb-A-14, and all groups showed good tolerability. Detailed results are shown in Table 6.

[1142] Table 6: Efficacy analysis of different antibody-drug conjugates in PC-9 cell tumor-bearing mouse model Note: V P0 V represents the average tumor volume during group administration; P35 The mean tumor volume is 35 days after drug administration.

[1143] 4.6 In vivo efficacy detection of different antibody-drug conjugates in the PC-9 model - 2

[1144] PC-9 human non-small cell lung cancer cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) with high EGFR and B7H3 expression were cultured in vitro as a monolayer in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were digested twice weekly with trypsin-EDTA and passaged. When the cells reached the exponential growth phase, the medium was removed for mycoplasma detection, and the cells were collected. 5 × 10⁵ cells were subcutaneously inoculated into the right scapula of each mouse. 6 PC-9 cells were suspended in 0.1 ml of serum-free culture medium containing 50% Matrigel. The cells were allowed to grow to an average tumor volume of 100–250 mm. 3 Mice with irregular tumor volumes or those that were too small or too large were excluded. The remaining mice were randomly divided into groups of 6 or 7 mice each, based on tumor volume and body weight. Each group received a single intravenous (iv) tail vein injection on the day of grouping. Dosage settings: BsAb04-E-01 was administered at 5 mg / kg, while BsAb05-E-01, BsAb06-E-01, and hIgG1-ME-01 were administered at the same molar dose as 5 mg / kg of BsAb04-E-01. Tumors were measured twice weekly using calipers after grouping, and tumor volume was calculated using the following formula: V = 0.5a × b 2 Where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily. Tumor growth inhibition rate (TGI) (%) was calculated using the formula: TGI (%) (tumor volume) = [1 - (T...] Vt -T V0 ) / (C Vt -C V0 []×100%; When tumor regression occurs, TGI(%) (tumor volume) = 100% - (T Vt -T V0 ) / T V0 ×100%. T V0 T represents the average tumor volume in the test drug group during the administration of the drug to the test drug group. Vt C represents the average tumor volume in the test drug group on day t after drug administration; V0 C represents the average tumor volume in the solvent group during the grouped drug administration. Vt The mean tumor volume of the solvent group is t days after drug administration.

[1145] The results showed that, with a single intravenous administration, BsAb04-E-01, BsAb05-E-01, and BsAb06-E-01 all exhibited significant efficacy against human non-small cell lung cancer PC-9 cell subcutaneous xenograft tumors in mice. The animals in each group tolerated the drugs well. Detailed results are shown in Table 7.

[1146] Table 7: Efficacy analysis of different antibody-drug conjugates in PC-9 cell tumor-bearing mouse model Note: TV, tumor volume; Px, refers to day x after drug administration.

[1147] 4.7 In vivo efficacy detection of different antibody-drug conjugates in the BxPC-3 model

[1148] Human pancreatic cancer cells BxPC-3 (purchased from Nanjing Kebai Biotechnology Co., Ltd.) with high EGFR expression and low B7H3 expression were cultured in vitro as a monolayer in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were digested twice weekly with trypsin-EDTA and passaged. When the cells reached the exponential growth phase, the medium was removed for mycoplasma detection, and the cells were collected. 5 × 10⁵ cells were subcutaneously seeded into the right scapula of each mouse. 6 BxPC-3 cells were suspended in 0.1 ml of serum-free culture medium containing 50% Matrigel. The tumors were allowed to grow to an average size of 100–250 mm. 3 Mice with irregular tumor volumes or tumors that were too small or too large were excluded. The remaining mice were randomly divided into groups of 5 mice each based on tumor volume and body weight. All groups received intravenous (iv) administration via tail vein, once a week for a total of 3 weeks (QW*3). Dosage settings: BsAb04-E-01 was administered at 3 mg / kg; BsAb05-E-01, BsAb06-E-01, and hIgG1-ME-01 were administered at an equivalent molar dose of 3 mg / kg of BsAb04-E-01. Tumors were measured twice weekly using calipers after grouping, and tumor volume was calculated using the following formula: V = 0.5a × b 2 Where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily. Tumor growth inhibition rate (TGI) (%) was calculated using the formula: TGI (%) (tumor volume) = [1 - (T...] Vt -T V0 ) / (C Vt -C V0 []×100%; When tumor regression occurs, TGI(%) (tumor volume) = 100% - (T Vt -T V0 ) / T V0 ×100%. T V0 T represents the average tumor volume in the test drug group during the administration of the drug to the test drug group. Vt C represents the average tumor volume in the test drug group on day t after drug administration; V0 C represents the average tumor volume in the solvent group during the grouped drug administration. Vt The mean tumor volume of the solvent group is t days after drug administration.

[1149] The res...

Claims

1. A bispecific antibody comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to B7H3.

2. The bispecific antibody according to 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 EGFR; 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.

3. The bispecific antibody according to claim 1 or 2, wherein, The first VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 6; and / or, the first VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 7; Preferably, the CDR is defined by the Chothia, AbM, Kabat, IMGT, MacCallum, or AHo numbering system.

4. The bispecific antibody according to any one of claims 1-3, wherein: The first VL includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 10, CDR-L2 containing the sequence shown in SEQ ID NO: 12, and CDR-L3 containing the sequence shown in SEQ ID NO: 14; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 11, CDR-L2 containing the sequence shown in SEQ ID NO: 13, and CDR-L3 containing the sequence shown in SEQ ID NO: 14; And / or, The first VH includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 16, CDR-H2 containing the sequence shown in SEQ ID NO: 20, and CDR-H3 containing the sequence shown in SEQ ID NO: 23; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 18, CDR-H2 containing the sequence shown in SEQ ID NO: 22, and CDR-H3 containing the sequence shown in SEQ ID NO: 23; (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; or, (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 17, CDR-H2 containing the sequence shown in SEQ ID NO: 21, and CDR-H3 containing the sequence shown in SEQ ID NO:

24.

5. The bispecific antibody according to any one of claims 1-4, wherein, The first VL contains an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; And / or, The first VH contains an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

6. The bispecific antibody according to any one of claims 1-5, wherein, The second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 8 or 67; and / or, the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 9 or 68; Preferably, the second antigen-binding domain comprises: (i) CDR-L1, CDR-L2 and CDR-L3 contained in VL as shown in SEQ ID NO: 8, and / or CDR-H1, CDR-H2 and CDR-H3 contained in VH as shown in SEQ ID NO: 9; (ii) CDR-L1, CDR-L2 and CDR-L3 contained in VL as shown in SEQ ID NO: 67, and / or CDR-H1, CDR-H2 and CDR-H3 contained in VH as shown in SEQ ID NO: 9; (iii) CDR-L1, CDR-L2, and CDR-L3 contained in VL as shown in SEQ ID NO: 67, and / or CDR-H1, CDR-H2, and CDR-H3 contained in VH as shown in SEQ ID NO: 68; or, (iv) CDR-L1, CDR-L2 and CDR-L3 contained in VL as shown in SEQ ID NO: 8, and / or CDR-H1, CDR-H2 and CDR-H3 contained in VH as shown in SEQ ID NO: 68; Preferably, the CDR is defined by the Chothia, AbM, Kabat, IMGT, MacCallum, or AHo numbering system.

7. The bispecific antibody according to any one of claims 1-6, wherein: (a) The second VL includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 25, CDR-L2 containing the sequence shown in SEQ ID NO: 27, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 26, CDR-L2 containing the sequence shown in SEQ ID NO: 28, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; And / or, The second VH includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 31, CDR-H2 containing the sequence shown in SEQ ID NO: 35, and CDR-H3 containing the sequence shown in SEQ ID NO: 38; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 33, CDR-H2 containing the sequence shown in SEQ ID NO: 37, and CDR-H3 containing the sequence shown in SEQ ID NO: 38; (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; or, (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 32, CDR-H2 comprising the sequence shown in SEQ ID NO: 36, and CDR-H3 comprising the sequence shown in SEQ ID NO:

39. or, (b) The second VL includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 69, CDR-L2 containing the sequence shown in SEQ ID NO: 71, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 70, CDR-L2 containing the sequence shown in SEQ ID NO: 72, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; And / or, The second VH includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 31, CDR-H2 containing the sequence shown in SEQ ID NO: 35, and CDR-H3 containing the sequence shown in SEQ ID NO: 38; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 33, CDR-H2 containing the sequence shown in SEQ ID NO: 37, and CDR-H3 containing the sequence shown in SEQ ID NO: 38; (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; or, (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 32, CDR-H2 comprising the sequence shown in SEQ ID NO: 36, and CDR-H3 comprising the sequence shown in SEQ ID NO:

39. or, (c) The second VL includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 69, CDR-L2 containing the sequence shown in SEQ ID NO: 71, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 70, CDR-L2 containing the sequence shown in SEQ ID NO: 72, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; And / or, The second VH includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 75, CDR-H2 containing the sequence shown in SEQ ID NO: 79, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 77, CDR-H2 containing the sequence shown in SEQ ID NO: 81, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (iii) CDR-H1 containing the sequence shown in SEQ ID NO: 74, CDR-H2 containing the sequence shown in SEQ ID NO: 78, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 76, CDR-H2 comprising the sequence shown in SEQ ID NO: 80, and CDR-H3 comprising the sequence shown in SEQ ID NO:

83. or (d) The second VL includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 25, CDR-L2 containing the sequence shown in SEQ ID NO: 27, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 26, CDR-L2 containing the sequence shown in SEQ ID NO: 28, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; And / or, The second VH includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 75, CDR-H2 containing the sequence shown in SEQ ID NO: 79, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 77, CDR-H2 containing the sequence shown in SEQ ID NO: 81, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (iii) CDR-H1 containing the sequence shown in SEQ ID NO: 74, CDR-H2 containing the sequence shown in SEQ ID NO: 78, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 76, CDR-H2 containing the sequence shown in SEQ ID NO: 80, and CDR-H3 containing the sequence shown in SEQ ID NO:

83.

8. The bispecific antibody according to any one of claims 1-7, wherein, (i) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; (ii) The second VL comprises the amino acid sequence shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or, the second VH comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or, (iii) The second VL comprises the amino acid sequence shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or, the second VH comprises the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or, (iv) The second VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

9. The bispecific antibody according to any one of claims 1-8, wherein, (i) The first VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 6, and the first VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO: 7; and the second VL includes CDR-L1, CDR-L2 and CDR-L3 contained in the VL shown in SEQ ID NO: 8, and the second VH includes CDR-H1, CDR-H2 and CDR-H3 contained in the VH shown in SEQ ID NO:

9. (ii) The first VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 6, and the first VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7; and the second VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 67, and the second VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:

9. (iii) The first VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 6, and the first VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7; and the second VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 67, and the second VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 68, or (iv) The first VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 6, and the first VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7; and the second VL includes CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 8, and the second VH includes CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO:

68. Preferably, the CDR is defined by the Chothia, AbM, Kabat, IMGT, MacCallum, or AHo numbering system.

10. The bispecific antibody according to any one of claims 1-9, wherein, (a) The first antigen-binding domain includes: The first VL, which includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 10, CDR-L2 containing the sequence shown in SEQ ID NO: 12, and CDR-L3 containing the sequence shown in SEQ ID NO: 14; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 11, CDR-L2 containing the sequence shown in SEQ ID NO: 13, and CDR-L3 containing the sequence shown in SEQ ID NO: 14; and, The first VH, which includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 16, CDR-H2 containing the sequence shown in SEQ ID NO: 20, and CDR-H3 containing the sequence shown in SEQ ID NO: 23; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 18, CDR-H2 containing the sequence shown in SEQ ID NO: 22, and CDR-H3 containing the sequence shown in SEQ ID NO: 23; (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 15, CDR-H2 comprising the sequence shown in SEQ ID NO: 19, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23; or, (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 17, CDR-H2 containing the sequence shown in SEQ ID NO: 21, and CDR-H3 containing the sequence shown in SEQ ID NO: 24; and (b) The second antigen-binding domain comprises: (b1) The second VL, which includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 25, CDR-L2 containing the sequence shown in SEQ ID NO: 27, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 26, CDR-L2 containing the sequence shown in SEQ ID NO: 28, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; and, The second VH, which includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 31, CDR-H2 containing the sequence shown in SEQ ID NO: 35, and CDR-H3 containing the sequence shown in SEQ ID NO: 38; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 33, CDR-H2 containing the sequence shown in SEQ ID NO: 37, and CDR-H3 containing the sequence shown in SEQ ID NO: 38; (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; or, (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 32, CDR-H2 containing the sequence shown in SEQ ID NO: 36, and CDR-H3 containing the sequence shown in SEQ ID NO: 39; or, (b2) The second VL, which includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 69, CDR-L2 containing the sequence shown in SEQ ID NO: 71, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 70, CDR-L2 containing the sequence shown in SEQ ID NO: 72, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; and, The second VH, which includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 31, CDR-H2 containing the sequence shown in SEQ ID NO: 35, and CDR-H3 containing the sequence shown in SEQ ID NO: 38; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 33, CDR-H2 containing the sequence shown in SEQ ID NO: 37, and CDR-H3 containing the sequence shown in SEQ ID NO: 38; (iii) CDR-H1 comprising the sequence shown in SEQ ID NO: 30, CDR-H2 comprising the sequence shown in SEQ ID NO: 34, and CDR-H3 comprising the sequence shown in SEQ ID NO: 38; or, (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 32, CDR-H2 comprising the sequence shown in SEQ ID NO: 36, and CDR-H3 comprising the sequence shown in SEQ ID NO:

39. or, (b3) The second VL, which includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 69, CDR-L2 containing the sequence shown in SEQ ID NO: 71, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 70, CDR-L2 containing the sequence shown in SEQ ID NO: 72, and CDR-L3 containing the sequence shown in SEQ ID NO: 73; and, The second VH, which includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 75, CDR-H2 containing the sequence shown in SEQ ID NO: 79, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 77, CDR-H2 containing the sequence shown in SEQ ID NO: 81, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (iii) CDR-H1 containing the sequence shown in SEQ ID NO: 74, CDR-H2 containing the sequence shown in SEQ ID NO: 78, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (iv) CDR-H1 comprising the sequence shown in SEQ ID NO: 76, CDR-H2 comprising the sequence shown in SEQ ID NO: 80, and CDR-H3 comprising the sequence shown in SEQ ID NO:

83. or (b4) The second VL, which includes: (i) CDR-L1 containing the sequence shown in SEQ ID NO: 25, CDR-L2 containing the sequence shown in SEQ ID NO: 27, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; or, (ii) CDR-L1 containing the sequence shown in SEQ ID NO: 26, CDR-L2 containing the sequence shown in SEQ ID NO: 28, and CDR-L3 containing the sequence shown in SEQ ID NO: 29; and, The second VH, which includes: (i) CDR-H1 containing the sequence shown in SEQ ID NO: 75, CDR-H2 containing the sequence shown in SEQ ID NO: 79, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (ii) CDR-H1 containing the sequence shown in SEQ ID NO: 77, CDR-H2 containing the sequence shown in SEQ ID NO: 81, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (iii) CDR-H1 containing the sequence shown in SEQ ID NO: 74, CDR-H2 containing the sequence shown in SEQ ID NO: 78, and CDR-H3 containing the sequence shown in SEQ ID NO: 82; (iv) CDR-H1 containing the sequence shown in SEQ ID NO: 76, CDR-H2 containing the sequence shown in SEQ ID NO: 80, and CDR-H3 containing the sequence shown in SEQ ID NO:

83.

11. The bispecific antibody according to any one of claims 1-10, wherein, (i) the first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and The second VL comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it. or, (ii) The first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or, the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and The second VL comprises the amino acid sequence shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it. or, (iii) The first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or, the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and The second VL comprises the amino acid sequence shown in SEQ ID NO: 67, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it. or (iv) The first VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or, the first VH comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and The second VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

12. The bispecific antibody 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 connected to the C-terminus or N-terminus of the heavy chain of the full-length antibody, either via a connector or not.

13. The bispecific antibody of claim 12, wherein, The first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), the second antigen-binding domain is an scFv, and the bispecific antibody comprises: (i) a peptide chain IA comprising 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; and (ii) A peptide chain IB comprising the VH of the first antigen-binding domain, the CH1 region of the heavy chain, an Fc domain monomer, and the second 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 bispecific antibody comprises two identical or different peptide chains IA and two identical or different peptide chains IB, wherein the two peptide chains IB form a dimer through their respective Fc domain monomers. Preferably, the second antigen-binding domain is connected to the N-terminus of the VH of the first antigen-binding domain, either through a connector or not; or, the second antigen-binding domain is connected to the C-terminus of the Fc domain monomer, either through a connector or not.

14. The bispecific antibody of claim 12, wherein, The second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), the first antigen-binding domain is an scFv, and the bispecific antibody comprises: (i) peptide chain IV-A, 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 (ii) Peptide chain IV-B, comprising VH of the second antigen-binding domain, CH1 region of the heavy chain, 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 bispecific antibody comprises two identical peptide chains IV-A and two identical peptide chains IV-B, wherein the two peptide chains IV-B form a dimer through their respective Fc domain monomers; Preferably, the first antigen-binding domain is connected to the N-terminus of the VH of the second antigen-binding domain via or without a connector; or, the first antigen-binding domain is connected to the C-terminus of the Fc domain monomer via or without a connector.

15. The bispecific antibody according to any one of claims 1-11, wherein, The first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the bispecific antibody comprises: (i) peptide chain II-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; and (ii) Peptide chain II-B, comprising a VH of the second antigen-binding domain, a VH of the first 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 bispecific antibody comprises two identical or different peptide chains II-A and two identical or different peptide chains II-B, wherein the two peptide chains II-B form a dimer through their respective Fc domain monomers.

16. The bispecific antibody of claim 15, wherein: The VL of the second antigen-binding domain is connected to the N-terminus of the VL of the first antigen-binding domain, either via a linker or not: and / or The VH of the second antigen-binding domain is connected to the N-terminus of the VH of the first antigen-binding domain, either through a connector or not.

17. The bispecific antibody according to any one of claims 1-11, wherein, 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, 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; and (ii) Peptide chain III-B, comprising the VH of the first antigen-binding domain, the VH of the second antigen-binding domain, the CH1 region of the heavy chain, 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 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.

18. The bispecific antibody of claim 17, wherein: The VL of the first antigen-binding domain is connected to the N-terminus of the VL of the second antigen-binding domain, either via a linker or not; and / or The VH of the first antigen-binding domain is connected to the N-terminus of the VH of the second antigen-binding domain, either through a connector or not.

19. The bispecific antibody according to any one of claims 12-14, 16 or 18, wherein, Each of the peptide linkers is independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or each of the peptide linkers is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example having a structure as shown in (GGGGS)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 40, 41 or 44-51); or each of the peptide linkers independently contains an amino acid sequence shown in any one of SEQ ID NO: 40, 41 or 44-52.

20. The bispecific antibody of claims 13-19, wherein the monomer of the Fc domain comprises a modification capable of altering effector function; Preferably, the modification that alters the effector function comprises the following mutations according to EU numbers: L234A / L235A / G237A; Preferably, the Fc domain monomer is as shown in sequence SEQ ID NO: 53 or 61.

21. The bispecific antibody according to any one of claims 1-20, wherein, The bispecific antibody comprises: (1) A peptide chain IA comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or a peptide chain IB comprising the amino acid sequence shown in SEQ ID NO: 2 or 5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; (2) A peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or a peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; (3) A peptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 62 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or a peptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 63 or 64 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or (4) A peptide chain III-A comprising the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or a peptide chain III-B comprising the amino acid sequence shown in SEQ ID NO: 66 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

22. The bispecific antibody according to any one of claims 1-21, wherein, The bispecific antibody is conjugated with at least one label; preferably, the label is selected from enzymes, fluorescent dyes, radioisotopes, biotin, and colloidal gold.

23. An antibody-drug conjugate comprising the bispecific antibody as described in any one of claims 1-21 and at least one therapeutic agent.

24. The antibody-drug conjugate of claim 23, wherein the therapeutic agent is a cytotoxic agent.

25. The antibody-drug conjugate of claim 24, comprising the structure shown below: Ab’-[M-L-E-D] x in: Ab' is the bispecific antibody according to any one of claims 1-21; M is the linker site that is connected to the bispecific antibody; L is a structural segment that connects the joint portions M and E; E is a structural segment connecting L and D; D is the cytotoxic agent or a fragment thereof; and x is any integer selected from 1 to 10.

26. The antibody-drug conjugate of claim 25, 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.

27. The antibody-drug conjugate of claim 25, 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.

28. The antibody-drug conjugate of claim 25, 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.

29. The antibody-drug conjugate of claim 25, wherein, M is selected from 30. The antibody-drug conjugate of claim 25, wherein, M is 31. The antibody-drug conjugate of claim 25, wherein, M is selected from 32. The antibody-drug conjugate of claim 25, wherein, M is selected from 33. The antibody-drug conjugate according to any one of claims 25-32, 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: 54)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 55)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 56)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 57)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 58))), 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.

34. The antibody-drug conjugate of claim 33, 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: 54)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 55)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 56)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 57)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 58)).

35. The antibody-drug conjugate according to any one of claims 25-32, 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: 54)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 55)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 56)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 57)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 58)), Where s is selected from integers from 1 to 20.

36. The antibody-drug conjugate according to any one of claims 25-32, wherein, The L is selected from one or more of the following structures:

37. The antibody-drug conjugate according to any one of claims 25-32, wherein, The L is selected from the following structures:

38. The antibody-drug conjugate according to any one of claims 25-32, wherein, The L is selected from the following structures:

39. The antibody-drug conjugate according to any one of claims 25-32, wherein, The L is selected from the following structures:

40. The antibody-drug conjugate according to any one of claims 25-32, wherein, The L is selected from the following structures:

41. The antibody-drug conjugate according to any one of claims 25-32, wherein, The L is selected from the following structures:

42. The antibody-drug conjugate according to any one of claims 25-41, wherein, E is a single bond or is selected from the following structures: -NHCH2-, -NHCH2-O-CH2-CO-, -CO-O-CH2-CO-, 43. The antibody-drug conjugate according to any one of claims 25-41, wherein, E represents a single bond, -NHCH2-, -NHCH2-O-CH2-CO-, 44. The antibody-drug conjugate according to any one of claims 25-41, wherein, E is -NHCH2- or 45. The antibody-drug conjugate according to any one of claims 25-41, wherein, E can be -NHCH2- or a single bond.

46. ​​The antibody-drug conjugate according to any one of claims 25-41, wherein, E is either -NHCH2- or -NHCH2-O-CH2-CO-.

47. The antibody-drug conjugate according to any one of claims 25-41, wherein, E is 48. The antibody-drug conjugate according to any one of claims 25-47, wherein, Selected from the following structures:

49. The antibody-drug conjugate according to any one of claims 25-47, wherein, Selected from the following structures:

50. The antibody-drug conjugate according to any one of claims 25-49, wherein, The cytotoxic drugs are selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

51. The antibody-drug conjugate of claim 50, wherein, The microtubule inhibitor is an olistatin or maytansine compound.

52. The antibody-drug conjugate of claim 51, wherein, The olistatin compounds are selected from the following:

53. The antibody-drug conjugate of claim 50, wherein, The DNA intercalating agent is pyrrolobenzodiazepine. (PBD) 54. The antibody-drug conjugate of claim 50, wherein, The DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.

55. The antibody-drug conjugate of claim 54, 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.

56. The antibody-drug conjugate of claim 50, wherein, The RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analogue thereof.

57. The antibody-drug conjugate according to any one of claims 25-49, 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, -CO-CH2OH, and R4 and R5 are each independently selected from H, halogen, hydroxyl, methyl, and amino; or R4 and R5 are connected to form a 5-6 membered oxygen-containing heterocycle; R6 is selected from hydrogen, C 2-6 alkenyl 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, -CO-C 1-6 Alkyl and -C 1-4 Alkylene-NR d R e Or, R a and R b The atoms connected to it may optionally be substituted with R f 5-6 membered nitrogen-containing heterocycles; among which, R d R e and R f Each time it appears, it is independently selected from H and C. 1-6 alkyl; Alternatively, R6 and R7, together with the carbon atoms they are attached to, form a 5-6 membered ring.

58. The antibody-drug conjugate according to any one of claims 25-49, 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 25.

59. The antibody-drug conjugate according to any one of claims 25-49, 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 25.

60. The antibody-drug conjugate of claim 58 or 59, wherein, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amine group on the cytotoxic drug.

61. The antibody-drug conjugate according to any one of claims 25-60, wherein, The antibody-drug conjugate is selected from: Wherein, Ab-(S-) is a bispecific antibody as defined in any one of claims 1-21; This indicates the specific linkage between the thiol group of the cysteine ​​residue in the bispecific antibody and the pyrimidin or succinimide group in the antibody-drug conjugate.

62. The antibody-drug conjugate of claim 61, wherein, The antibody-drug conjugate is obtained by forming a thioether bond between the thiol group in the bispecific antibody and the pyrimidin group or succinimide group in the antibody-drug conjugate through an addition reaction or a substitution reaction.

63. The antibody-drug conjugate of claim 61 or 62, wherein, The Ab' is a bispecific antibody selected from BsAb01, BsAb02, BsAb03, BsAb04, BsAb05, or BsAb06.

64. A composition comprising one or more antibody-drug conjugates as described in any one of claims 23-63.

65. The composition of claim 64, wherein, The composition has a DAR (drug-antibody conjugate ratio) value of 1 to 10.

66. The composition of claim 65, wherein, The DAR value of the composition is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10.

67. The composition of claim 65, wherein, The composition has a DAR value of 3 to 9.

68. The composition of claim 65, wherein, The composition has a DAR value of 4 to 8.

69. The composition of claim 65, wherein, The DAR values ​​of the compositions are 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 to 5.5, 3.5 to 6.0, 3.5 to 6.5, 3.5 to 7.0, 3.5 to 7.5, 3.5 to 8.0, 4.0 to 4.5, 4.0 to 5.0, 4.0 to 5.5, 4.0 to 6.0, 4.0 to 6.5, 4.0 to 7.0, 4.0 to 7.5, 4.0 to 8.0, 4.5 to 5.0, and 4.5 to 5. 5, 4.5 to 6.0, 4.5 to 6.5, 4.5 to 7.0, 4.5 to 7.5, 4.5 to 8.0, 5.0 to 5.5, 5.0 to 6.0, 5.0 to 6.5, 5.0 to 7.0, 5.0 to 7.5, 5.0 to 8.0, 5.5 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 5.5 to 7.5, 5.5 to 8.0, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.5, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.5, 7.0 to 7.5, 7.0 to 9.0 or 7.5 to 9.

0.

70. An isolated nucleic acid molecule or group of nucleic acid molecules comprising a nucleotide sequence encoding a bispecific antibody according to any one of claims 1-21.

71. A vector comprising the isolated nucleic acid molecule or nucleic acid molecule group as described in claim 70; Preferably, the vector comprises nucleotide sequences encoding each peptide chain of the bispecific antibody according to any one of claims 1-21, and the nucleotide sequences encoding each peptide chain are present on the same or different vectors.

72. A host cell comprising the isolated nucleic acid molecule or group of nucleic acid molecules as described in claim 70, or the vector as described in claim 71.

73. A method for preparing the bispecific antibody according to any one of claims 1-21, comprising culturing the host cell according to claim 72 under conditions that allow expression of the bispecific antibody, and recovering the bispecific antibody from the cultured host cell culture.

74. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1-21, or an isolated nucleic acid molecule or group of nucleic acid molecules according to claim 70, or a carrier according to claim 71, or a host cell according to claim 72, or an antibody-drug conjugate according to any one of claims 23-63, or a composition according to any one of claims 64-69, and a pharmaceutically acceptable pharmaceutical carrier and / or excipient.

75. The pharmaceutical composition of claim 74, 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.

76. Use of the bispecific antibody of any one of claims 1-22, or the isolated nucleic acid molecule or nucleic acid molecule group of claims 70, or the vector of claim 71, or the host cell of claim 72, or the antibody-drug conjugate of any one of claims 23-63, or the composition of any one of claims 64-69, or the pharmaceutical composition of claims 74 or 75 in the preparation of a product for use in the prevention and / or treatment and / or as an adjuvant for the treatment of diseases related to EGFR and / or B7H3 in subjects, and / or for use in vitro or in subjects to inhibit the activity of EGFR and / or B7H3, and / or for detection of the presence or level of EGFR and / or B7H3 in a sample, and / or for inhibition of cell proliferation.

77. The use as described in claim 76, wherein, The diseases associated with EGFR and / or B7H3 are tumors, and / or The cells are cells expressing EGFR and / or B7H3, further tumor cells expressing EGFR and / or B7H3, and even further tumor cells overexpressing EGFR and / or B7H3.

78. The use as described in claim 77, wherein, The tumor is B7H3 and / or EGFR positive; preferably, the tumor is lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma. Preferably, the cancer is lung cancer, pancreatic cancer, colorectal cancer, or melanoma; More preferably, the cancer is lung cancer, such as non-small cell lung cancer.

79. A method for preventing and / or treating and / or treating, as an adjunct therapy, a subject with a disease associated with EGFR and / or B7H3, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any one of claims 1-21, or an isolated nucleic acid molecule or group of nucleic acid molecules of claim 70, or a vector of claim 71, or a host cell of claim 72, or an antibody-drug conjugate of any one of claims 23-63, or a composition of any one of claims 64-69, or a pharmaceutical composition of claim 74 or 75.

80. The method of claim 79, wherein, The diseases associated with EGFR and / or B7H3 are tumors.

81. The method of claim 80, wherein, The tumor is B7H3 and / or EGFR positive; preferably, the tumor is lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymphoma, pancreatic cancer, head and neck cancer, and Ewing's sarcoma; preferably, the cancer is lung cancer, prostate cancer, breast cancer, ovarian cancer, or melanoma. Preferably, the cancer is lung cancer, pancreatic cancer, colorectal cancer, or melanoma; More preferably, the cancer is lung cancer, such as non-small cell lung cancer.

82. A diagnostic or therapeutic kit comprising a bispecific antibody of any one of claims 1-22, or an isolated nucleic acid molecule or nucleic acid molecule group of claim 70, or a vector of claim 71, or a host cell of claim 72, or an antibody-drug conjugate of any one of claims 23-63, or a composition of any one of claims 64-69, or a pharmaceutical composition of claim 74 or 75, and optionally instructions for use and / or a delivery device.

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