Antibody-drug conjugate, pharmaceutical composition thereof, and use thereof

Abstract

Provided are an antibody-drug conjugate, a pharmaceutical composition thereof, and use thereof. Specifically, provided are an antibody-drug conjugate represented by formula (I), a drug linker for preparing the antibody-drug conjugate, and use of the antibody-drug conjugate in anti-tumor.

Description

Antibody-drug conjugates, pharmaceutical compositions and applications thereof

[0001] This application is based on the application with CN application number 202311871776.6 and application date December 29, 2023, and the application with CN application number 202411292297.3 and application date September 14, 2024, and claims their priority. The disclosed contents of these CN applications are hereby introduced into this application as a whole. Technical Field

[0002] The present invention relates to the field of medicine, and specifically relates to a class of antibody-drug conjugates, their pharmaceutical compositions and applications. Background Art

[0003] Antibody-drug conjugates (ADCs) couple antibodies and small molecule cytotoxic drugs through specific linkers. Antibodies act as carriers to transport small molecule drugs into target cells, thereby reducing systemic exposure and improving safety. ADCs are a research and development hotspot for targeted tumor therapy.

[0004] With the advancement of ADC technology, immunostimulating antibody conjugates (ISACs) have been developed. ISACs conjugate immunostimulatory agents or immunomodulatory agents, such as TLR7 and / or TLR8 agonists, to antibodies via cleavable or non-cleavable linkers. These conjugates combine the precise targeting of antibodies with the immune activation and long-lasting immune memory of immunostimulatory agents, achieving the safety of systemic administration and eliciting anti-tumor immune responses.

[0005] In anti-cancer research, a clear trend is shifting from monotherapy to combination therapy. From a clinical perspective, an ideal combination therapy can enhance clinical efficacy, reduce drug toxicity, and delay or prevent the development of drug resistance. Combination therapy involves combining two existing drugs, such as a cytotoxic drug with an immunostimulant, or combining two active ingredients to create a new single drug.

[0006] By leveraging the targeting properties of antibodies, bifunctional immunostimulatory antibody drug conjugates (iADCs) obtained by conjugating cytotoxic drugs and immunostimulatory agents to antibodies can simultaneously enter tumor cells and immune cells in the tumor microenvironment, exerting the dual effects of tumor killing by cytotoxic drugs and local immune activation by immunostimulatory agents, thereby improving the efficacy of antibody drug conjugates in treating cancer.

[0007] B7H3, also known as CD276, is an immune checkpoint molecule and co-stimulatory / co-inhibitory immunomodulatory protein that plays a dual role in the immune system. B7H3 is a type I transmembrane protein of the B7 family and has two isoforms: 2Ig-B7H3 and 4Ig-B7H3. In humans, the membrane-bound form of 4Ig-B7H3 is predominant. B7H3 is abnormally overexpressed in a variety of solid tumors, including head and neck cancer, kidney cancer, lung cancer, breast cancer, pancreatic cancer, and melanoma. It is expressed at lower levels in normal tissues, with low-to-moderate expression only detected in tissues such as the pancreas, liver, colon, and skin. B7H3 participates in the biological processes of multiple cancer development, including promoting tumor proliferation / migration, epithelial-mesenchymal transition, tumor angiogenesis, and treatment resistance, leading to poor patient prognosis. Therefore, B7H3 has become a promising therapeutic target for anti-cancer treatment. Summary of the Invention

[0008] The present application relates to an antibody-drug conjugate for treating cancers associated with B7H3 expression (e.g., B7H3-positive cancers). The conjugate is a bifunctional immunostimulatory antibody-drug conjugate obtained by conjugating a cytotoxic drug and a TLR agonist to an antibody. The conjugate can simultaneously enter tumor cells and immune cells in the tumor microenvironment, exerting the dual effects of tumor killing by the cytotoxic drug and local immune activation by the TLR agonist. Compared with antibody-drug conjugates conjugated with a cytotoxic drug alone, the conjugate has an improved cancer treatment effect.

[0009] In one aspect, the present invention provides an antibody-drug conjugate as shown in formula (I):

[0010] in,

[0011] Ab' is an antibody or antigen-binding fragment thereof that specifically binds to B7H3;

[0012] M is a linker site connected to the antibody or antigen-binding fragment thereof;

[0013] X is a linker connecting M and Aa;

[0014] Aa is selected from the group consisting of amino acid fragments and peptide fragments formed from two or more amino acids;

[0015] L 1 To connect Aa and D 1 The linker;

[0016] L 2 To connect Aa and D 2 The linker;

[0017] D 1 It is the cytotoxic drug part;

[0018] D2 is the TLR agonist portion;

[0019] m is selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0020] In another aspect, the present invention provides a composition of an antibody drug conjugate, comprising the above-mentioned antibody drug conjugate, wherein the DAR value of the composition is 1.0-10.0, preferably 1.0-5.0, for example, 1.0-1.5, 1.0-2.0, 1.0-2.5, 1.0-3.0, 1.0-3.5, 1.0-4.0, 1.0-4.5, 1.0-5.0, 2.0-2.5, 2.0-3.0 , 2.0~3.5,2.0~4.0,2.0~4.5,2.0~5.0,2.5~3.0,2.5~3.5,2.5~4.0,2.5~4.5,2.5~5.0,3.0~3.5,3.0~4.0,3.0~4.5,3.0~5.0,3.5~4.0,3.5~4.5,3.5~5.0,4.0~4.5,4.0~5.0,4.5~5.0.

[0021] In another aspect, the present invention provides a compound as shown in formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0022] in,

[0023] X, Aa, L 1 、L 2 、D 1 and D 2 As defined in any of the preceding clauses,

[0024] M 1 A precursor of a linker site for connection to an antibody or antigen-binding fragment thereof;

[0025] Preferably, M 1 Selected from the following structures:

[0026] More preferably, M 1 Selected from the following structures:

[0027] More preferably, M 1 Selected from the following structures:

[0028] wherein each a is independently selected from an integer of 0-6, and b is selected from an integer of 1-10;

[0029] LG represents a leaving group, preferably, LG is independently selected from halogen (such as F, Cl, Br, I), halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halogenated phenoxy, hydroxyl, thiol, amino, nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments, the halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 The alkylsulfoxide group, halophenoxy group, alkenyl group, alkynyl group and alkynyl group-containing structural fragments are optionally substituted with one or more suitable substituents;

[0030] Preferably, LG are each independently selected from halogen (such as F, Cl, Br, I), halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkylsulfoxide, halophenoxy, hydroxyl, sulfhydryl, amino, nitro, azide, cyano, alkenyl, alkynyl, and structural fragments containing alkynyl;

[0031] More preferably, LG is C 1-6 Alkylsulfonyl is, for example, methylsulfonyl.

[0032] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to B7H3, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) shown in SEQ ID NO: 23 or 1 or a variant thereof, and / or a light chain variable region (VL) shown in SEQ ID NO: 2 or a variant thereof.

[0033] In another aspect, the present invention provides a pharmaceutical composition comprising any of the above-described antibody-drug conjugates, compounds, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites or prodrugs, combinations of antibody-drug conjugates, antibodies or antigen-binding fragments thereof, and one or more pharmaceutical excipients.

[0034] In another aspect, the present invention provides use of any of the above compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites or prodrugs, antibodies or antigen-binding fragments thereof in the preparation of antibody-drug conjugates, in particular for the preparation of any of the above antibody-drug conjugates.

[0035] In another aspect, the present invention provides use of any of the above-described antibody-drug conjugates, compounds, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites or prodrugs, compositions of antibody-drug conjugates, antibodies or antigen-binding fragments thereof, or pharmaceutical compositions in the preparation of a medicament, in particular, in the preparation of a medicament for treating and / or preventing cancer (e.g., cancers associated with B7H3 expression, such as B7H3-positive cancers).

[0036] In another aspect, the present invention provides a method for treating and / or preventing cancer (e.g., cancer associated with B7H3 expression, e.g., B7H3-positive cancer), comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of any of the foregoing antibody-drug conjugates, compounds (e.g., drug linkers, TLR agonists, or cytotoxic drugs), or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites, or prodrugs, antibody-drug conjugate compositions, antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions.

[0037] In another aspect, the present invention also provides an intermediate for preparing any of the above-mentioned antibody drug conjugates, compounds, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites or prodrugs. DETAILED DESCRIPTION

[0038] Definition of terms

[0039] Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the techniques used herein is intended to refer to techniques generally understood in the art, including variations of those techniques that are obvious to those skilled in the art or replacements with equivalent techniques. Furthermore, laboratory procedures such as genomics, nucleic acid chemistry, and molecular biology used herein are conventional procedures widely used in the corresponding fields. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0040] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing 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 is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, 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 from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

[0041] The term "antibody" also includes embodiments in which the heavy chain constant region comprises 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 to a pyroglutamate. Thus, in a composition comprising the antibodies disclosed herein, each antibody therein may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid, or have the N-terminal amino acid cyclized to a pyroglutamate.

[0042] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0043] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, such as those defined by the Kabat, Chothia, IMGT, or AbM numbering systems. In certain embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

[0044] The following general rules (published at www.bioinf.org.uk: Professor Andrew CR Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with amino acids that make up the epitope to which the antibody binds. In rare cases, these generally constant features do not appear; however, Cys residues are the most conserved feature.

[0045] V H The entire amino acid sequence of a V is generally numbered according to Kabat, and the three CDRs within the variable region may be defined according to any of the above numbering systems. H The amino acid positions in the sequence may be numbered sequentially starting from amino acid position 1 to the end of the sequence, or according to Kabat numbering. H and V L The amino acid positions in are defined according to sequential numbering.

[0046] Amino acid positions in the heavy chain constant region can be numbered sequentially starting from amino acid position 1 and continuing to the end of the sequence, or 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 begins at position 118 and ends at position 447. Unless otherwise indicated, amino acid positions in the heavy and light chains described herein are defined according to sequential numbering.

[0047] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0048] The term "antigen-binding fragment" of an antibody refers to polypeptides that are fragments of an antibody, such as polypeptides that are fragments of a full-length antibody, which retain the ability to specifically bind to the same antigen bound by the full-length antibody and / or compete with the full-length antibody for specific binding to the antigen, and are also referred to as "antigen-binding portions." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0049] The term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0050] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0051] The term "Fc" refers to an antibody fragment formed by 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 is not involved in antigen binding.

[0052] The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 25) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 26) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, a VH-VH-COOH domain comprising NH2-VH-VH-COOH, NH 2- VL-VL-COOH scFv.

[0053] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0054] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured in terms of the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC). 50 )express.

[0055] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived. In some embodiments, the variants of the invention have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which they are derived, or have one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which they are derived; preferably, the substitutions are conservative substitutions.

[0056] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0057] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0058] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0059] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0060] The term "murine antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for murine hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells in mice after antigen invasion.

[0061] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0062] The twenty conventional amino acids referred to herein are denoted according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0063] The terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0064] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched saturated hydrocarbon group, for example, "C 1-20 Alkyl", "C 1-10 Alkyl", "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 alkyl”, 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.

[0065] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight-chain or branched saturated hydrocarbon group, for example, "C 1-20Alkylene", "C 1-10 Alkylene", "C 3-10 Alkylene", "C 5-8 Alkylene", "C 1-6 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.

[0066] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocyclic alkyl and bicyclic alkyl (such as spirocyclic alkyl, bicyclic alkyl and bridged cycloalkyl). 3-10 The term "cycloalkyl" refers to a cycloalkyl group having 3 to 10 ring carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0067] The term "heterocyclyl" 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, 3-12 membered heterocyclyl, 3-8 membered heterocyclyl, 5-6 membered heterocyclyl, and the like, such as tetrahydrofuranyl, pyrrolidinyl, piperidinyl, tetrahydropyranyl, and the like.

[0068] The term "heteroaryl" 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, 5-10 membered heteroaryl, 5-6 membered heteroaryl, and the like, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, and the like.

[0069] The term "aryl" refers to a group obtained by removing a hydrogen atom from the aromatic carbon atom of an aromatic hydrocarbon molecule. 6-10 Specific examples of aryl include, but are not limited to, phenyl, naphthyl, anthracenyl, and the like.

[0070] As used herein, the term "fragment" or "structural fragment" refers to the portion of a compound molecule remaining after losing one or more atoms or groups of atoms. For example, a "cytotoxic drug fragment" refers to the portion of the cytotoxic drug remaining after the cytotoxic drug described herein loses a hydrogen atom or hydroxyl group and is linked to the linker in the antibody-drug conjugate, also referred to as a "cytotoxic drug portion". In some specific embodiments, the cytotoxic drug fragment / portion is D 1Similarly, "TLR agonist fragment" means the remaining portion of the TLR agonist obtained by losing one or more atoms or groups of atoms from the TLR agonist described herein and connecting to the linker in the antibody drug conjugate, also referred to as "TLR agonist portion". In some specific embodiments, the TLR agonist fragment / portion is represented by D 2 express.

[0071] If a substituent or value is described as being "independently selected" from a group or value, each substituent or value is selected independently of the other. Thus, each substituent or value may be the same as or different from another (other) substituent or value.

[0072] The present invention also includes all pharmaceutically acceptable isotopically labeled compounds which are identical to the compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of suitable isotopes for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H. 3 H, deuterium D, tritium T); carbon isotopes (such as 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 35 S). Certain isotopically labeled compounds of the invention (e.g., those incorporating radioactive isotopes) are useful in drug and / or substrate tissue distribution studies (e.g., assays). The radioactive isotope tritium (i.e., 3 H) and carbon-14 (i.e. 14 C) are particularly useful for this purpose because they are easy to incorporate and easy to detect. 11 C. 18 F. 15 O and 13N) substitution can be used to examine substrate receptor occupancy in positron emission tomography (PET) studies. Isotopically labeled compounds of the present invention can be prepared by methods analogous to those described in the accompanying schemes and / or examples and preparations by using appropriate isotopically labeled reagents instead of the non-labeled reagents previously employed. Pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent is isotopically substituted, for example, D2O, acetone-d6 or DMSO-d6.

[0073] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., 1, 2, 3, or 4) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0074] As used herein, the term "suitable substituent" refers to modifications that can be made to a compound by one skilled in the art according to the needs of the compound substituent. "Suitable substituents" include oxo (=O), halogen, cyano, NR 8 R 9 , carboxyl, thiol, hydroxyl, ester (e.g. -C 1-6 Alkyl-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10 Aryl, benzyl, hydroxy substituted benzyl, indolylmethylene and C 1-6 Haloalkoxy, R 8 、R 9 Each independently selected from H, C 1-6 Alkyl, C 3-6Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Haloalkoxy, halogen, hydroxy, carboxyl and ester groups (e.g. -C 1-6 Alkyl-C(=O)-OC 1-6 alkyl).

[0075] Whether explicitly stated or not, the numerical values ​​in this application are modified by the term “about.” The term “about” means within ±20%, ±10%, ±5%, or ±2% of the numerical value.

[0076] The ranges provided herein are to be understood as shorthand for all values ​​within that range. For example, a range of "1-10" is to be understood to include any number, combination of numbers, or subrange selected from that range. Specifically, "an integer selected from 1-10" refers to an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and "an integer selected from 0-6" refers to an integer selected from 0, 1, 2, 3, 4, 5, or 6.

[0077] The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

[0078] It should also be understood that certain compounds of the present invention may be used therapeutically in free form or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites, or prodrugs that, upon administration to a patient in need thereof, are capable of directly or indirectly providing a compound of the present invention or a metabolite or residue thereof. Therefore, when reference is made herein to a "compound of the present invention," such various derivative forms of the compound are also intended to be encompassed.

[0079] Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof. Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts. For a review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0080] The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the crystal lattice of the compound. The amount of the polar solvent, especially water, may be present in a stoichiometric or non-stoichiometric ratio.

[0081] Those skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides, as nitrogen requires an available lone pair of electrons to oxidize to an oxide; those skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include oxidation of heterocycles and tertiary amines with peroxyacids such as peracetic acid and meta-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate, and dioxirane such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see for example: TL Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp 748-750; AR Katritzky and AJ Boulton, Eds., Academic Press; and GWH Cheeseman and ESGWerstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392, AR Katritzky and AJ Boulton, Eds., Academic Press.

[0082] Also included within the scope of the present invention are metabolites of the compounds of the invention, i.e., substances formed in vivo upon administration of the compounds of the invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Thus, the present invention includes metabolites of the compounds of the invention, including compounds produced by contacting a compound of the invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0083] The present invention further includes within its scope prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have little or no pharmacological activity, and when administered to the body or thereon, can be converted into the compounds of the present invention having the desired activity by, for example, hydrolytic cleavage. Typically, such prodrugs will be functional group derivatives of the compounds that are readily converted into the desired therapeutically active compounds in vivo. Further information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems," Volume 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 (E.B. Roche, ed., American Pharmaceutical Association). The prodrugs of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present invention with certain moieties known to those skilled in the art as "pro-moieties" (e.g., as described in "Design of Prodrugs," H. Bundgaard (Elsevier, 1985)).

[0084] The term "about" means within ±10%, preferably within ±5%, and more preferably within ±2% of the stated numerical value.

[0085] In this article, the antibody drug conjugates of the present invention When the targeting moiety is an antibody or antigen-binding fragment, it indicates the specific connection method between the thiol group in the antibody or antigen-binding fragment and the drug linker.

[0086] In this article, the antibody drug conjugates of the present invention When the targeting moiety is an antibody or antigen-binding fragment, it indicates the specific connection method between the amino group in the antibody or antigen-binding fragment and the drug linker.

[0087] Antibody drug conjugates are also characterized by the average loading of the drug moiety (e.g., cytotoxic drugs and TLR agonists) to the antibody binding moiety, often referred to as the drug-to-antibody ratio (DAR) of the conjugated sample. For example, the average DAR of an ADC can be calculated from LC-MS data of reduced and deglycosylated samples, based on the average loading of the LC and HC chains. The DAR of a given antibody drug conjugate sample represents the average number of drug (payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains.

[0088] While the drug-to-antibody ratio has an exact value for a particular antibody drug conjugate molecule (e.g., m in Formula (I)), it will be understood that when used to describe a sample containing many molecules, this value will often be an average value due to some degree of heterogeneity typically associated with the conjugation step. The average loading of an antibody drug conjugate sample is referred to herein as the drug-to-antibody ratio or "DAR."

[0089] In some embodiments, the structure of the antibody-drug conjugate of the present application can be represented by Formula I, where m refers to the number of small molecule drug fragments attached to each antibody molecule. During the preparation of the antibody-drug conjugate, each antibody molecule may be attached to a different number of small molecule drug fragments. Therefore, in general, the composition of the antibody-drug conjugate is a mixture of antibody-drug conjugates with different drug-antibody conjugation ratios. In practice, DAR is usually used to represent the average number of drugs attached to the antibody.

[0090] In some embodiments, the DAR value (drug / antibody ratio of the conjugated sample) of the antibody drug conjugate composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8 ~9, 8~10, or 9~10, preferably 3~8, for example, 3.0~3.5, 3.0~4.0, 3.0~4.5, 3.0~5.0, 3.0~5.5, 3.0~6.0, 3.5~4.0, 3.5~4.5, 3.5~5.0, 3.5~5.5, 3.5~6.0, 3.5~6.5, 3.5~7.0, 3.5~7.5, 3.5~8.0, 4.0~4.5, 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4. 5 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 8.0, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.0, 7.0 to 7.5, 7.0 to 8.0 or 7.5 to 8.0, or preferably 1.0 to 5.0, for example, 1.0 to 1 .5,1.0~2.0,1.0~2.5,1.0~3.0,1.0~3.5,1.0~4.0,1.0~4.5,1.0~5.0,2.0~2.5,2.0~3.0,2.0~3.5,2.0~4.0,2.0~4.5,2.0~5.0,2.5~3.0,2.5~3.5,2.5~4.0,2.5~4.5,2.5~5.0,3.0~3.5,3.0~4.0,3.0~4.5,3.0~5.0,3.5~4.0,3.5~4.5,3.5~5.0,4.0~4.5,4.0~5.0,4.5~5.0.

[0091] In the present invention, "treating" generally refers to the partial or complete stabilization or cure of a disease and / or side effects resulting from the disease. As used herein, "treating" encompasses any treatment of a patient's disease that: (a) suppresses the symptoms of the disease, i.e., arrests its progression; or (b) alleviates the symptoms of the disease, i.e., causes regression of the disease or its symptoms.

[0092] In the present invention, "prevention" refers to the inhibition and delay of the onset of a disease, and includes not only prevention before the development of the disease but also prevention of recurrence of the disease after treatment.

[0093] In the present invention, "subject" refers to a vertebrate. In some embodiments, the vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, the mammal refers to a human.

[0094] In the present invention, an "effective amount" refers to an amount that is effective to achieve the desired therapeutic effect at the necessary dosage and time. The "therapeutically effective amount" may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the active ingredient to elicit the desired response in the individual. A therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the active ingredient outweigh any toxic or deleterious consequences. In cancer treatment, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor volume; inhibit (i.e., slow down to a certain extent, preferably stop) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent.

[0095] Antibody Drug Conjugates

[0096] One aspect of the present invention provides an antibody drug conjugate as shown in formula (I):

[0097] in,

[0098] Ab' is an antibody or antigen-binding fragment thereof that specifically binds to B7H3;

[0099] M is a linker site connected to the antibody or antigen-binding fragment thereof;

[0100] X is a linker connecting M and Aa;

[0101] Aa is selected from the group consisting of amino acid fragments and peptide fragments formed from two or more amino acids;

[0102] L 1 To connect Aa and D 1 The linker;

[0103] L2 To connect Aa and D 2 The linker;

[0104] D 1 It is the cytotoxic drug part;

[0105] D 2 is the TLR agonist portion;

[0106] m is selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0107] In some embodiments, m is 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-16, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10.

[0108] In some embodiments, m is an integer.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0110] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0111] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof;

[0112] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0113] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 14 or a variant thereof, CDR-H2 of SEQ ID NO: 15 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof;

[0114] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0115] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 8 or a variant thereof, CDR-H2 of SEQ ID NO: 9 or a variant thereof, and CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 3 or a variant thereof, CDR-L2 of SEQ ID NO: 4 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof; or

[0116] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0117] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 16 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 5 or a variant thereof.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0119] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0120] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof;

[0121] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0122] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 14 or a variant thereof, CDR-H2 of SEQ ID NO: 15 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof;

[0123] (3) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0124] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 8 or a variant thereof, CDR-H2 of SEQ ID NO: 9 or a variant thereof, and CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 3 or a variant thereof, CDR-L2 of SEQ ID NO: 4 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof; or

[0125] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0126] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 16 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 5 or a variant thereof.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof comprises: VH or a variant thereof shown in SEQ ID NO: 23, and / or VL or a variant thereof shown in SEQ ID NO: 2; preferably, the antibody or antigen-binding fragment thereof comprises: VH or a variant thereof shown in SEQ ID NO: 23 and a heavy chain of a heavy chain constant region (CH) or a variant thereof shown in SEQ ID NO: 18, and / or VL or a variant thereof shown in SEQ ID NO: 2 and a light chain of a light chain constant region (CL) or a variant thereof shown in SEQ ID NO: 19; preferably, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 22, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 21.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof comprises: VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; preferably, the antibody or antigen-binding fragment thereof comprises: VH or a variant thereof shown in SEQ ID NO: 1 and a heavy chain of a heavy chain constant region (CH) or a variant thereof shown in SEQ ID NO: 18, and / or VL or a variant thereof shown in SEQ ID NO: 2 and a light chain of a light chain constant region (CL) or a variant thereof shown in SEQ ID NO: 19; preferably, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 21.

[0129] In some embodiments, the N-terminal glutamine of the heavy chain or heavy chain variable region and / or the light chain or light chain variable region undergoes cyclization to form pyroglutamate or pyroglutamate; and / or, the heavy chain or heavy chain constant region (CH) or variant thereof does not have a C-terminal lysine.

[0130] In some embodiments, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0131] In some embodiments, Formula (I) is further wherein Ab-(Y-) represents the antibody or antigen-binding fragment thereof as described above, and -(Y-) represents the connecting portion between the amino acid residues in the antibody or antigen-binding fragment thereof and M.

[0132] In some embodiments, the amino acid residue is a cysteine, lysine, serine, or threonine residue.

[0133] In some embodiments, Y is S, NH, or O.

[0134] In some embodiments, M is a covalent bond or is selected from the following structures:

[0135] wherein each a is independently selected from an integer of 0-6, b is selected from an integer of 1-10, and the 1 position of M is connected to Ab', and the 2 position is connected to X.

[0136] In some embodiments, each a is independently selected from 0, 1, 2, 3, 4, 5, and 6. In some embodiments, each a is independently selected from 1, 2, 3, 4, 5, and 6. In some embodiments, each a is independently selected from 2, 3, 4, 5, and 6.

[0137] In some embodiments, b is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0138] In some embodiments, M is a covalent bond or is selected from the following structures:

[0139] The 1 position of M is connected to Ab', and the 2 position is connected to X.

[0140] In some embodiments, X is a covalent bond or is selected from C 1-6 Alkylene, -NH-(CH2) c -C(O)-,

[0141] wherein each c is independently selected from an integer of 0-6, each d is independently selected from an integer of 1-10; the 3-position of X is connected to M, and the 4-position is connected to Aa.

[0142] In some embodiments, each c is independently selected from 0, 1, 2, 3, 4, 5, and 6. In some embodiments, each c is independently selected from 1, 2, 3, 4, 5, and 6. In some embodiments, c is 2.

[0143] In some embodiments, each d is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0144] In some embodiments, X is a covalent bond or Among them, the 3-position of X is connected to M, and the 4-position is connected to Aa.

[0145] In some embodiments, X is a covalent bond.

[0146] In some embodiments, X is Among them, the 3-position of X is connected to M, and the 4-position is connected to Aa.

[0147] In some embodiments, Aa is selected from the group consisting of fragments of amino acids: Gly, Phe, Ala, Val, Cys, Asp, Glu, Lys, Nle, and Arg.

[0148] In some embodiments, Aa is selected from the group consisting of fragments of the following amino acids: Cys, Asp, Glu, Lys, and Nle.

[0149] In some embodiments, Aa is selected from the following structures:

[0150] Among them, the 5 position of Aa is connected to X, and the 6 position is connected to L 1 Connected, 7 position and L 2 connected.

[0151] In some embodiments, L 1 It is a covalent bond or is selected from a non-cleavable linker and a cleavable linker, wherein the cleavable linker is cleaved by an enzyme present in a pathological environment, wherein the enzyme is selected from a protease, a phosphatase, a pyrophosphatase, a β-glucuronidase, a β-galactosidase and a sulfatase.

[0152] In some embodiments, L 1 -L a -L b -L c -,in:

[0153] L a is a covalent bond or selected from C 1-6Alkylene, -NH-(CH2) e -C(O)-, wherein each e is independently selected from an integer of 0-6, and each f is independently selected from an integer of 1-10;

[0154] L b is a covalent bond or is selected from an amino acid fragment and a peptide fragment formed by two or more amino acids, wherein the amino acid is selected from Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn;

[0155] L c is a covalent bond or is selected from -NH-CH2- and the following structures:

[0156] In some embodiments, L a is a covalent bond or selected from C 1-6 Alkylene, -NH-(CH2) e -C(O)-,

[0157] In some embodiments, L a Selected from covalent bonds,

[0158] In some embodiments, L a It is a covalent bond.

[0159] In some embodiments, each e is independently selected from 0, 1, 2, 3, 4, 5, and 6. In some embodiments, each e is independently selected from 1, 2, 3, 4, 5, and 6. In some embodiments, e is 2.

[0160] In some embodiments, each f is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0161] In some embodiments, L b Selected from Val-Cit, Ala-Ala-Ala, Val-Lys-Gly and Gly-Gly-Phe-Gly (SEQ ID NO: 24).

[0162] In some embodiments, L b is a covalent bond or is selected from the following structures:

[0163] In some embodiments, L c -NH-CH2- or

[0164] In some embodiments, L 1 is a covalent bond or is selected from the following structures:

[0165] wherein each e is independently selected from an integer of 0-6, each f is independently selected from an integer of 1-10; L 1 The 8th position is connected to Aa, and the 9th position is connected to D 1 connected.

[0166] In some embodiments, L 1 for:

[0167] Among them, L 1 The 8th position is connected to Aa, and the 9th position is connected to D 1 connected.

[0168] In some embodiments, L 2 The invention relates to a method for preparing a cleavable linker selected from a non-cleavable linker and a cleavable linker, wherein the cleavable linker is cleaved by an enzyme present in a pathological environment, the enzyme being selected from a protease, a phosphatase, a pyrophosphatase, a β-glucuronidase, a β-galactosidase and a sulfatase.

[0169] In some embodiments, L 2 Selected from the following structures:

[0170] wherein each g is independently selected from an integer of 0-6, each h is independently selected from an integer of 1-10; L 2 The 10th position is connected to Aa, and the 11th position is connected to D 2 connected.

[0171] In some embodiments, each g is independently selected from 0, 1, 2, 3, 4, 5, and 6. In some embodiments, each g is independently selected from 1, 2, 3, 4, 5, and 6. In some embodiments, g is 2.

[0172] In some embodiments, each h is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0173] In some embodiments, L 2 Selected from the following structures:

[0174] Among them, L 2 The 10th position is connected to Aa, and the 11th position is connected to D 2 connected.

[0175] In some embodiments, D 1Selected from cytotoxic drug parts. In some embodiments, the cytotoxic drugs include but are not limited to tubulin inhibitors, DNA damaging agents and topoisomerase inhibitors. In some embodiments, the tubulin inhibitors include but are not limited to dolastatin and auristatin, maytansine, tubulysins and cryptomycins. In some embodiments, the tubulin inhibitor is selected from dolastatin 10, MMAE, MMAF, maytansine, DM1, DM3 and DM4. In some embodiments, the topoisomerase inhibitors include but are not limited to camptothecin and its derivatives, epipodophyllotoxins, anthracyclines and anthraquinones. In some embodiments, the topoisomerase inhibitor is selected from camptothecin, SN-38, exitecan, topotecan, belotecan, rubitecan, diflutecan, 10-hydroxycamptothecin, 9-aminocamptothecin, Dxd, T030, doxorubicin, epirubicin and PNU-159682. In some embodiments, the DNA damaging agent includes but is not limited to pyrrolobenzodiazepine (Pyrrolobenzodiazepine), duocarmycin and calicheamicin.

[0176] In some embodiments, D 1 Selected from the following structures:

[0177] In some embodiments, D 2 is a TLR agonist moiety, wherein the TLR agonist is selected from a TLR2 agonist, a TLR4 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist, and a TLR9 agonist. In some embodiments, the TLR agonist is selected from a TLR7 agonist, a TLR8 agonist, and a TLR7 / 8 agonist.

[0178] In some embodiments, D 2 Selected from the following structures:

[0179] In some embodiments, Selected from:

[0180] (For example ),

[0181] Wherein, e is selected from an integer of 0-6, f is selected from an integer of 1-10; the 5th position is connected to X, the 7th position is connected to L 2 Connected, 9 position and D 1 connected.

[0182] In some embodiments, Selected from:

[0183] wherein each g is independently selected from an integer of 0-6, each h is independently selected from an integer of 1-10; the 5 position is connected to X, the 6 position is connected to L 1 Connected, 11 position and D 2 connected.

[0184] In some embodiments, Selected from:

[0185] Among them, position 5 is connected to X, position 6 is connected to L 1 Connected, 11 position and D 2 connected.

[0186] In some embodiments, Selected from:

[0187] wherein each g is independently selected from an integer of 0-6, each h is independently selected from an integer of 1-10; the 5 position is connected to X, the 9 position is connected to D 1 Connected, 11 position and D 2 connected.

[0188] In some embodiments, Selected from:

[0189] The 5 position is connected to X, and the 9 position is connected to D 1 Connected, 11 position and D 2 connected.

[0190] In some embodiments, Selected from:

[0191] (For example ),

[0192] wherein each e is independently selected from an integer of 0-6, and each f is independently selected from an integer of 1-10.

[0193] In some embodiments, Selected from:

[0194] wherein each g is independently selected from an integer of 0-6, and each h is independently selected from an integer of 1-10.

[0195] In some embodiments, Selected from:

[0196] In some embodiments, Selected from:

[0197] wherein each a is independently selected from an integer of 0-6, each c is independently selected from an integer of 0-6, and each d is independently selected from an integer of 1-10; the 1 position is connected to Ab', the 6 position is connected to L 1 Connected, 7 position and L 2 connected.

[0198] In some embodiments, Selected from:

[0199] Among them, position 1 is connected to Ab', position 6 is connected to L 1 Connected, 7 position and L 2 connected.

[0200] In some embodiments, Selected from:

[0201] wherein each a is independently selected from an integer of 0-6, each c is independently selected from an integer of 0-6, each d is independently selected from an integer of 1-10, each e is independently selected from an integer of 0-6, each f is independently selected from an integer of 1-10, each g is independently selected from an integer of 0-6, and each h is independently selected from an integer of 1-10; the 1 position is connected to Ab', the 9 position is connected to D 1 Connected, 11 position and D 2 connected.

[0202] In some embodiments, Selected from:

[0203] Among them, position 1 is connected to Ab', position 9 is connected to D 1 Connected, 11 position and D 2 connected.

[0204] In some embodiments, Selected from:

[0205] wherein each g is independently selected from an integer of 0-6, each h is independently selected from an integer of 1-10; and the 5 position is connected to X.

[0206] In some embodiments, Selected from:

[0207] The 5 position is connected to X.

[0208] In some embodiments, the antibody drug conjugate is selected from the following structures:

[0209] Each m is independently selected from 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0210] In some embodiments, Ab-(S-) in each antibody drug conjugate is an antibody or antigen-binding fragment thereof as defined in any of the preceding items, and the S in Ab-(S- is derived from a sulfhydryl group in the antibody or antigen-binding fragment thereof. In some embodiments, Ab-(S-) in each antibody drug conjugate is an antibody or antigen-binding fragment thereof comprising a VH as shown in SEQ ID NO: 23 or a VH as shown in SEQ ID NO: 1 and a VL as shown in SEQ ID NO: 2, for example, Ab-01 (which comprises: a heavy chain as shown in SEQ ID NO: 22, and a light chain as shown in SEQ ID NO: 21), for example, Ab-02 (which comprises: a heavy chain as shown in SEQ ID NO: 20, and a light chain as shown in SEQ ID NO: 21).

[0211] In some embodiments, the N-terminal glutamine of the heavy chain or heavy chain variable region and / or the light chain or light chain variable region of the antibody or antigen-binding fragment thereof undergoes cyclization to form pyroglutamate or pyroglutamate; and / or, the heavy chain or heavy chain constant region (CH) or variant thereof does not have a C-terminal lysine.

[0212] In some embodiments, the present invention provides a composition of an antibody drug conjugate, comprising an antibody drug conjugate as defined in any one of the preceding items, wherein the DAR value of the composition is 1.0-10.0, preferably 1.0-5.0, for example, 1.0-1.5, 1.0-2.0, 1.0-2.5, 1.0-3.0, 1.0-3.5, 1.0-4.0, 1.0-4.5, 1.0-5.0, 2.0-2.5, 2.0-3.0, 2.0-3.5, 2.0-4.0, 2.0-4.5, 2.0-5.0, 2.5-3.0, 2.5-3.5, 2.5-4.0, 2.5-4.5, 2.5-5.0, 3.0-3.5, 3.0-4.0 , 3.0-4.5, 3.0-5.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 4.0-4.5, 4.0-5.0, 4.5-5.0, for example, about 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.0 9, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.3, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, about 1.5, about 1.51, about 1.52, about 1.53, about 1.54, about 1.55, about 1.56, about 1.57, about 1.58, about 1.59, about 1.6, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.7, about 1.71, about 1.72, about 1.73, about 1.74, about 1.75, about 1.76, about 1.77, about 1.78, about 1.79, about 1.8, about 1.81, about 1.82, about 1.83, about 1.84, about 1.85, about 1.86, about 1.87, about 1.88, about 1.89, about 1.9, about 1.91, about 1.92, about 1.93, about 1 .94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99, about 2.0, about 2.01, about 2.02, about 2.03, about 2.04, about 2.05, about 2.06, about 2.07, about 2.08, about 2.09, about 2.1, about 2.11, about 2.12, about 2.13, about 2.14, about 2.15, about 2.16, about 2.17, about 2.18, about 2.19, about 2.2, about 2.21, about 2.22, about 2.23, about 2.24, about 2.25, about 2.26, about 2.27, about 2.28, about 2.29, about 2.3, about 2.31, about 2.32, about 2.33, about 2.34, about 2.35, about 2. 36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, about 2.53, about 2.54, about 2.55, about 2.56, about 2.5 7, about 2.58, about 2.59, about 2.6, about 2.61, about 2.62, about 2.63, about 2.64, about 2.65, about 2.66, about 2.67, about 2.68, about 2.69, about 2.7, about 2.71, about 2.72, about 2.73, about 2.74, about 2.75, about 2.76, about 2.77, about 2.7 8, about 2.79, about 2.8, about 2.81, about 2.82, about 2.83, about 2.84, about 2.85, about 2.86, about 2.87, about 2.88, about 2.89, about 2.9, about 2.91, about 2.92, about 2.93, about 2.94, about 2.95, about 2.96, about 2.97, about 2.98, about 2.99 , about 3.0, about 3.01, about 3.02, about 3.03, about 3.04, about 3.05, about 3.06, about 3.07, about 3.08, about 3.09, about 3.1, about 3.11, about 3.12, about 3.13, about 3.14, about 3.15, about 3.16, about 3.17, about 3.18, about 3.19, about 3.2, about 3.21, about 3.22, about 3.23, about 3.24, about 3.25, about 3.26, about 3.27, about 3.28, about 3.29, about 3.3, about 3.31, about 3.32, about 3.33, about 3.34, about 3.35, about 3.36, about 3.37, about 3.38, about 3.39, about 3.4, about 3.41, about 3.42, about 3.43, about 3.44, about 3.45, about 3.46, about 3.47, about 3.48, about 3.49, about 3.5, about 3.51, about 3.52, about 3.53, about 3.54, about 3.55, about 3.56, about 3.57, about 3.58, about 3.59, about 3.6, about 3.61, about 3.62, about 3 .63, about 3.64, about 3.65, about 3.66, about 3.67, about 3.68, about 3.69, about 3.7, about 3.71, about 3.72, about 3.73, about 3.74, about 3.75, about 3.76, about 3.77, about 3.78, about 3.79, about 3.8, about 3.81, about 3.82, about 3.83, about 3.84, about 3.85, about 3.86, about 3.87, about 3.88, about 3.89, about 3.9, about 3.91, about 3.92, about 3.93, about 3.94, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4.0, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.06, about 4.07, about 4.08, about 4.09, about 4.1, about 4.11, about 4.12, about 4. 13, about 4.14, about 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.2, about 4.21, about 4.22, about 4.23, about 4.24, about 4.25, about 4.26, about 4.27, about 4.28, about 4.29, about 4.3, about 4.31, about 4.32, about 4.33, about 4.34, about 4.35, about 4.36, about 4.37, about 4.38, about 4.39, about 4.4, about 4.41, about 4.4 2, about 4.43, about 4.44, about 4.45, about 4.46, about 4.47, about 4.48, about 4.49, about 4.5, about 4.51, about 4.52, about 4.53, about 4.54, about 4.55, about 4.56, about 4.57, about 4.58, about 4.59, about 4.6, about 4.61, about 4.62, about 4.63, about 4.64, about 4.65, about 4.66, about 4.67, about 4.68, about 4.69, about 4.7, about 4.71 , about 4.72, about 4.73, about 4.74, about 4.75, about 4.76, about 4.77, about 4.78, about 4.79, about 4.8, about 4.81, about 4.82, about 4.83, about 4.84, about 4.85, about 4.86, about 4.87, about 4.88, about 4.89, about 4.9, about 4.91, about 4.92, about 4.93, about 4.94, about 4.95, about 4.96, about 4.97, about 4.98, about 4.99, about 5.0. .

[0213] In some embodiments, the antibody drug conjugate is selected from iADC-1 to iADC-25, wherein each m is independently selected from 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, Ab-(S-) is an antibody or antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO: 23 or a VH as set forth in SEQ ID NO: 1 and a VL as set forth in SEQ ID NO: 2, e.g., Ab-01 (which comprises: a heavy chain as set forth in SEQ ID NO: 22, and a light chain as set forth in SEQ ID NO: 21), e.g., Ab-02 (which comprises: a heavy chain as set forth in SEQ ID NO: 20, and a light chain as set forth in SEQ ID NO: 21), and S in Ab-(S-) is derived from a sulfhydryl group in the antibody or antigen-binding fragment thereof, e.g., a sulfhydryl group of a cysteine ​​residue in the antibody or antigen-binding fragment thereof.

[0214] In some embodiments, the antibody drug conjugate is selected from iADC-8a to iADC-12a:

[0215] wherein Ab′ is an antibody or antibody binding fragment thereof as described above, and each m is independently selected from 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0216] In some embodiments, the antibody drug conjugate is selected from iADC-8a to iADC-12a, wherein each m is independently selected from 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and Ab' is an antibody or antigen-binding fragment thereof comprising a VH as shown in SEQ ID NO: 23 or a VH as shown in SEQ ID NO: 1 and a VL as shown in SEQ ID NO: 2, e.g., Ab-01 (which comprises: a heavy chain as shown in SEQ ID NO: 22, and a light chain as shown in SEQ ID NO: 21), e.g., Ab-02 (which comprises: a heavy chain as shown in SEQ ID NO: 20, and a light chain as shown in SEQ ID NO: 21).

[0217] In some embodiments, Ab' is linked to form a conjugate through one or more sulfhydryl groups of a cysteine ​​residue, one or more amino groups of a lysine residue, one or more hydroxyl groups of a threonine residue, or one or more hydroxyl groups of a serine residue in the antibody.

[0218] In some embodiments, Ab' is linked to form a conjugate through one or more sulfhydryl groups of cysteine ​​residues in the antibody.

[0219] In some embodiments, Ab' is linked through one or more amino groups of lysine residues in the antibody to form a conjugate.

[0220] In some embodiments, Ab' is linked through one or more hydroxyl groups of a threonine residue in the antibody to form a conjugate.

[0221] In some embodiments, Ab' is linked through one or more hydroxyl groups of a serine residue in the antibody to form a conjugate.

[0222] In some embodiments, Ab' is linked to form a conjugate through m sulfhydryl groups of a cysteine ​​residue, m amino groups of a lysine residue, m hydroxyl groups of a threonine residue, or m hydroxyl groups of a serine residue in the antibody.

[0223] In some embodiments, Ab' is linked through m sulfhydryl groups of cysteine ​​residues in the antibody.

[0224] In some embodiments, Ab' is linked through m amino groups of lysine residues in the antibody.

[0225] In some embodiments, Ab' is linked through the m hydroxyl groups of a threonine residue in the antibody.

[0226] In some embodiments, Ab' is linked through the m hydroxyl groups of a serine residue in the antibody.

[0227] In some embodiments, the antibody drug conjugates described herein are optionally substituted with one or more suitable substituents.

[0228] In some embodiments, the antibody-drug conjugates of the present invention have improved cancer treatment efficacy. In some embodiments, the antibody-drug conjugates of the present invention have good physicochemical properties. In some embodiments, the antibody-drug conjugates of the present invention have good safety.

[0229] Drug-linker

[0230] In another aspect, the present invention provides a drug linker simultaneously coupled with a cytotoxic drug and a TLR agonist, which can be used to prepare the aforementioned antibody-drug conjugate.

[0231] Specifically, the present invention provides a compound as represented by formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0232] in,

[0233] X, Aa, L 1 、L 2 、D 1 and D 2 As defined in any of the preceding clauses,

[0234] M 1 It is a precursor of the linker site that is connected to an antibody or its antigen-binding fragment.

[0235] In some embodiments, the antibody or antigen-binding fragment thereof is as defined in any one of the preceding items.

[0236] In some embodiments, M 1 Selected from the following structures:

[0237] wherein each a is independently selected from an integer of 0-6, b is selected from an integer of 1-10, and LG represents a leaving group.

[0238] In some embodiments, M 1 Selected from the following structures:

[0239] In some embodiments, LG are each independently selected from halogen (eg, F, Cl, Br, I), halo 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1- 6-alkylsulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halogenated phenoxy, hydroxyl, thiol, amino, nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments, the halogenated C 1-6 Alkyl, C 1- 6-alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 The alkylsulfoxide group, halophenoxy group, alkenyl group, alkynyl group, and alkynyl group-containing structural fragments are optionally substituted with one or more suitable substituents.

[0240] In some embodiments, LG are each independently selected from halogen (eg, F, Cl, Br, I), halo 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1- 6-alkylsulfonate group, C 1-6 Alkyl sulfinate group, C 1-6Alkylsulfoxide, halophenoxy, hydroxyl, thiol, amino, nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments.

[0241] In some embodiments, LG is C 1-6 Alkylsulfonyl is, for example, methylsulfonyl.

[0242] In some embodiments, M 1 Selected from the following structures:

[0243] wherein each a is independently selected from an integer of 0-6, and b is independently selected from an integer of 1-10.

[0244] In some embodiments, M 1 for a is an integer selected from 0 to 6;

[0245] L2 is wherein g is selected from an integer of 0-6, h is selected from an integer of 1-10, and L 2 The 10th position is connected to Aa, and the 11th position is connected to D 2 connected;

[0246] D 2 for

[0247] In some embodiments, M 1 for a is an integer selected from 0-6.

[0248] In some embodiments, M 1 for

[0249] In some embodiments, L2 is

[0250] In some embodiments, Selected from: Wherein a is selected from an integer of 0-6; 6 position and L 1 Connected, 7 position and L 2 connected.

[0251] In some embodiments, Selected from: wherein a is selected from an integer of 0-6; the 6th position is L 1 Connected, 7 position and L 2 connected.

[0252] In some embodiments, Selected from: Among them, position 6 is L1 Connected, 7 position and L 2 connected.

[0253] In some embodiments, Selected from: wherein a is selected from an integer of 0-6, g is selected from an integer of 0-6, h is selected from an integer of 1-10; 9 position is selected from D 1 Connected, 11 position and D 2 Connected, LG is as defined in the previous item.

[0254] In some embodiments, Selected from: wherein a is selected from an integer of 0-6, g is selected from an integer of 0-6, h is selected from an integer of 1-10; 9 position is selected from D 1 Connected, 11 position and D 2 connected.

[0255] In some embodiments, Selected from:

[0256] The 9 position is D 1 Connected, 11 position and D 2 connected.

[0257] In some embodiments, the compound represented by formula (II) is selected from the following structures:

[0258] In some embodiments, the drug linkers described herein are optionally substituted with one or more suitable substituents.

[0259] In the above-mentioned Formula I and Formula II of the present invention, the groups of all embodiments can be appropriately selected and combined to obtain different general formula ranges or specific embodiments. These ranges and embodiments are all within the present invention. The present invention covers compounds or antibody drug conjugates obtained by combining any of the various embodiments.

[0260] Intermediate / Linker Unit

[0261] In some embodiments, the present invention provides a compound as represented by formula (III) or a salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof:

[0262] in,

[0263] X, Aa, L 1 、L 2 、M 1 As defined in any of the preceding clauses,

[0264] G 1 is p-nitrophenoxy or C 1-6 Alkanoyloxy;

[0265] G 2 It is a hydroxyl group or an oxo group.

[0266] In some embodiments, when L 1 The 9-position structure is When G 1 It is p-nitrophenoxy.

[0267] In some embodiments, when L 1 The 9-position structure is When G 1 C 1-6 Alkanoyloxy.

[0268] In some embodiments, when L 2 The 11-position structure is When G 2 It is an oxo group.

[0269] In some embodiments, when L 2 The 11-position structure is When G 2 It is a hydroxyl group.

[0270] In some embodiments, the compound represented by formula (III) is selected from:

[0271] In some embodiments, the present invention provides a compound as shown below, or a salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof:

[0272] Wherein, PG1 is each independently H or an amino protecting group, and the amino protecting group is preferably an alkoxycarbonyl amino protecting group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl (or ethyl)oxycarbonyl; an acyl amino protecting group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa ), o-(p-)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; alkyl amino protecting groups, such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn).

[0273] In some embodiments, the present invention provides a compound as shown below, or a salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof:

[0274] The compounds shown above or their salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites or prodrugs can be used to prepare the drug linkers or antibody drug conjugates herein.

[0275] In some embodiments, the present invention provides a linking unit having A compound of the structure or its salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, wherein X, Aa, L 1 、L 2 、M 1 As defined in any of the preceding clauses. In some embodiments, The compound of structure is selected from:

[0276] In some embodiments, the present invention provides a linking unit, which is a compound having a structure represented by formula (IV) or a salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0277] Among them, X, Aa, L 1 , a, g and h are as defined in any of the above items.

[0278] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof is provided, which comprises a linker unit of the structure shown in formula (IV) and LG or D 1 At least one of the connection units and LG or D 1 Connection, where D 1 Connect to position 9 of the connection unit; LG connects to position 1 of the connection unit; where D 1 As defined in any of the above items, LG is as defined in any of the above items.

[0279] In some embodiments, the present invention provides a linking unit, which is a compound having a structure represented by Formula (IVa) or a salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0280] Among them, LG, X, Aa, L 1 , a, g and h are as defined in any of the above items.

[0281] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof is provided, which comprises a linker unit of the structure shown in formula (IVa) and optionally D 1 , the connecting unit and D 1 Connection, where D 1 Connect to position 9 of the connection unit; D 1 As defined in any of the above.

[0282] In some embodiments, the present invention provides a linking unit having the following structure:

[0283] Among them, M, M 1 ,X,Aa,L 1 、L 2 、D 1 and D 2 As defined in any of the above.

[0284] In some embodiments, the intermediates described herein are optionally substituted with one or more suitable substituents.

[0285] Antibodies or antigen-binding fragments thereof

[0286] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to B7H3, wherein the antibody or antigen-binding fragment thereof comprises:

[0287] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0288] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof;

[0289] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0290] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 14 or a variant thereof, CDR-H2 of SEQ ID NO: 15 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof;

[0291] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0292] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 8 or a variant thereof, CDR-H2 of SEQ ID NO: 9 or a variant thereof, and CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 3 or a variant thereof, CDR-L2 of SEQ ID NO: 4 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof; or

[0293] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0294] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 16 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 5 or a variant thereof.

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

[0296] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0297] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof;

[0298] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0299] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 14 or a variant thereof, CDR-H2 of SEQ ID NO: 15 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof;

[0300] (3) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0301] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 8 or a variant thereof, CDR-H2 of SEQ ID NO: 9 or a variant thereof, and CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 3 or a variant thereof, CDR-L2 of SEQ ID NO: 4 or a variant thereof, and CDR-L3 of SEQ ID NO: 5 or a variant thereof; or

[0302] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0303] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 16 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 5 or a variant thereof.

[0304] In some embodiments, the antibody or antigen-binding fragment thereof comprises: VH shown in SEQ ID NO: 23 or 1 or a variant thereof, and / or, VL shown in SEQ ID NO: 2 or a variant thereof.

[0305] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain comprising a VH or a variant thereof shown in SEQ ID NO: 23 or 1 and a heavy chain constant region (CH) or a variant thereof shown in SEQ ID NO: 18, and / or a light chain comprising a VL or a variant thereof shown in SEQ ID NO: 2 and a light chain constant region (CL) or a variant thereof shown in SEQ ID NO: 19.

[0306] In some embodiments, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 22 or 20, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 21.

[0307] In some embodiments, the N-terminal glutamine of the heavy chain or heavy chain variable region and / or the light chain or light chain variable region undergoes cyclization to form pyroglutamate or pyroglutamate; and / or, the heavy chain or heavy chain constant region (CH) or variant thereof does not have a C-terminal lysine.

[0308] In some embodiments, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0309] In some embodiments, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:22, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:21.

[0310] In some embodiments, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:20, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:21.

[0311] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

[0312] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0313] In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, wherein each antibody or antigen-binding fragment may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, the N-terminal amino acid cyclized to pyroglutamic acid, or the N-terminal amino acid cyclized to pyroglutamate.

[0314] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain) (conversion of the N-terminal glutamine or glutamic acid in the heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., CHO cells) or during purification / storage.

[0315] In certain embodiments, the N-terminal glutamine of the VH comprising the sequence as shown in SEQ ID NO: 23 or 1 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate; and / or the N-terminal glutamate of the VL comprising the sequence as shown in SEQ ID NO: 2 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate.

[0316] In some embodiments, the N-terminal glutamine of the heavy chain shown in SEQ ID NO: 22 or 20 and / or the light chain shown in SEQ ID NO: 21 undergoes cyclization to form pyroglutamate or pyroglutamate; and / or, the heavy chain constant region (CH) shown in SEQ ID NO: 18 or its variant does not have a C-terminal lysine.

[0317] Conjugate

[0318] In another aspect, the present application provides a conjugate comprising a compound (e.g., a drug linker, a TLR agonist, or a cytotoxic drug) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, or a linker unit as described in any of the foregoing. In some embodiments, the conjugate is an antibody-drug conjugate, wherein the compound (e.g., a drug linker, a TLR agonist, or a cytotoxic drug) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof is linked to an antibody or antigen-binding fragment thereof.

[0319] In some embodiments, the present application provides an antibody-drug conjugate comprising any of the above antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or antigen-binding fragment thereof is linked to a bioactive molecule (e.g., a TLR agonist and / or a cytotoxic drug) via a linker.

[0320] Pharmaceutical composition

[0321] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate, compound (e.g., drug linker, TLR agonist, or cytotoxic drug) described above, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, and one or more pharmaceutical excipients.

[0322] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known or will be apparent to those skilled in the art based on the disclosure of the present invention. As described in REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, EW, ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients.

[0323] Treatment methods and applications

[0324] In another aspect, the present invention provides use of any of the above compounds (e.g., drug linkers, TLR agonists, or cytotoxic drugs) or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites, or prodrugs thereof in the preparation of antibody-drug conjugates, in particular, the preparation of any of the above antibody-drug conjugates.

[0325] In another aspect, the present invention provides use of any of the foregoing antibody-drug conjugates, compounds (e.g., drug linkers, TLR agonists, or cytotoxic drugs) or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites, or prodrugs thereof, compositions of antibody-drug conjugates, antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions in the preparation of a medicament, in particular, in the preparation of a medicament for treating and / or preventing cancer (e.g., cancer associated with B7H3 expression).

[0326] In another aspect, the present invention provides an antibody-drug conjugate, compound (e.g., drug linker, TLR agonist or cytotoxic drug) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, a composition of an antibody-drug conjugate, an antibody or antigen-binding fragment thereof, or a pharmaceutical composition as described above for use in treating and / or preventing a disease, such as cancer (e.g., a cancer associated with B7H3 expression).

[0327] In another aspect, the present invention provides a method for treating and / or preventing cancer (e.g., cancer associated with B7H3 expression), comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of any of the foregoing antibody-drug conjugates, compounds (e.g., drug linkers, TLR agonists, or cytotoxic drugs), or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites, or prodrugs, antibody-drug conjugate compositions, antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions.

[0328] In some embodiments, the cancer is selected from breast cancer (eg, ductal carcinoma), gastric cancer, lung cancer (eg, lung adenocarcinoma), and colon cancer.

[0329] Example

[0330] In order to make the purpose and technical scheme of the present invention clearer, the embodiments of the present invention are described in detail below in conjunction with embodiment.But those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.Unindicated specific conditions in the examples are all carried out according to the conditions of normal conditions or manufacturer's advice.Reagents used or instruments not indicated by manufacturer are all conventional products that can be obtained commercially.

[0331] The structures of the compounds were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS). 1 H NMR measurements were performed on a JEOL Eclipse 400 nuclear magnetometer. The solvents used were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6). The internal standard was tetramethylsilane (TMS). Chemical shifts (δ) were given in parts per million (ppm).

[0332] The MS measuring instrument is an Agilent (ESI) mass spectrometer, manufactured by Agilent, model: Agilent 6120B.

[0333] Preparation method for preparative high performance liquid chromatography:

[0334] Instrument model: Agilent 1260, chromatographic column: Waters SunFire Prep C18OBD (19 mm × 150 mm × 5.0 μm); column temperature: 25°C; flow rate: 20.0 mL / min; detection wavelength: 214 nm; elution gradient: (0 min: 10% A, 90% B; 16.0 min: 90% A, 10% B); mobile phase A: acetonitrile; mobile phase B: 0.05% formic acid in water.

[0335] Aluminum plates (20×20 cm) produced by Merck were used for thin layer chromatography silica gel plates (TLC), and the specifications used for thin layer chromatography separation and purification were GF 254 (1 mm) produced in Yantai.

[0336] The reaction is monitored by thin layer chromatography (TLC) or LC-MS; the developing solvent systems used include: dichloromethane and methanol system, n-hexane and ethyl acetate system, and petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound or by adding triethylamine.

[0337] Preparation method of reverse phase column chromatography:

[0338] Preparation method A:

[0339] Instrument model: Biotage fast medium-pressure preparative chromatography, chromatographic column: Agela C18 reversed-phase column (Spherical; 20-35 μm; 100A); column temperature: 25°C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: water;

[0340] Preparation method B:

[0341] Instrument model: Biotage fast medium-pressure preparative chromatography, chromatographic column: Agela C18 reversed-phase column (Spherical; 20-35 μm; 100A); column temperature: 25°C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: 0.05% formic acid in water;

[0342] Preparation Method C:

[0343] Instrument model: Biotage fast medium-pressure preparative chromatography, chromatographic column: Agela C18 reversed-phase column (Spherical; 20-35 μm; 100A); column temperature: 25°C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: 0.05% ammonium bicarbonate aqueous solution;

[0344] Microwave reaction was carried out using Biotage Initiator+ (400W, RT-300°C) microwave reactor.

[0345] Column chromatography generally uses 200-300 mesh silica gel as a carrier. Eluent systems include: dichloromethane and methanol systems, and petroleum ether and ethyl acetate systems. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine can also be added for adjustment.

[0346] Unless otherwise specified in the examples, the reaction temperature is room temperature (20°C to 35°C).

[0347] The reagents used in the present invention were purchased from Acros Organics, Aldrich Chemical Company, Teber Chemical and other companies.

[0348] In general synthesis methods, examples, and intermediate synthesis examples, the meanings of the abbreviations are as follows.

[0349] The information of the sequences involved in the present invention is described in the following table:

[0350] 1. Compound Examples

[0351] Intermediate Preparation Example 1: Preparation of 2-((1-((4-((4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamoyl)cyclohexyl)methyl)-2,5-dioxopyrrolidin-3-yl)thio)acetic acid (Compound Int1)

[0352] Step 1: Preparation of N-(4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl-1-carboxamide

[0353] 1-(4-Aminobutyl)-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine (200 mg, 0.60 mmol) and N,N-diisopropylethylamine (155 mg, 1.2 mmol) were added to N,N-dimethylformamide (2 mL), and a solution of 2,5-dioxopyrrolidin-1-yl-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl-1-carboxylate (240 mg, 0.72 mmol) in dichloromethane (1 mL) was added dropwise. The reaction mixture was stirred at 25 ° C for 12 hours. Water was added to the reaction system, and the mixture was extracted three times with dichloromethane. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1 (v / v)) to give the title compound of this step (282 mg, yield: 85.4%).

[0354] MS m / z(ESI):551.3[M+H] + .

[0355] Step 2: Preparation of 2-((1-((4-((4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)carbamoyl)cyclohexyl)methyl)-2,5-dioxopyrrolidin-3-yl)thio)acetic acid (Int1)

[0356] N-(4-(4-amino-2-butyl-7-methyl-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl)butyl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl-1-carboxamide (282 mg, 0.51 mmol) and thioglycolic acid (94 mg, 1.02 mmol) were added to dichloromethane (50 mL), and the reaction mixture was stirred at 25°C for 30 minutes. The reaction mixture was concentrated to obtain a yellow solid, which was slurried with methyl tert-butyl ether to obtain the title compound (237 mg, yield: 72.3%).

[0357] MS m / z(ESI):643.3[M+H] + .

[0358] Intermediate Preparation Example 2: Preparation of Compound Int2

[0359] Step 1: Preparation of compound Int2-2

[0360] N-tert-Butyloxycarbonyl-S-trityl-L-cysteine ​​(384 mg, 0.83 mmol) and 2,6-azido-3,6,9,12,15,18,21,24-octaoxahexacosacontan-1-amine (435 mg, 0.99 mmol) were dissolved in N,N-dimethylformamide (4 mL). 1-Hydroxybenzotriazole (167.58 mg, 1.24 mmol), N,N-diisopropylethylamine (320.43 mg, 2.48 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (238 mg, 1.24 mmol) were added under ice-cooling. The mixture was stirred at 20°C for 3 hours. The reaction solution was added dropwise to water and extracted with dichloromethane. The organic phase was dried and concentrated to give the title compound (0.69 g, yield: 78.4%).

[0361] MS m / z(ESI):884.4[M+H] + .

[0362] Step 2: Preparation of compound Int2-3

[0363] Compound Int2-2 (0.69 g, 0.78 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (10 mL) was added dropwise. The mixture was stirred at room temperature for 2 hours, concentrated, and diluted with dichloromethane (20 mL). The organic phase was washed with sodium bicarbonate solution, dried, and concentrated to obtain the title compound of this step (0.6 g, yield: 98%).

[0364] MS m / z(ESI):784.4[M+H] + .

[0365] Step 3: Preparation of compound Int2-4

[0366] Compound Int1 (136 mg, 211 mmol) and compound Int2-3 (182 mg, 233 mmol) were dissolved in N,N-dimethylformamide (4 mL), and 1-hydroxybenzotriazole (43.2 mg, 0.32 mmol), N,N-diisopropylethylamine (81.6 mg, 0.63 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (61.3 mg, 0.32 mmol) were added under ice bath. The mixture was stirred at 20°C for 3 hours. The reaction solution was dropped into water, extracted with dichloromethane, and the organic phase was dried and concentrated to obtain the title compound of this step (0.23 g, yield: 95.8%).

[0367] MS m / z(ESI):1408.6[M+H] + .

[0368] Step 4: Preparation of compound Int2-5

[0369] Compound Int2-4 (0.23 g, 0.16 mmol) was dissolved in dichloromethane (2 mL) and triethylsilane (0.52 mL), and trifluoroacetic acid (1 mL) was added dropwise. The mixture was stirred at room temperature for 1 hour, concentrated, and diluted with dichloromethane (20 mL). The organic phase was washed with sodium bicarbonate solution, dried, and concentrated to obtain the title compound of this step (0.15 g, yield: 78.9%).

[0370] MS m / z(ESI):1166.5[M+H] + .

[0371] Step 5: Preparation of compound Int2

[0372] Compound Int2-5 (0.15 g, 0.13 mmol) was dissolved in N,N-dimethylformamide (5 mL), and MC-VC-PABC-MMAE (0.17 g, 0.13 mmol) and N,N-diisopropylethylamine (50.3 mg, 0.39 mmol) were added. The mixture was stirred at room temperature for 1 hour, and an aqueous solution of ammonium chloride (10 mL) was added. The mixture was extracted three times with dichloromethane (10 mL). The organic phase was dried and concentrated, and the crude product was purified by preparative thin-layer silica gel plate (eluent: dichloromethane / methanol = 10 / 1 (v / v)) to give the title compound of this step (150 mg, yield: 47.0%).

[0373] MS m / z(ESI):1241.7[M / 2+H] + .

[0374] Intermediate Preparation Example 3: Preparation of Compound Int3

[0375] Step 1: Preparation of compound Int3-1

[0376] Compound Int2-3 (192 mg, 0.244 mmol) and 2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26-octaoxa-5-nonacosane-29-oic acid (110.53 mg, 0.222 mmol) were dissolved in N,N-dimethylformamide (3 mL), and 1-hydroxybenzotriazole (45 mg, 0.333 mmol) and N,N-diisopropylethylamine (85.9 mg) were added under ice-cooling. , 0.666mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (63.8mg, 0.333mmol), stirred at 20°C for 6 hours, the reaction solution was dropped into water (10mL), extracted three times with dichloromethane (10mL), the organic phase was dried and concentrated, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1 (v / v)) to give the title compound of this step (145mg, yield: 51.6%).

[0377] MS m / z(ESI):1263.6[M+H] + .

[0378] Step 2: Preparation of compound Int3-2

[0379] Compound Int3-1 (145 mg, 0.11 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added dropwise. The mixture was stirred at room temperature for 2 hours, concentrated, and diluted with dichloromethane (10 mL). The organic phase was washed with sodium bicarbonate solution, dried, and concentrated to give the title compound of this step (130 mg, yield: 97.0%).

[0380] MS m / z(ESI):1163.6[M+H] + .

[0381] Step 3: Preparation of compound Int3-3

[0382] Compound Int1 (65 mg, 0.10 mmol) and compound Int3-2 (130 mg, 0.11 mmol) were dissolved in N,N-dimethylformamide (5 mL), and 1-hydroxybenzotriazole (20.3 mg, 0.15 mmol), N,N-diisopropylethylamine (38.7 mg, 0.3 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (28.8 mg, 0.15 mmol) were added under ice bath, and stirred at 20°C for 3 hours. The reaction solution was added dropwise to water (10 mL), extracted three times with dichloromethane (10 mL), and the organic phase was dried and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1 (v / v)) to obtain the title compound of this step (110 mg, yield: 60.8%).

[0383] MS m / z(ESI):1787.8[M+H] + .

[0384] Step 4: Preparation of compound Int3-4

[0385] Compound Int3-3 (110 mg, 0.06 mmol) was dissolved in dichloromethane (2 mL) and triethylsilane (0.4 mL), and trifluoroacetic acid (1 mL) was added dropwise. The mixture was stirred at room temperature for 1 hour, concentrated, and diluted with dichloromethane (20 mL). The organic phase was washed with sodium bicarbonate solution, dried, and concentrated to give the title compound of this step (95 mg, yield: 100.0%).

[0386] MS m / z(ESI):1545.7[M+H] + .

[0387] Step 5: Preparation of compound Int3

[0388] Compound Int3-4 (95 mg, 0.06 mmol) was dissolved in N,N-dimethylformamide (25 mL), and MC-VC-PABC-MMAE (79 mg, 0.06 mmol) and N,N-diisopropylethylamine (23.2 mg, 0.18 mmol) were added. The mixture was stirred at room temperature for 1 hour, and an aqueous solution of ammonium chloride (10 mL) was added. The mixture was extracted three times with dichloromethane (10 mL). The organic phase was dried and concentrated, and the crude product was purified by preparative thin-layer silica gel plate (eluent: dichloromethane / methanol = 8 / 1 (v / v)) to give the title compound of this step (73 mg, yield: 41.4%).

[0389] MS m / z(ESI):1431.3[M / 2+H] + .

[0390] Intermediate Preparation Example 4: Preparation of Compound Int4

[0391] Step 1: Preparation of compound Int4-1

[0392] VC-PABC-MMAE (155 mg, 131.07 μmol) and 2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26-octaoxa-5-nonacosane-29-oic acid (68.65 mg, 131.07 μmol) were dissolved in anhydrous N,N-dimethylformamide (4 mL), and N,N-diisopropylethylamine (42.78 mg, 327.68 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (75.53 mg, 196.61 μmol) were added thereto, and the reaction was carried out at 25 ° C for 5 hours. The reaction solution was concentrated and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 (v / v)) to give the title compound of this step (193 mg, yield: 87.3%).

[0393] MS m / z(ESI):1603.0[M+H] + .

[0394] Step 2: Preparation of compound Int4-2

[0395] Under nitrogen, compound Int4-1 (165 mg, 97.79 μmol) and zinc bromide (139.08 mg, 586.72 μmol) were added to anhydrous dichloromethane (2 mL) in sequence and reacted at 25°C for 2 hours. The reaction mixture was concentrated to obtain the crude title compound, which was used directly in the next step.

[0396] MS m / z(ESI):1502.9[M+H] + .

[0397] Step 3: Preparation of compound Int4-3

[0398] Compound Int4-2 (114 mg, 72.06 μmol) and N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 1-(tert-Butoxycarbonyl)-L-lysine (37.43 mg, 72.06 μmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (23.52 mg, 180.16 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (41.53 mg, 108.09 μmol) were added. The mixture was allowed to react at 25°C for 5 hours. The reaction mixture was concentrated to obtain a residue which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 (v / v)) to obtain the title compound (110 mg, yield: 70.4%).

[0399] MS m / z(ESI):1953.1[M+H] + .

[0400] Step 4: Preparation of compound Int4-4

[0401] Compound Int4-3 (100 mg, 46.07 μmol) was dissolved in dichloromethane (3 mL), and diethylamine (67.40 mg, 921.47 μmol) was added. The mixture was allowed to react at 20°C for 10 hours. The reaction mixture was concentrated to obtain a residue which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 (v / v)) to obtain the title compound (40 mg, yield: 42.8%).

[0402] MS m / z(ESI):1731.1[M+H] + .

[0403] Step 5: Preparation of compound Int4-6

[0404] Compound Int4-4 (100 mg, 54.88 μmol) and compound Int4-5 (46.64 mg, 54.88 μmol) were added to anhydrous N,N-dimethylformamide (2 mL). N,N-diisopropylethylamine (10.75 mg, 82.31 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (25.29 mg, 65.85 μmol) were added, and the mixture was reacted at 25°C for 5 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (60 mg, yield: 40.7%).

[0405] MS m / z(ESI):1277.8[M / 2+H] + .

[0406] Step 4: Preparation of compound Int4

[0407] Under nitrogen, compound Int4-6 (45 mg, 7.04 μmol) and zinc bromide (6.68 mg, 28.18 μmol) were added to anhydrous dichloromethane (2 mL) in sequence and reacted at 25°C for 2 hours. The reaction solution was concentrated to obtain the crude title compound, which was used directly in the next step.

[0408] MS m / z(ESI):1227.8[M / 2+H] + .

[0409] Intermediate Preparation Example 5: Preparation of Compound Int5

[0410] Step 1: Preparation of compound Int5-2

[0411] Compound Int5-1 (186 mg, 243.74 μmol), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (131 mg, 292.49 μmol), N,N-diisopropylethylamine (66.32 mg, 487.48 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (136.58 mg, 341.24 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) in sequence and reacted at 0°C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (Preparation Method A, elution gradient: A% = 25%-35%) to obtain the title compound of this step (270 mg, yield: 92.9%).

[0412] MS m / z(ESI):1132.6[M+H] + .

[0413] Step 2: Preparation of compound Int5-3

[0414] Compound Int5-2 (340 mg, 285.23 μmol) and trifluoroacetic acid (2 mL) were added to dichloromethane (6 mL) and reacted at 25°C for 2 hours. The reaction solution was concentrated to obtain a residue, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 4 / 1 (v / v)) to obtain the title compound (250 mg, yield: 77.4%).

[0415] MS m / z(ESI):1076.6[M+H] + .

[0416] Step 3: Preparation of compound Int5-4

[0417] Compound Int5-3 (150 mg, 132.40 μmol), VC-PABC-MMAE (156.57 mg, 132.40 μmol), N,N-diisopropylethylamine (25.93 mg, 198.60 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (61.02 mg, 158.88 μmol) were added sequentially to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0°C for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 35% - 45%) to obtain the title compound (250 mg, yield: 82.2%).

[0418] MS m / z(ESI):1091.6[M / 2+H] + .

[0419] Step 4: Preparation of compound Int5

[0420] Compound Int5-4 (250 mg, 8.86 μmol) and piperidine (37.45 mg, 435.44 μmol) were added to N,N-dimethylformamide (4 mL) and reacted at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method A, elution gradient: A% = 20% - 30%) to obtain the title compound (185 mg, yield: 82.4%).

[0421] MS m / z(ESI):980.6[M / 2+H] + .

[0422] Intermediate Preparation Example 6: Preparation of Compound Int6

[0423] Step 1: Preparation of compound Int6-2

[0424] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (85.29 mg, 200.46 μmol), N,N-diisopropylethylamine (45.2 mg, 346.26 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (103.94 mg, 270.22 μmol), and compound Int6-1 (150 mg, 182.24 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) in sequence and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation Method B, elution gradient: A% = 25%-35%) to obtain the title compound of this step (170 mg, yield: 79.8%).

[0425] MS m / z(ESI):1160.6[M+H] + .

[0426] Step 2: Preparation of compound Int6-3

[0427] Compound Int6-2 (170 mg, 138.15 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (6 mL) and trifluoroacetic acid (3 mL) and reacted at 25°C for 2 hours. The reaction solution was concentrated to obtain the title compound (133 mg, yield: 86.3%).

[0428] MS m / z(ESI):1104.6[M+H] + .

[0429] Step 3: Preparation of compound Int6-4

[0430] Compound Int6-3 (51 mg, 46.19 μmol), VC-PABC-MMAE (51.88 mg, 46.19 μmol), N,N-diisopropylethylamine (24.37 mg, 187.03 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (19.14 mg, 49.88 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 1.5 hours. The reaction system was poured into ice water (10 mL) and extracted with ethyl acetate (5 mL x 3). The organic phases were combined and backwashed with saturated brine (3 mL x 2). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the title compound (86 mg, yield: 84.3%).

[0431] MS m / z(ESI):1105.7[M / 2+H] + .

[0432] Step 4: Preparation of compound Int6

[0433] Compound Int6-4 (86 mg, 38.93 μmol) and piperidine (5.36 mg, 62.27 μmol) were added to N,N-dimethylformamide (1 mL) and allowed to react at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 25%) to obtain the title compound (47 mg, yield: 60.8%).

[0434] MS m / z(ESI):1988.2[M+H] + .

[0435] Intermediate Preparation Example 7: Preparation of Compound Int7

[0436] Step 1: Preparation of compound Int7-1

[0437] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (91.93 mg, 216.06 μmol), compound Int7-4 (148.2 mg, 180.05 μmol), N,N-diisopropylethylamine (46.54 mg, 360.11 μmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (75.26 mg, 198.06 μmol) were added to N,N-dimethylformamide (2 mL) and stirred at 25°C for 2 hours. The reaction solution was purified by reverse phase column chromatography (Preparation Method B, elution gradient: A% = 30%-40%) to obtain the title compound of this step (152 mg, yield: 68.6%).

[0438] MS m / z(ESI):1230.7[M+H] + .

[0439] Step 2: Preparation of compound Int7-2

[0440] Compound Int7-1 (133 mg, 108.08 μmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at 25° C. for 2 hours. The reaction mixture was concentrated to obtain the crude title compound, which was used directly in the next step.

[0441] MS m / z(ESI):1174.6[M+H] + .

[0442] Step 3: Preparation of compound Int7-3

[0443] Compound Int7-2 (47.5 mg, 40.44 μmol), VC-PABC-MMAE (54.52 mg, 48.53 μmol), N,N-diisopropylethylamine (10.45 mg, 80.89 μmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (16.91 mg, 44.49 μmol) were added to N,N-dimethylformamide (3 mL) and stirred at 25°C for 2 hours. The reaction solution was diluted with N,N-dimethylformamide and purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 40% - 50%) to obtain the title compound (57.8 mg, 62.7% yield).

[0444] MS m / z(ESI):1140.7[M / 2+H] + .

[0445] Step 4: Preparation of compound Int7

[0446] Compound Int7-3 (57.8 mg, 25.35 μmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (9.27 mg, 126.76 μmol) was added. The mixture was stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method A, elution gradient: A% = 40% - 50%) to obtain the title compound (39.6 mg, 75.9% yield).

[0447] MS m / z(ESI):1029.6[M / 2+H] + .

[0448] Intermediate Preparation Example 8: Preparation of Compound Int8

[0449] Step 1: Preparation of compound Int8-2

[0450] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (276.97 mg, 644.45 μmol), N,N-diisopropylethylamine (209.93 mg, 1610 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (296.84 mg, 773.34 μmol), and compound Int8-1 (604 mg, 644.45 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) and stirred at 0°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation Method B, elution gradient: A% = 25% - 35%) to obtain the title compound of this step (747.9 mg, yield: 95.6%).

[0451] MS m / z(ESI):1214.6[M+H] + .

[0452] Step 2: Preparation of compound Int8-3

[0453] Compound Int8-2 (460 mg, 378.75 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (6 mL) and trifluoroacetic acid (3 mL) and stirred at 25° C. for 2 hours. The reaction mixture was concentrated to obtain the title compound (400 mg, yield: 91.2%).

[0454] MS m / z(ESI):1158.6[M+H] + .

[0455] Step 3: Preparation of compound Int8-4

[0456] Compound Int8-3 (113.74 mg, 91.19 μmol), N,N-diisopropylethylamine (23.24 mg, 178.03 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (51.28 mg, 133.52 μmol), and VC-PABC-MMAE (95 mg, 84.56 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) and stirred at 0°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 55% - 65%) to obtain the title compound (170 mg, yield: 93.5%).

[0457] MS m / z(ESI):1132.7[M / 2+H] + .

[0458] Step 4: Preparation of compound Int8

[0459] Compound Int8-4 (170 mg, 75.09 μmol) and diethylamine (27.74 mg, 375.47 μmol) were added to N,N-dimethylformamide (4 mL) and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 55% - 65%) to obtain the title compound (110 mg, yield: 71.9%).

[0460] MS m / z(ESI):1021.6[M / 2+H] + .

[0461] Intermediate Preparation Example 9: Preparation of Compound Int9

[0462] Step 1: Preparation of compound Int9-1

[0463] Compound Int8-3 (440 mg, 379.83 μmol), N,N-diisopropylethylamine (98.18 mg, 759.66 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (173.31 mg, 455.8 μmol), and GGFG-Dxd (319.38 mg, 379.83 μmol) were added to anhydrous N,N-dimethylformamide (6 mL) and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 25% - 35%) to obtain the title compound (250 mg, yield: 35.0%).

[0464] MS m / z(ESI):991.5[M / 2+H] + .

[0465] Step 2: Preparation of compound Int9

[0466] Compound Int9-1 (205 mg, 103.47 μmol) and diethylamine (36.31 mg, 491.49 μmol) were added to N,N-dimethylformamide (2 mL) and reacted at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 25% - 35%) to obtain the title compound (160 mg, yield: 93.1%).

[0467] MS m / z(ESI):586.9[M / 3+H] + .

[0468] Intermediate Preparation Example 10: Preparation of Compound Int10

[0469] Step 1: Preparation of compound Int10-2

[0470] Compound Int8-3 (75.54 mg, 61.78 μmol), N,N-diisopropylethylamine (15.97 mg, 123.56 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (30.54 mg, 80.32 μmol), and compound Int10-1 (50 mg, 67.96 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 25% - 35%) to obtain the title compound (85 mg, yield: 77.2%).

[0471] MS m / z(ESI):1876.9[M+H] + .

[0472] Step 2: Preparation of compound Int10

[0473] Compound Int10-2 (85 mg, 45.3 μmol) and diethylamine (15.9 mg, 215.2 μmol) were added to N,N-dimethylformamide (2 mL) and reacted at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 45% - 55%) to obtain the title compound (70 mg, yield: 98.3%).

[0474] MS m / z(ESI):1653.8[M+H] + .

[0475] Intermediate Preparation Example 11: Preparation of Compound Int11

[0476] Step 1: Preparation of compound Int11-2

[0477] Compound Int8-3 (51.95 mg, 44.85 μmol), N,N-diisopropylethylamine (11.09 mg, 85.84 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (22.85 mg, 60.08 μmol), and compound Int11-1 (30 mg, 40.77 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) in sequence and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 45%-50%) to obtain the title compound (56 mg, yield: 73.2%).

[0478] MS m / z(ESI):1876.9[M+H] + .

[0479] Step 2: Preparation of compound Int11

[0480] Compound Int11-2 (57 mg, 30.38 μmol) and diethylamine (11.22 mg, 151.91 μmol) were added to N,N-dimethylformamide (2 mL) and reacted at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 45% - 55%) to obtain the title compound (30 mg, yield: 59.7%).

[0481] MS m / z(ESI):827.4[M / 2+H] + .

[0482] Intermediate Preparation Example 12: Preparation of Compound Int12

[0483] Step 1: Preparation of compound Int12-1

[0484] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (37.29 mg, 87.65 μmol), N,N-diisopropylethylamine (14.16 mg, 109.56 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38.88 mg, 102.25 μmol), and compound Int6-1 (55 mg, 73.04 μmol) were added to anhydrous N,N-dimethylformamide (5 mL) in sequence and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation Method B, elution gradient: A% = 25%-35%) to obtain the title compound of this step (80 mg, yield: 99.4%).

[0485] MS m / z(ESI):1160.6[M+H] + .

[0486] Step 2: Preparation of compound Int12-2

[0487] Compound Int12-1 (50 mg, 43.09 μmol) was dissolved in a mixture of anhydrous dichloromethane (3 mL) and trifluoroacetic acid (1.5 mL) and reacted at 25°C for 4 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 35% - 45%) to obtain the title compound (45 mg, yield: 62.2%).

[0488] MS m / z(ESI):1104.6[M+H] + .

[0489] Step 3: Preparation of compound Int12-3

[0490] Compound Int12-2 (45 mg, 40.74 μmol), N,N-diisopropylethylamine (7.58 mg, 58.07 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (20.81 mg, 54.19 μmol), and VC-PABC-MMAE (45.78 mg, 40.74 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 30%) to obtain the title compound (30 mg, yield: 35.1%).

[0491] MS m / z(ESI):1105.7[M / 2+H] + .

[0492] Step 4: Preparation of compound Int12

[0493] Compound Int12-3 (30 mg, 13.44 μmol) and diethylamine (4.58 mg, 53.76 μmol) were added to N,N-dimethylformamide (2 mL) and reacted at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 35% - 45%) to obtain the title compound (25 mg, yield: 93.6%).

[0494] MS m / z(ESI):994.6[M / 2+H] + .

[0495] Intermediate Preparation Example 13: Preparation of Compound Int13

[0496] Step 1: Preparation of compound Int13-1

[0497] Compound Int4-5 (150 mg, 169.23 μmol) and N-tert-butyloxycarbonyllysine methyl ester hydrochloride (46.37 mg, 169.23 μmol) were dissolved in anhydrous N,N-dimethylformamide (4 mL). N,N-diisopropylethylamine (44.18 mg, 338.46 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (78.01 mg, 203.08 μmol) were added sequentially, and the mixture was reacted at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 6 / 1 (v / v)) to obtain the title compound (160 mg, yield: 95.8%).

[0498] MS m / z(ESI):1084.6[M+H] + .

[0499] Step 2: Preparation of compound Int13-2

[0500] Compound Int13-1 (185 mg, 162.08 μmol) was dissolved in a mixture of tetrahydrofuran (1 mL), methanol (1 mL), and water (1 mL). Lithium hydroxide monohydrate (28.63 mg, 648.31 μmol) was added and reacted at 25°C for 3 hours. The reaction solution was concentrated, and N,N-dimethylformamide (1 mL) was added. The resulting solution was purified by reverse-phase column chromatography (Preparation Method A, elution gradient: A% = 25% - 35%) to obtain the title compound (160 mg, yield: 87.6%).

[0501] MS m / z(ESI):1070.6[M+H] + .

[0502] Step 3: Preparation of compound Int13-3

[0503] Compound Int13-2 (50 mg, 42.04 μmol), VC-PABC-MMAE (52.48 mg, 42.04 μmol), N,N-diisopropylethylamine (13.70 mg, 105.11 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (19.37 mg, 50.45 μmol) were added to N,N-dimethylformamide (1.5 mL) and reacted at room temperature for 1.5 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 45% - 55%) to obtain the title compound (41 mg, yield: 40.3%).

[0504] MS m / z(ESI):1088.7[M / 2+H]+ .

[0505] Step 4: Preparation of compound Int13

[0506] Compound Int13-3 (38 mg, 15.72 μmol) and anhydrous zinc bromide (17.88 mg, 78.59 μmol) were added to anhydrous dichloromethane (3 mL) and reacted at room temperature for 5 hours. The reaction solution was concentrated to obtain a crude product, which was purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 30%) to obtain the title compound (30 mg, yield: 82.8%).

[0507] MS m / z(ESI):1038.6[M / 2+H] + .

[0508] Intermediate Preparation Example 14: Preparation of Compound Int14

[0509] Step 1: Preparation of compound Int14-1

[0510] VC-PABC-MMAE (150 mg, 133.52 μmol), N,N-diisopropylethylamine (22.43 mg, 173.58 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (66 mg, 173.58 μmol) and N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 -(tert-Butoxycarbonyl)-L-lysine (62.56 mg, 133.52 μmol) was added to anhydrous N,N-dimethylformamide (5 mL) and reacted at 0°C for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 60% - 70%) to obtain the title compound (170 mg, yield: 85.1%).

[0511] MS m / z(ESI):1573.9[M+H] + .

[0512] Step 2: Preparation of compound Int14-2

[0513] Compound Int14-1 (85 mg, 53.46 μmol) and diethylamine (19.75 mg, 267.32 μmol) were added to N,N-dimethylformamide (4 mL) and reacted at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 25% - 35%) to obtain the title compound (73 mg, yield: 98.6%).

[0514] MS m / z(ESI):1351.8[M+H] + .

[0515] Step 3: Preparation of compound Int14-4

[0516] Compound Int14-3 (55.52 mg, 63.81 μmol), N,N-diisopropylethylamine (10.32 mg, 79.07 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (28.34 mg, 73.8 μmol), and compound Int14-2 (75 mg, 55.48 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) and reacted at 0°C for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 25% - 40%) to obtain the title compound (38 mg, yield: 32.7%).

[0517] MS m / z(ESI):1102.7[M / 2+H] + .

[0518] Step 4: Preparation of compound Int14

[0519] Compound Int14-4 (38 mg, 17.24 μmol) and zinc bromide (29.81 mg, 131.05 μmol) were added to anhydrous dichloromethane (3 mL) and reacted at 25°C for 3 hours. The reaction solution was concentrated to obtain a crude product, which was purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 30%) to provide the title compound (61 mg, yield: 78.8%).

[0520] MS m / z(ESI):702.1[M / 3+H] + .

[0521] Intermediate Preparation Example 15: Preparation of Compound Int15

[0522] Step 1: Preparation of compound Int15-1

[0523] Compound Int6-1 (60 mg, 79.78 μmol), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid (32.78 mg, 79.78 μmol), N,N-diisopropylethylamine (25.73 mg, 199.46 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (36.38 mg, 95.73 μmol) were added to N,N-dimethylformamide (1 mL) in sequence and stirred at room temperature for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (Preparation B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (52 mg, yield: 56.9%).

[0524] MS m / z(ESI):1146.7[M+H] + .

[0525] Step 2: Preparation of compound Int15-2

[0526] Compound Int15-1 (50 mg, 43.60 μmol) and 48% aqueous hydrobromic acid (0.5 mL) were added to acetonitrile (1 mL) and stirred at room temperature for 3 hours. The reaction mixture was concentrated to obtain the title compound (35 mg, yield: 73.4%).

[0527] MS m / z(ESI):1090.6[M+H] + .

[0528] Step 3: Preparation of compound Int15-3

[0529] Compound Int15-2 (35 mg, 32.09 mmol), VC-PABC-MMAE (36.04 mg, 32.09 μmol), N,N-diisopropylethylamine (10.34 mg, 80.22 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (14.63 mg, 38.50 μmol) were added to N,N-dimethylformamide (1 mL) and stirred at room temperature for 2 hours. The reaction mixture was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 40% - 45%) to obtain the title compound (63 mg, yield: 89.3%).

[0530] MS m / z(ESI):1098.6[M / 2+H] + .

[0531] Step 4: Preparation of compound Int15

[0532] Compound Int15-3 (63 mg, 28.7 μmol) and piperidine (4.82 mg, 57.40 μmol) were added to N,N-dimethylformamide (1 mL) and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 25% - 35%) to obtain the title compound (28 mg, yield: 49.5%).

[0533] MS m / z(ESI):1974.2[M+H] + .

[0534] Intermediate Preparation Example 16: Preparation of Compound Int16

[0535] Step 1: Preparation of compound Int16-1

[0536] (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid (35.52 mg, 86.33 μmol), N,N-diisopropylethylamine (14.16 mg, 109.56 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38.88 mg, 102.25 μmol), and compound Int6-1 (65 mg, 86.33 μmol) were added to anhydrous N,N-dimethylformamide (5 mL) in sequence and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation Method B, elution gradient: A% = 25%-35%) to obtain the title compound of this step (50 mg, yield: 53.2%).

[0537] MS m / z(ESI):1146.6[M+H] + .

[0538] Step 2: Preparation of compound Int16-2

[0539] Compound Int16-1 (50 mg, 43.18 μmol) was dissolved in a mixture of anhydrous dichloromethane (3 mL) and trifluoroacetic acid (1.5 mL) and reacted at 25°C for 4 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 35% - 45%) to obtain the title compound (45 mg, yield: 89.2%).

[0540] MS m / z(ESI):1090.5[M+H] + .

[0541] Step 3: Preparation of compound Int16-3

[0542] Compound Int16-2 (41.53 mg, 38.09 μmol), N,N-diisopropylethylamine (6.82 mg, 52.37 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (18.07 mg, 47.05 μmol), and VC-PBAC-MMAE (42.8 mg, 38.09 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 25%) to obtain the title compound (52 mg, yield: 65.4%).

[0543] MS m / z(ESI):1098.6[M / 2+H] + .

[0544] Step 4: Preparation of compound Int16

[0545] Compound Int16-3 (52 mg, 23.69 μmol) and piperidine (6.52 mg, 76.54 μmol) were added to N,N-dimethylformamide (2 mL) and reacted at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 35% - 45%) to obtain the title compound (31 mg, yield: 82.9%).

[0546] MS m / z(ESI):658.7[M / 3+H] + .

[0547] Intermediate Preparation Example 17: Preparation of Compound Int17

[0548] Step 1: Preparation of compound Int17-1

[0549] VC-PABC-MMAE (60 mg, 53.41 μmol), N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 62-(tert-Butoxycarbonyl)-L-lysine (25 mg, 53.41 μmol), N,N-diisopropylethylamine (17.2 mg, 133.52 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (24.35 mg, 64.09 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 1 hour. The reaction system was poured into ice water (10 mL) and extracted with ethyl acetate (5 mL*3). The organic phases were combined and backwashed with saturated brine (3 mL*2). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the title compound (82 mg, crude product).

[0550] MS m / z(ESI):1574.0[M+H] + .

[0551] Step 2: Preparation of compound Int17-2

[0552] Compound Int17-1 (82.00 mg, crude) and piperidine (8.07 mg, 93.78 μmol) were added sequentially to N,N-dimethylformamide (1 mL) and allowed to react at room temperature for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 25% - 35%) to obtain the title compound (41 mg, total yield for two steps: 56.8%).

[0553] MS m / z(ESI):1351.9[M+H] + .

[0554] Step 3: Preparation of compound Int17-3

[0555] Compound Int17-2 (41.00 mg, 30.32 μmol), compound Int4-5 (25.52 mg, 30.32 μmol), N,N-diisopropylethylamine (9.78 mg, 75.81 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (17.28 mg, 45.48 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 45%-50%) to obtain the title compound (35 mg, yield: 53.1%).

[0556] MS m / z(ESI):1088.7[M / 2+H] + .

[0557] Step 4: Preparation of compound Int17

[0558] Compound Int17-3 (35 mg, 16.09 μmol) and anhydrous zinc bromide (14.28 mg, 64.36 μmol) were added to dichloromethane (2 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 25% - 30%) to obtain the title compound (25 mg, yield: 74.9%).

[0559] MS m / z(ESI):1038.6[M / 2+H] + .

[0560] Intermediate Preparation Example 18: Preparation of Compound Int18

[0561] Step 1: Preparation of compound Int18-1

[0562] Compound Int14-1 (87 mg, 55.27 μmol) and zinc bromide (95.56 mg, 420.09 μmol) were added to anhydrous dichloromethane (3 mL) and reacted at 25°C for 3 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 35% - 45%) to obtain the title compound (61 mg, yield: 78.8%).

[0563] MS m / z(ESI):1473.8[M+H] + .

[0564] Step 2: Preparation of compound Int18-2

[0565] Compound Int14-3 (21.25 mg, 24.42 μmol), N,N-diisopropylethylamine (4.54 mg, 34.81 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.48 mg, 32.49 μmol), and compound Int18-1 (36 mg, 24.42 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0°C for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 35%) to obtain the title compound (45 mg, yield: 83.4%).

[0566] MS m / z(ESI):1163.7[M / 2+H] + .

[0567] Step 3: Preparation of compound Int18

[0568] Compound Int18-2 (40 mg, 17.03 μmol) and diethylamine (6.29 mg, 85.13 μmol) were added to N,N-dimethylformamide (4 mL) and reacted at 25°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 25% - 35%) to obtain the title compound (30 mg, yield: 83.8%).

[0569] MS m / z(ESI):1052.7[M / 2+H] + .

[0570] Intermediate Preparation Example 19: Preparation of Compound Int19

[0571] Step 1: Preparation of compound Int19-2

[0572] Compound Int19-1 (800 mg, 1.67 mmol) and piperidine (287.11 mg, 3.34 mmol) were added to N,N-dimethylformamide (15 mL) and reacted at room temperature for 2 hours. After concentration, the reaction solution was added with methyl tert-butyl ether (20 mL). After sonication for 5 minutes, the mixture was allowed to stand, filtered, and the solid was dried to obtain the title compound (315 mg, 73.5% yield).

[0573] MS m / z(ESI):253.1[M+H] + .

[0574] Step 2: Preparation of compound Int19-3

[0575] Compound Int19-2 (90 mg, 321.09 μmol), N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 1-(tert-Butoxycarbonyl)-L-lysine (202.53 mg, 321.09 μmol), N,N-diisopropylethylamine (104.60 mg, 802.72 μmol), 1-hydroxybenzotriazole (49.23 mg, 353.20 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (77.43 mg, 401.36 μmol) were added to N,N-dimethylformamide (3 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 50% - 55%) to obtain the title compound (142 mg, yield: 47.4%).

[0576] MS m / z(ESI):802.4[M+H] + .

[0577] Step 3: Preparation of compound Int19-4

[0578] Compound Int19-3 (142 mg, 159.37 μmol), ammonium formate (50.75 mg, 796.84 μmol), and 10% palladium on carbon (15 mg) were added to anhydrous methanol (4 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 30% - 35%) to obtain the title compound (101 mg, yield: 80.1%).

[0579] MS m / z(ESI):712.4[M+H] + .

[0580] Step 4: Preparation of compound Int19-5

[0581] Compound Int19-4 (95 mg, 133.46 μmol), exitecan mesylate (70.94 mg, 133.46 μmol), N,N-diisopropylethylamine (43.04 mg, 333.66 μmol), and bis(2-oxo-3-oxazolidinyl)phosphinoyl chloride (50.85 mg, 200.20 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 3 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 45% - 55%) to obtain the title compound (105 mg, yield: 69.7%).

[0582] MS m / z(ESI):1129.5[M+H] + .

[0583] Step 5: Preparation of compound Int19-6

[0584] Compound Int19-5 (105 mg, 92.98 μmol) and piperidine (15.83 mg, 185.97 μmol) were added to N,N-dimethylformamide (1 mL) and allowed to react at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method A, elution gradient: A% = 27% - 33%) to obtain the title compound (46 mg, yield: 54.5%).

[0585] MS m / z(ESI):907.4[M+H] + .

[0586] Step 6: Preparation of compound Int19-7

[0587] Compound Int8-3 (50 mg, 41.85 μmol), compound Int19-6 (37.95 mg, 41.85 μmol), N,N-diisopropylethylamine (13.63 mg, 104.61 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (19.27 mg, 50.21 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method A, elution gradient: A% = 40%-48%) to obtain the title compound (43 mg, yield: 50.2%).

[0588] MS m / z(ESI):1024.5[M / 2+H] + .

[0589] Step 7: Preparation of compound Int19

[0590] Compound Int19-7 (40 mg, 19.54 μmol) and piperidine (3.33 mg, 39.07 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. After concentration, the reaction solution was added with methyl tert-butyl ether (5 mL). After sonication for 5 minutes, the mixture was allowed to stand, filtered, and the solid dried to provide the title compound (28 mg, 70.7% yield).

[0591] MS m / z(ESI):1824.9[M+H] + .

[0592] Intermediate Preparation Example 20: Preparation of Compound Int20

[0593] Step 1: Preparation of compound Int20-2

[0594] Compound Int20-1 (460 mg, 808.9 μmol) and N-tert-butyloxycarbonyl-L-cysteine ​​ethyl ester (242 mg, 970.67 μmol) were dissolved in N,N-dimethylformamide (6 mL). Potassium iodide (13.43 mg, 80.89 μmol) and N,N-diisopropylethylamine (150 mg, 1152.68 μmol) were added and reacted at 25°C for 10 hours. The reaction solution was concentrated, and the resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 5 / 1 (v / v)) to obtain the title compound (320 mg, yield: 64.5%).

[0595] MS m / z(ESI):646.1[M+H] + .

[0596] Step 2: Preparation of compound Int20-3

[0597] Compound Int20-2 (220 mg, 337.26 μmol) and Dess-Martin periodinane (216.66 mg, 505.89 μmol) were added to dichloromethane (15 mL) and reacted at 25°C for 10 hours. Saturated sodium thiosulfate solution (5 mL) and saturated aqueous sodium bicarbonate solution (10 mL) were added to the reaction solution and stirred until clear. The solution was then extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 (v / v)) to obtain the title compound (185 mg, yield: 85.2%).

[0598] MS m / z(ESI):644.3[M+H] + .

[0599] Step 3: Preparation of compound Int20-4

[0600] Compound Int20-3 (146.31 mg, 227.26 μmol), 2-butyl-7-methyl-1-(piperidin-4-ylmethyl)-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-4-amine (65 mg, 181.81 μmol), sodium cyanoborohydride (31.56 mg, 502.2 mmol), and glacial acetic acid (31.4 mg, 518.16 mmol) were added sequentially to a mixture of 1,2-dichloroethane (4 mL) and methanol (2 mL) and reacted at 25°C for 4 hours. The crude product obtained by concentration of the reaction solution was dissolved in methanol (3 mL) and purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 30%) to obtain the title compound (109 mg, yield: 63.8%).

[0601] MS m / z(ESI):985.5[M+H] + .

[0602] Step 4: Preparation of compound Int20-5

[0603] Compound Int20-4 (100 mg, 101.49 μmol) was dissolved in methanol (4 mL), and a solution of lithium hydroxide monohydrate (21.31 mg, 507.46 μmol) in water (1 mL) was added. The mixture was allowed to react at 25°C for 4 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 35% - 45%) to obtain the title compound (85 mg, yield: 87.5%).

[0604] MS m / z(ESI):957.5[M+H] + .

[0605] Step 5: Preparation of compound Int20-6

[0606] Compound Int20-5 (65 mg, 67.91 μmol), N,N-diisopropylethylamine (12.63 mg, 96.76 μmol, 16.15 μL), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (34.69 mg, 90.31 μmol), and VC-PABC-MMAE (76.28 mg, 67.91 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 30% - 45%) to obtain the title compound (85 mg, yield: 63.9%).

[0607] MS m / z(ESI):1032.1[M / 2+H] + .

[0608] Step 6: Preparation of compound Int20

[0609] Compound Int20-6 (38 mg, 18.42 μmol) and zinc bromide (31.85 mg, 140.01 μmol) were added to anhydrous dichloromethane (2 mL) and reacted at 25°C for 4 hours. The reaction solution was concentrated to obtain a crude product, which was purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 30%) to provide the title compound (30 mg, yield: 87.0%).

[0610] MS m / z(ESI):982.1[M / 2+H] + .

[0611] Drug-linker Preparation Example 1-1: Preparation of Compound C-1

[0612] Compound Int2 (50 mg, 0.02 mmol) was dissolved in DMSO (6 mL) and water (0.6 mL). Under nitrogen protection, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(prop-2-yn-1-yl)hexanamide (5.99 mg, 0.024 mmol), sodium ascorbate (5.94 mg, 0.03 mmol) and anhydrous copper sulfate (6.4 mg, 0.04 mmol) were added. The mixture was stirred at room temperature for 1 hour, extracted with water and ethyl acetate, and the organic phase was dried and concentrated. The crude product was purified by preparative high performance liquid chromatography to obtain the title compound of this step (15 mg, yield: 27.2%).

[0613] MS m / z(ESI):1365.7[M / 2+H] + .

[0614] Drug Linker Preparation Example 1-2: Preparation of Compound C-2

[0615] The synthetic route of Example 1-1 was adopted, and the reaction raw material compound Int2 was replaced by compound Int3 to obtain the title compound of this step (10 mg, yield: 6%).

[0616] MS m / z(ESI):1555.3[M / 2+H] + .

[0617] Drug Linker Preparation Example 1-3: Preparation of Compound C-3

[0618] Compound Int4 (20 mg, 7.74 μmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propionate (2.17 mg, 7.74 μmol), and N,N-diisopropylethylamine (1.52 mg, 11.61 μmol) were added sequentially to anhydrous N,N-dimethylformamide (1.5 mL) and reacted at 25°C for 3 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (0.64 mg, 3% yield).

[0619] MS m / z(ESI):1303.8[M / 2+H] + .

[0620] Drug Linker Preparation Example 1-4: Preparation of Compound C-4

[0621] Compound Int5 (50 mg, 24.24 μmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propionate (20.38 mg, 72.72 μmol), and N,N-diisopropylethylamine (4.75 mg, 36.36 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (11 mg, yield: 20.2%).

[0622] MS m / z(ESI):1056.1[M / 2+H] + .

[0623] Drug Linker Preparation Example 1-5: Preparation of Compound C-5

[0624] Compound Int5 (60 mg, 29.09 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (9.44 mg, 09.09 μmol), and N,N-diisopropylethylamine (45.70 mg, 43.63 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (11 mg, 16.3% yield).

[0625] MS m / z(ESI):1077.1[M / 2+H] + .

[0626] Drug Linker Preparation Example 1-6: Preparation of Compound C-6

[0627] Compound Int5 (60 mg, 29.09 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (22.38 mg, 58.18 μmol), N,N-diisopropylethylamine (5.7 mg, 43.63 μmol), and 1-hydroxybenzotriazole (5.09 mg, 14.55 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) and reacted at 25°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (4 mg, 5.8% yield).

[0628] MS m / z(ESI):1105.6[M / 2+H] + .

[0629] Drug Linker Preparation Example 1-7: Preparation of Compound C-7

[0630] Compound Int6 (47 mg, 23.66 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (12.99 mg, 35.49 μmol), and N,N-diisopropylethylamine (5.55 mg, 42.57 μmol) were added to N,N-dimethylformamide (0.5 mL) and reacted at room temperature for 1.5 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 35%) to obtain the title compound (37.38 mg, yield: 70.6%).

[0631] MS m / z(ESI):1119.6[M / 2+H] + .

[0632] Drug Linker Preparation Example 1-8: Preparation of Compound C-8

[0633] Compound Int7 (50 mg, 24.31 μmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propionate (7.76 mg, 29.16 μmol), and N,N-diisopropylethylamine (4.52 mg, 34.63 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (21.5 mg, 41.3% yield).

[0634] MS m / z(ESI):1105.2[M / 2+H] + .

[0635] Drug Linker Preparation Example 1-9: Preparation of Compound C-9

[0636] Compound Int8 (113.67 mg, 55.68 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (22.27 mg, 60.95 μmol), and N,N-diisopropylethylamine (11.63 mg, 89.08 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (55 mg, 53.9% yield).

[0637] MS m / z(ESI):1146.6[M / 2+H] + .

[0638] Drug-linker Preparation Example 1-10: Preparation of Compound C-10

[0639] Compound Int7 (70 mg, 30.62 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (18.64 mg, 45.93 μmol), and N,N-diisopropylethylamine (7.98 mg, 61.24 μmol) were added sequentially to anhydrous N,N-dimethylformamide (1 mL) and reacted at 25°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (42 mg, 55.4% yield).

[0640] MS m / z(ESI):1154.7[M / 2+H] + .

[0641] Drug Linker Preparation Example 1-11: Preparation of Compound C-11

[0642] Compound Int9 (90 mg, 51.17 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (23.02 mg, 63.01 μmol), and N,N-diisopropylethylamine (12.02 mg, 92.1 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (27 mg, 29.2% yield).

[0643] MS m / z(ESI):1005.5[M / 2+H] + .

[0644] Drug Linker Preparation Example 1-12: Preparation of Compound C-12

[0645] Compound Int10 (70 mg, 42.33 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (20.1 mg, 55.02 μmol), and N,N-diisopropylethylamine (7.87 mg, 60.31 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (2.53 mg, 7.1% yield).

[0646] MS m / z(ESI):1903.8[M+H] + .

[0647] Drug Linker Preparation Example 1-13: Preparation of Compound C-13

[0648] Compound Int11 (30 mg, 18.14 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (8.62 mg, 23.58 μmol), and N,N-diisopropylethylamine (3.55 mg, 27.21 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (11 mg, 31.8% yield).

[0649] MS m / z(ESI):952.4[M / 2+H] + .

[0650] Drug-linker Preparation Example 1-14: Preparation of Compound C-14

[0651] Compound Int12 (25 mg, 12.58 μmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propionate (10.05 mg, 37.74 μmol), and N,N-diisopropylethylamine (2.34 mg, 17.92 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (8 mg, yield: 31.3%).

[0652] MS m / z(ESI):1070.1[M / 2+H] + .

[0653] Drug Linker Preparation Example 1-15: Preparation of Compound C-15

[0654] Compound Int12 (28.80 mg, 14.49 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (5.88 mg, 14.49 μmol), and N,N-diisopropylethylamine (3.78 mg, 28.98 μmol) were added to N,N-dimethylformamide (0.5 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 35%) to obtain the title compound (13.57 mg, yield: 41.8%).

[0655] MS m / z(ESI):1119.6[M / 2+H] + .

[0656] Drug-linker Preparation Example 1-16: Preparation of Compound C-16

[0657] Compound Int13 (30 mg, 13.01 μmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propionate (5.25 mg, 19.51 μmol), and N,N-diisopropylethylamine (2.54 mg, 19.51 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 1.5 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 25% - 40%) to obtain the title compound (9.81 mg, yield: 31.5%).

[0658] MS m / z(ESI):1114.2[M / 2+H] + .

[0659] Drug Linker Preparation Example 1-17: Preparation of Compound C-17

[0660] Compound Int14 (20 mg, 9.50 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (5.56 mg, 15.21 μmol), and N,N-diisopropylethylamine (1.77 mg, 13.67 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (6 mg, 28.2% yield).

[0661] MS m / z(ESI):1177.7[M / 2+H] + .

[0662] Drug Linker Preparation Example 1-18: Preparation of Compound C-18

[0663] Compound Int15 (28 mg, 14.19 μmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propionate (5.66 mg, 21.28 μmol), and N,N-diisopropylethylamine (2.75 mg, 21.28 μmol) were added to N,N-dimethylformamide (0.5 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 20% - 35%) to obtain the title compound (7.64 mg, yield: 25.3%).

[0664] MS m / z(ESI):1063.1[M / 2+H] + .

[0665] Drug Linker Preparation Example 1-19: Preparation of Compound C-19

[0666] Compound Int16 (23.15 mg, 11.73 μmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propionate (6.25 mg, 23.46 μmol), and N,N-diisopropylethylamine (2.18 mg, 16.72 μmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (12 mg, 50.7% yield).

[0667] MS m / z(ESI):1063.1[M / 2+H] + .

[0668] Drug-linker Preparation Example 1-20: Preparation of Compound C-20

[0669] Compound Int16 (24.7 mg, 12.52 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (5.08 mg, 12.52 μmol), and N,N-diisopropylethylamine (3.26 mg, 25.03 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation B, elution gradient: A% = 20% - 35%) to obtain the title compound (17.16 mg, yield: 61.7%).

[0670] MS m / z(ESI):1112.6[M / 2+H] + .

[0671] Drug Linker Preparation Example 1-21: Preparation of Compound C-21

[0672] Compound Int17 (25 mg, 12.05 μmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propionate (4.37 mg, 16.26 μmol), and N,N-diisopropylethylamine (3.12 mg, 24.10 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 30% - 45%) to obtain the title compound (11.1 mg, yield: 41.4%).

[0673] MS m / z(ESI):1114.2[M / 2+H] + .

[0674] Drug Linker Preparation Example 1-22: Preparation of Compound C-22

[0675] Compound Int18 (20 mg, 9.50 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (5.56 mg, 15.21 μmol), and N,N-diisopropylethylamine (1.77 mg, 13.67 μmol) were added sequentially to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (8 mg, 37.6% yield).

[0676] MS m / z(ESI):1177.7[M / 2+H] + .

[0677] Drug-linker Preparation Example 1-23: Preparation of Compound C-23

[0678] Step 1: Preparation of compound 23-1

[0679] Dissolve (tert-Butoxycarbonyl)-L-lysine (500 mg, 1.93 mmol) in saturated aqueous sodium bicarbonate (10 mL) at 0°C, add methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (314.87 mg, 1.93 mmol), and react at 0°C for 40 minutes and then at 25°C for 50 minutes. Cool to 0°C, adjust the pH to approximately 3.0 with concentrated sulfuric acid, and extract with ethyl acetate. The organic phases are combined, washed with brine, and concentrated to give a crude product, which is purified by silica gel column chromatography (eluent: dichloromethane / methanol = 5 / 1 (v / v)) to afford the title compound (320 mg, yield: 50.9%).

[0680] MS m / z(ESI):349.1[M+Na] + .

[0681] Step 2: Preparation of compound 23-2

[0682] Compound 23-1 (23.75 mg, 72.78 μmol), VC-PABC-MMAE (81.76 mg, 72.78 μmol), N,N-diisopropylethylamine (14.25 mg, 109.25 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (39.13 mg, 101.89 μmol) were added sequentially to anhydrous N,N-dimethylformamide (3 mL) and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 30% - 35%) to obtain the title compound (80 mg, yield: 72.9%).

[0683] MS m / z(ESI):1431.8[M+H] + .

[0684] Step 3: Preparation of compound 23-3

[0685] Compound 23-2 (56 mg, 39.11 μmol) was dissolved in anhydrous dichloromethane (3 mL), and zinc bromide (71.18 mg, 2312.9 μmol) was added. The mixture was allowed to react at 25°C for 2 hours. The residue obtained by concentration of the reaction solution was dissolved in methanol and purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 30% - 35%) to obtain the title compound (28.5 mg, yield: 54.7%).

[0686] MS m / z(ESI):1331.7[M+H] + .

[0687] Step 4: Preparation of compound C-23

[0688] Compound Int14-3 (9.31 mg, 10.7 μmol), compound 23-3 (14.25 mg, 10.7 μmol), N,N-diisopropylethylamine (2.1 mg, 16.05 μmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.34 mg, 13.91 μmol) were added to anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method B, elution gradient: A% = 20%-30%) to obtain the title compound (15 mg, yield: 64.2%).

[0689] MS m / z(ESI):1092.7[M / 2+H] + .

[0690] Drug Linker Preparation Example 1-24: Preparation of Compound C-24

[0691] Step 1: Preparation of compound 24-1

[0692] Compound Int19 (28 mg, 15.34 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (8.41 mg, 23.01 μmol), and N,N-diisopropylethylamine (4.00 mg, 30.68 μmol) were added to N,N-dimethylformamide (1 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation A, elution gradient: A% = 35% - 40%) to obtain the title compound (30 mg, yield: 94.2%).

[0693] MS m / z(ESI):1038.5[M / 2+H] + .

[0694] Step 2: Preparation of compound C-24

[0695] Compound 24-1 (30 mg, 13.01 μmol) and anhydrous zinc bromide (11.72 mg, 52.04 μmol) were added to anhydrous dichloromethane (2 mL) and reacted at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (Preparation Method A, elution gradient: A% = 25% - 35%) to obtain the title compound (4.65 mg, yield: 16.8%).

[0696] MS m / z(ESI):1975.9[M+H] + .

[0697] Drug Linker Preparation Example 1-25: Preparation of Compound C-25

[0698] Compound Int20 (30 mg, 15.28 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (8.94 mg, 24.46 μmol), and N,N-diisopropylethylamine (2.84 mg, 21.75 μmol, 3.64 μL) were dissolved in anhydrous N,N-dimethylformamide (2 mL) and reacted at 0°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (20 mg, 62.2% yield).

[0699] MS m / z(ESI):1107.1[M / 2+H] + .

[0700] 2. Preparation of Immunostimulatory Antibody Drug Conjugates (iADC)

[0701] The B7H3 monoclonal antibody (Ab-01) stock solution was adjusted to the target pH value with 1 M disodium hydrogen phosphate solution. 0.1 M EDTA and TCEP (10 mM) were added to the resulting B7H3 monoclonal antibody (Ab-01) buffer solution, and the mixture was shaken and reacted at room temperature for 2 hours. DMSO solutions (10 mM) of drug linkers C-1 to C-25 were then added thereto, shaken and reacted at room temperature for 2 hours. The mixture was then purified using a NAP-5 gel column and washed with a histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solutions for immunostimulatory antibody drug conjugates (iADC) iADC-1 to iADC-25, which were stored at -20°C.

[0702] III. Drug / Antibody Ratio of Immunostimulatory Antibody Drug Conjugates (iADC): Determination of DAR Value

[0703] The molecular weight of iADC was determined by LC-MS, and the drug / antibody ratio (DAR) was calculated.

[0704] Chromatographic determination conditions:

[0705] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0706] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[0707] Flow rate: 0.25 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 1 μl;

[0708] Mass spectrometry conditions:

[0709] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0710] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5s;

[0711] m / z 600-4000; Time bins to sum 40.

[0712] The DAR values ​​of immunostimulatory antibody drug conjugates (iADCs) were calculated by CE-SDS, as shown in Table 1:

[0713] Table 1. Average DAR value test results of immunostimulatory antibody drug conjugates (iADC)

[0714] Biological tests

[0715] Experimental Example 1: Inhibitory Effect of Immunomodulatory Antibody Drug Conjugate (iADC) on the Proliferation of B7H3-Positive Tumor Cells

[0716] Experimental steps:

[0717] 1. Collect B7H3-positive tumor cells HCC1954 (Connolly) and NCI-N87 (ATCC) in the logarithmic growth phase and count them.

[0718] 2. The two tumor cells were seeded into 96-well plates (Corning), with 2000 cells (HCC1954) or 3000 cells (NCI-N87) per well in an 80 μL volume. The plates were cultured in a 37°C, 5% CO2 incubator overnight to allow the cells to adhere (18-24 h).

[0719] 3. Prepare a 5x drug working solution using a gradient concentration according to the experimental design. Remove the 96-well cell plate and add 20 μL of the working solution from the compound plate to the cell plate. Incubate the cell plate in a 37°C, 5% CO2 incubator for 72 hours.

[0720] 4. Test plate: Take out the cell plate and observe the cell status under a microscope. After the observation, follow the CellCounting- 2.0 kit (Novozymes) instructions, the cell plate was added with detection solution, and raw data were obtained using PHERA Star FS.

[0721] 5. Calculate the inhibition rate (IR%) = 1-(mean luminescence value of experimental wells / mean luminescence value of DMSO control wells) * 100%. Graphpad Prism 8.0 software was used for data analysis and graphing. The IC was calculated by fitting a four-parameter curve based on the logarithm of cell viability and drug concentration. 50 value.

[0722] Experimental results:

[0723] The inhibitory effect of each immunostimulatory antibody drug conjugate (iADC) on tumor cell proliferation was determined according to the above method, as shown in Table 2 and Table 3:

[0724] Table 2. Inhibitory effect of immunostimulatory antibody drug conjugates (iADCs) on the proliferation of HCC1954 tumor cells

[0725] Table 3. Inhibitory effect of immunostimulatory antibody drug conjugates (iADCs) on the proliferation of tumor cells NCI-N87

[0726] The results showed that each iADC had a strong proliferation inhibitory effect on HCC1954 cells and NCI-N87 cells.

[0727] Experimental Example 2: Inhibitory Effect of Immunomodulatory Antibody Drug Conjugate (iADC) on the Proliferation of Calu-6 Tumor Cells with Low HER2 Expression and B7H3 Positive Expression

[0728] Experimental steps:

[0729] 1. Collect HER2 low-expressing B7H3-positive tumor cells Calu-6 (Nanjing Kebai) in the logarithmic growth phase.

[0730] 2. Tumor cells were seeded into 96-well plates (Corning), with 8,000 cells per well in a volume of 80 μL. The plates were placed in a 37° C., 5% CO 2 incubator and cultured overnight to allow the cells to adhere (18 to 24 h).

[0731] 3. Prepare a 5x drug working solution using a gradient concentration according to the experimental design. Remove the 96-well cell plate and add 20 μL of the working solution from the compound plate to the cell plate. Incubate the cell plate in a 37°C, 5% CO2 incubator for 72 hours.

[0732] 4. Test plate: Take out the cell plate and observe the cell status under a microscope. After the observation, follow the CellCounting- 2.0 kit (Novozymes) instructions, the cell plate was added with detection solution, and raw data were obtained using PHERA Star FS.

[0733] Inhibition rate (IR%) was calculated as 1-(mean luminescence value of experimental wells / mean luminescence value of DMSO control wells)*100%. Graphpad Prism 8.0 software was used for data analysis and graphing. IC was calculated by fitting a four-parameter curve based on the logarithm of inhibition rate and drug concentration. 50 value.

[0734] Experimental results:

[0735] The proliferation inhibitory effect of each immunostimulatory antibody drug conjugate (iADC) on tumor cells Calu-6 was determined according to the above method. The results are shown in Table 4:

[0736] Table 4. Inhibitory effect of immunostimulatory antibody drug conjugates (iADCs) on the proliferation of tumor cells Calu-6

[0737] The results showed that each iADC had a strong inhibitory effect on the proliferation of Calu-6 cells.

[0738] Experimental Example 3: Determination of the ability of immunomodulatory antibody drug conjugates (iADCs) to stimulate TNF-α secretion from peripheral blood mononuclear cells (PBMCs) in the presence of tumor cells

[0739] Experimental steps:

[0740] 1. HCC1954 tumor cells (Connotai) in the logarithmic growth phase were collected, washed twice with PBS, and resuspended in RPMI1640 + 10% heat-inactivated FBS complete medium to adjust the cell density to 2×10 5 HCC1954 tumor cells were seeded in 96-well plates (Corning) at 1×10 cells / mL. 4 cells.

[0741] 2. Resuscitate PBMC (Sai ​​Li Biotechnology) and resuspend the cells in RPMI1640+10% heat-inactivated FBS complete medium to adjust the density to 6×10 5 10 cells / mL were added to a 96-well plate inoculated with tumor cells, with 3×10 cells / well 4 PBMC cells.

[0742] 3. Add 100 μL of the drug to be tested at different concentrations to the well plate to make the final concentrations 500 nM, 166.7 nM, 55.6 nM, 18.52 nM, 6.17 nM, 2.06 nM, and 0.69 nM, respectively. Add 100 μL of RPMI1640 + 10% heat-inactivated FBS complete medium to the blank wells.

[0743] 4. Incubate in a 5% CO2, 37°C incubator. After 24 hours, remove 50 μL of supernatant from each well and incubate with the primary antibody and HRP-conjugated secondary antibody according to the human TNF-α ELISA kit (Invitrogen). Develop the color and read the absorbance at 450 nm. Calculate the TNF-α concentration using Graphpad Prism 8.0 software based on the standard curve.

[0744] Experimental results:

[0745] The ability of each immunostimulatory antibody drug conjugate iADC to stimulate human peripheral blood mononuclear cells (PBMC) to secrete TNF-α in the presence of HCC1954 tumor cells was determined according to the above method. The results are shown in Table 5:

[0746] Table 5 Results of immunostimulatory antibody drug conjugates (iADC) stimulating TNF-α secretion

[0747] The results showed that each iADC could effectively stimulate PBMC to secrete TNF-α in the in vitro co-incubation system of HCC1954 tumor cells and PBMC. Other iADCs in this application also have similar ability to stimulate PBMC to secrete TNF-α, such as iADC-1, iADC-8, iADC-10, iADC-11, iADC-13, iADC-18, iADC-23, iADC-24, etc., all have EC values ​​less than 600nM. 50 value.

[0748] Experimental Example 4: In vivo efficacy study of immunomodulatory antibody drug conjugate (iADC) in a CT26-hB7H3 cell subcutaneous transplant tumor model

[0749] Experimental steps:

[0750] 1. Each Balb / c mouse (Chengdu Yaokang) was subcutaneously inoculated with 3×10 5 CT26-hB7H3 cells (suspended in 0.1 ml PBS).

[0751] 2. Wait until the average tumor volume grows to about 100mm3 When the time came, mice with irregular, too small or too large tumor volumes were excluded. The remaining mice were randomly grouped according to tumor volume and animal body weight, and were respectively given 0.9% sodium chloride injection (vehicle control) by intravenous injection (i.v.), iADC-8 (20 mg / kg, administered once a week for a total of two times), iADC-9 (20 mg / kg, administered once), iADC-10 (23.81 mg / kg, administered once). The efficacy of the test compound on this tumor-bearing mouse model and the tolerance of the animals to the test compound were observed.

[0752] 3. Weigh the mice and measure the tumor volume twice a week during the experiment, and record the data.

[0753] 4. Data statistics, the calculation formula for tumor volume (V): V = 1 / 2 × a × b 2 , where a and b represent the length and width respectively. The anti-tumor drug efficacy is evaluated by the tumor growth inhibition rate TGI (%), and the calculation formula: TGI (%) (瘤体积) = [1 - (T Vt - T V0 ) / (C Vt - C V0 )] × 100%, T V0 is the average tumor volume of the test compound group when grouped and administered; T Vt is the average tumor volume of the test compound group at t days after administration; C V0 is the average tumor volume of the vehicle group when grouped and administered; C Vt is the average tumor volume of the vehicle group at t days after administration. When the tumor shows regression, TGI (%) (瘤体积) = 100% - (T Vt - T V0 ) / T V0 × 100%. If the tumor shrinks compared to the initial volume, that is, when V t < V0, it is defined as partial regression (PR) of the tumor; if the tumor completely disappears, it is defined as complete regression (CR) of the tumor.

[0754] Experimental results:

[0755] After administration, each test substance was able to significantly inhibit tumor growth. Compared with the solvent control group, the TGI of iADC-8 17 days after administration was 60.72%, P < 0.05; the TGI of iADC-9 18 days after administration was 145.74%, P < 0.001; and the TGI of iADC-10 18 days after administration was 98.02%, P < 0.01 (Table 6). The results showed that iADC-8, iADC-9, and iADC-10 had significant tumor inhibition effects in the CT26-hB7H3 cell subcutaneous transplant tumor model. In addition, no mice died in any of the test groups, and the mice did not lose significant weight (Table 7), indicating that the mice tolerated the test substances well.

[0756] Table 6. Efficacy of immunostimulatory antibody drug conjugates in CT26-hB7H3 cell subcutaneous xenografts

[0757] PR: indicates partial regression.

[0758] Table 7. Changes in body weight of mice during in vivo drug efficacy of CT26-hB7H3 cell subcutaneous transplanted tumors

[0759] +: weight gain; -: weight loss.

[0760] Experimental Example 5: In vivo efficacy study of immunostimulatory antibody drug conjugate (iADC) in a MC38-hB7H3 cell subcutaneous transplant tumor model

[0761] Experimental steps:

[0762] 1. Each C57BL / 6J mouse (Chengdu Yaokang) was subcutaneously inoculated with 1×10 6 MC38-hB7H3 cells (suspended in 0.1 ml PBS).

[0763] 2. Wait until the average tumor volume grows to about 100mm 3 At the same time, mice with irregular, too small, or too large tumors were eliminated. The remaining mice were randomly divided into groups according to tumor volume and animal weight and given 0.9% sodium chloride injection (vehicle control), iADC-11 (10 mg / kg, single dose), or iADC-12 (21.2 mg / kg, single dose) via tail vein (iv) injection. The efficacy of the test compounds in the tumor-bearing mouse model and the animals' tolerance to the test compounds were observed.

[0764] 3. During the experiment, weigh the mice and measure the tumor volume twice a week, and record the data.

[0765] 4. Data statistics, the calculation formula for tumor volume (V): V = 1 / 2 × a × b 2 , where a and b represent the length and width respectively. Evaluation of the efficacy of anti-tumor drugs by tumor growth inhibition rate TGI (%), the calculation formula: TGI (%) (瘤体积) = [1 - (T Vt - T V0 ) / (C Vt - C V0 )] × 100%, T V0 is the average tumor volume of the test compound group during grouped administration, T Vt is the average tumor volume of the test compound group at t days after administration; C V0 is the average tumor volume of the vehicle group during grouped administration; C Vt is the average tumor volume of the vehicle group at t days after administration. When the tumor regresses, TGI (%) (瘤体积) = 100% - (T Vt - T V0 ) / T V0 × 100%. If the tumor shrinks compared to the initial volume, that is, when V t < V0, it is defined as partial regression (PR) of the tumor; if the tumor completely disappears, it is defined as complete regression (CR) of the tumor.

[0766] Experimental results:

[0767] After administration, both iADC-11 and iADC-12 could significantly inhibit tumor growth. Compared with the vehicle control group, the TGI of iADC-11 at 32 days after administration was 94.33%, P < 0.01; the TGI of iADC-12 at 32 days after administration was 60.18%, P < 0.01 (Table 8). The results showed that in the subcutaneous xenograft tumor model of MC38-hB7H3 cells, iADC-11 and iADC-12 had anti-tumor effects. In addition, no mice died in each test group, and there was no significant weight loss in mice (Table 9), indicating that the mice had good tolerance to each test substance.

[0768] Table 8. Pharmacodynamic effects of immunostimulatory antibody-drug conjugates on subcutaneous xenograft tumors of MC38-hB7H3 cells

[0769] PR: Indicates partial regression; CR: Indicates complete regression.

[0770] Table 9. Changes in mouse body weight in the in vivo pharmacodynamic effects of subcutaneous xenograft tumors of MC38-hB7H3 cells

[0771] +: Weight gain; -: Weight loss.

[0772] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims

1. An antibody-drug conjugate represented by Formula (I):Iwherein,Ab' represents an antibody or antigen-binding fragment thereof that specifically binds to B7H3;M represents a linking site connected to the antibody or antigen-binding fragment thereof;X represents a linker connecting M and Aa;Aa is an amino acid fragment or a peptide fragment formed from two or more amino acids;L1 represents a linker connecting Aa and D1;L2 represents a linker connecting Aa and D2;D1 represents a cytotoxic drug moiety;D2 represents a TLR agonist moiety;m ranges from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

2. The antibody-drug conjugate according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises:(1) the following heavy chain variable region (VH) and / or light chain variable region (VL):a heavy chain variable region (VH) comprising the following 3 CDRs: a CDR-H1 having a sequence as set forth in SEQ ID NO: 11 or a variant thereof, a CDR-H2 having a sequence as set forth in SEQ ID NO: 12 or a variant thereof, and a CDR-H3 having a sequence as set forth in SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: a CDR-L1 having a sequence as set forth in SEQ ID NO: 6 or a variant thereof, a CDR-L2 having a sequence as set forth in SEQ ID NO: 7 or a variant thereof, and a CDR-L3 having a sequence as set forth in SEQ ID NO: 5 or a variant thereof;(2) the following heavy chain variable region (VH) and / or light chain variable region (VL):a heavy chain variable region (VH) comprising the following 3 CDRs: a CDR-H1 having a sequence as set forth in SEQ ID NO: 14 or a variant thereof, a CDR-H2 having a sequence as set forth in SEQ ID NO: 15 or a variant thereof, and a CDR-H3 having a sequence as set forth in SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: a CDR-L1 having a sequence as set forth in SEQ ID NO: 6 or a variant thereof, a CDR-L2 having a sequence as set forth in SEQ ID NO: 7 or a variant thereof, and a CDR-L3 having a sequence as set forth in SEQ ID NO: 5 or a variant thereof;(3) the following heavy chain variable region (VH) and / or light chain variable region (VL):a heavy chain variable region (VH) comprising the following 3 CDRs: a CDR-H1 having a sequence as set forth in SEQ ID NO: 8 or a variant thereof, a CDR-H2 having a sequence as set forth in SEQ ID NO: 9 or a variant thereof, and a CDR-H3 having a sequence as set forth in SEQ ID NO: 10 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: a CDR-L1 having a sequence as set forth in SEQ ID NO: 3 or a variant thereof, a CDR-L2 having a sequence as set forth in SEQ ID NO: 4 or a variant thereof, and a CDR-L3 having a sequence as set forth in SEQ ID NO: 5 or a variant thereof; or(4) the following heavy chain variable region (VH) and / or light chain variable region (VL):a heavy chain variable region (VH) comprising the following 3 CDRs: a CDR-H1 having a sequence as set forth in SEQ ID NO: 16 or a variant thereof, a CDR-H2 having a sequence as set forth in SEQ ID NO: 17 or a variant thereof, and a CDR-H3 having a sequence as set forth in SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: a CDR-L1 having a sequence as set forth in SEQ ID NO: 6 or a variant thereof, a CDR-L2 having a sequence as set forth in SEQ ID NO: 7 or a variant thereof, and a CDR-L3 having a sequence as set forth in SEQ ID NO: 5 or a variant thereof.

3. The antibody-drug conjugate according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises:a VH as set forth in SEQ ID NO: 23 or 1 or a variant thereof, and / or, a VL as set forth in SEQ ID NO: 2 or a variant thereof;preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain comprising a VH as set forth in SEQ ID NO: 23 or 1 or a variant thereof, and a heavy chain constant region (CH) as set forth in SEQ ID NO: 18 or a variant thereof, and / or, a light chain comprising a VL as set forth in SEQ ID NO: 2 or a variant thereof, and a light chain constant region (CL) as set forth in SEQ ID NO: 19 or a variant thereof;preferably, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 22 or 20, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 21;optionally, the N-terminal glutamine of the heavy chain or heavy chain variable region and / or the light chain or light chain variable region undergoes cyclization to form a pyroglutamic acid or pyroglutamate; and / or, the heavy chain or heavy chain constant region (CH) or variant thereof lacks a C-terminal lysine.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein M is a covalent bond or is selected from the group consisting of the following structures:, , , , and ,wherein, each a is independently an integer ranging from 0 to 6, b is an integer ranging from 1 to 10, and the position 1 of M is connected to Ab', and the position 2 is connected to X;preferably, M is a covalent bond or is selected from the group consisting of the following structures: , , and .

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein X is a covalent bond or is selected from the group consisting of C1-6 alkylene, -NH-(CH2)c-C(O)-, , , , , , and , preferably a covalent bond or ;wherein, each c is independently an integer ranging from 0 to 6, and each d is independently an integer ranging from 1 to 10; the position 3 of X is connected to M, and the position 4 is connected to Aa.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein Aa is selected from the group consisting of the following amino acid fragments: Gly, Phe, Ala, Val, Cys, Asp, Glu, Lys, Nle, and Arg;preferably, Aa is selected from the group consisting of the following structures:, , , , , , , , , , , , , , , , , , , , , , , , , , and ;wherein, the position 5 of Aa is connected to X, the position 6 is connected to L1, and the position 7 is connected to L2.

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein L1 is a covalent bond or is selected from the group consisting of non-cleavable linkers and cleavable linkers, wherein the cleavable linker is cleavable by an enzyme present in a pathological environment, and the enzyme is selected from the group consisting of a protease, a phosphatase, a pyrophosphatase, β-glucuronidase, β-galactosidase, and a sulfatase;preferably, L1 is -La-Lb-Lc-, wherein:La is a covalent bond or is selected from the group consisting of C1-6 alkylene, -NH-(CH2)e-C(O)-, , , , , , , , and ; wherein, each e is independently an integer ranging from 0 to 6, and each f is independently an integer ranging from 0 to 10;Lb is a covalent bond or is an amino acid fragment or a peptide fragment formed by two or more amino acids, wherein the amino acid is selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala, and Asn; Lc is a covalent bond or is selected from the group consisting of -NH-CH2- and the following structures:, , , , and ;preferably, La is a covalent bond or is selected from the group consisting of C1-6 alkylene, -NH-(CH2)e-C(O)-, , , , , , , and ;preferably, Lb is a covalent bond or is selected from the group consisting of the following structures:, , , , , , , , , , , , , , , , , , , , , , , , , and .

8. The antibody-drug conjugate according to any one of claims 1 to 7, wherein L1 is a covalent bond or is selected from the group consisting of the following structures:, , , , , , , , , , , , and ;wherein, each e is independently an integer ranging from 0 to 6, and each f is independently an integer ranging from 1 to 10; the position of L1 is connected to Aa, and the position 9 is connected to D1;preferably, L1 is:, , , , , and ;wherein, the position 8 of L1 is connected to Aa, and the position is connected to D1.

9. The antibody-drug conjugate according to any one of claims 1 to 8, wherein L2 is selected from the group consisting of non-cleavable linkers and cleavable linkers, the cleavable linker is cleavable by an enzyme present in a pathological environment, and the enzyme is selected from the group consisting of a protease, a phosphatase, a pyrophosphatase, β-glucuronidase, β-galactosidase, and a sulfatase.

10. The antibody-drug conjugate according to any one of claims 1 to 9, wherein L2 is selected from the group consisting of the following structures:, , , , , , , , and ;wherein each g is independently an integer ranging from 0 to 6, and each h is independently an integer ranging from 1 to 10; the position 10 of L2 is connected to Aa, and the position 11 is connected to D2;preferably, L2 is selected from the group consisting of the following structures:, , , , and ;wherein, the position 10 of L2 is connected to Aa, and the position 11 is connected to D2.

11. The antibody-drug conjugate according to any one of claims 1 to 10, wherein D1 is selected from the group consisting of cytotoxic drug moieties, wherein the cytotoxic drug includes tubulin inhibitors, DNA damaging agents, and topoisomerase inhibitors; the tubulin inhibitors include dolastatin and auristatins, maytansines, tubulysins, and cryptomycins; the DNA damaging agents include pyrrolobenzodiazepines, duocarmycins, and calicheamicins; and the topoisomerase inhibitors include camptothecin and derivatives thereof, epipodophyllotoxins, anthracyclines, and anthraquinones;preferably, the tubulin inhibitor is selected from the group consisting of dolastatin 10, MMAE, MMAF, maytansine, DM1, DM3, and DM4; and the topoisomerase inhibitor is selected from the group consisting of camptothecin, SN-38, exatecan, topotecan, belotecan, rubitecan, diflomotecan, 10-hydroxycamptothecin, 9-aminocamptothecin, Dxd, T030, doxorubicin, epirubicin, and PNU-159682;preferably, D1 is selected from the group consisting of the following structures:, , , and .

12. The antibody-drug conjugate according to any one of claims 1 to 11, wherein D2 is a TLR agonist moiety, and the TLR agonist is selected from the group consisting of a TLR2 agonist, a TLR4 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist, and a TLR9 agonist;preferably, the TLR agonist is selected from the group consisting of a TLR7 agonist, a TLR8 agonist, and a TLR7 / 8 agonist;preferably, D2 is selected from the group consisting of the following structures:、、、、、、、、、、 and .

13. The antibody-drug conjugate according to any one of claims 1 to 12, wherein D2 is selected from the group consisting of the following structures:, (for example ), , , , , , , , , , , , and ,wherein, e is an integer ranging from 0 to 6, and f is an integer ranging from 1 to 10; the position 5 is connected to X, the position 7 is connected to L2, and the position 9 is connected to D1.

14. The antibody-drug conjugate according to any one of claims 1 to 13, wherein is selected from the group consisting of:, , , , , , , , , , and ; wherein, each g is independently an integer ranging from 0 to 6, and each h is independently an integer ranging from 1 to 10;preferably, is selected from the group consisting of:, , , , , , , , , , and ,wherein, the position 5 is connected to X, the position 6 is connected to L1, and the position 11 is connected to D2.

15. The antibody-drug conjugate according to any one of claims 1 to 14, wherein is selected from the group consisting of:, , , , , , , , , , , , , , , and , wherein, each g is independently an integer ranging from 0 to 6, and each h is independently an integer ranging from 1 to 10;preferably, is selected from the group consisting of:, , , , , , , , , , , , , , , and ;wherein, the position 5 is connected to X, the position 9 is connected to D1, and the position 11 is connected to D2.

16. The antibody-drug conjugate according to any one of claims 1 to 15, wherein is selected from the group consisting of:, (for example ), , , , , and ,wherein, each e is independently an integer ranging from 0 to 6, and each f is independently an integer ranging from 1 to 10.

17. The antibody-drug conjugate according to any one of claims 1 to 16, wherein is selected from the group consisting of:, , , , , , , , , and ; wherein, each g is independently an integer ranging from 0 to 6, and each h is independently an integer ranging from 1 to 10;preferably, conjugate is selected from the group consisting of:, , , , , , , , , and .

18. The antibody-drug conjugate according to any one of claims 1 to 17, wherein is selected from the group consisting of:, , , , , , , , , , , , , and ,wherein each a is independently an integer ranging from 0 to 6, each c is independently an integer ranging from 0 to 6, and each d is independently an integer ranging from 1 to 10;preferably, is selected from the group consisting of:, , , , , , , , , , , , , , and ;wherein the position 1 is connected to Ab', the position 6 is connected to L1, and the position 7 is connected to L2.

19. The antibody-drug conjugate according to any one of claims 1 to 18, wherein is selected from the group consisting of:, , , , , , , , , , , , , , , , , , , , , and , wherein, each a is independently an integer ranging from 0 to 6, each c is independently an integer ranging from 0 to 6, each d is independently an integer ranging from 1 to 10, each e is independently an integer ranging from 0 to 6, each f is independently an integer ranging from 1 to 10, each g is independently an integer ranging from 0 to 6, and each h is independently an integer ranging from 1 to 10; preferably, is selected from the group consisting of:, , , , , , , , , , , , , , , , , , , , , and ,wherein, the position 1 is connected to Ab', the position 9 is connected to D1, and the position 11 is connected to D2.

20. The antibody-drug conjugate according to any one of claims 1 to 19, wherein is selected from the group consisting of: and ,wherein, each g is independently an integer ranging from 0 to 6, and each h is independently an integer ranging from 1 to 10;preferably, is selected from the group consisting of: and wherein, the position 5 is connected to X.

21. The antibody-drug conjugate according to any one of claims 1 to 20, which has a structure selected from the group consisting of:, , , , , , , , , , , , , , , , , , , , , , , and ;each m independently ranges from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Ab-(S- is the antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 20;preferably, Ab-(S- in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO: 23 or a VH as set forth in SEQ ID NO: 1 and a VL as set forth in SEQ ID NO: 2, for example, Ab-01 (which comprises a heavy chain as set forth in SEQ ID NO: 22 and a light chain as set forth in SEQ ID NO: 21), for example, Ab-02 (which comprises a heavy chain as set forth in SEQ ID NO: 20 and a light chain as set forth in SEQ ID NO: 21); optionally, the N-terminal glutamine of the heavy chain or heavy chain variable region and / or the light chain or light chain variable region undergoes cyclization to form a pyroglutamic acid or pyroglutamate; and / or, the heavy chain or heavy chain constant region (CH) or a variant thereof lacks C-terminal lysine.

22. A composition, which comprises the antibody-drug conjugate according to any one of claims 1 to 21, wherein the composition has a DAR value of 1.0 to 10.0, preferably 1.0 to 5.0, for example, 1.0 to 1.5, 1.0 to 2.0, 1.0 to 2.5, 1.0 to 3.0, 1.0 to 3.5, 1.0 to 4.0, 1.0 to 4.5, 1.0 to 5.0, 2.0 to 2.5, 2.0 to 3.0, 2.0 to 3.5, 2.0 to 4.0, 2.0 to 4.5, 2.0 to 5.0, 2.5 to 3.0, 2.5 to 3.5, 2.5 to 4.0, 2.5 to 4.5, 2.5 to 5.0, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 4.0 to 4.5, 4.0 to 5.0, 4.5 to 5.0, for example, about 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.30, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.40, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, about 1.5, about 1.51, about 1.52, about 1.53, about 1.54, about 1.55, about 1.56, about 1.57, about 1.58, about 1.59, about 1.6, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.7, about 1.71, about 1.72, about 1.73, about 1.74, about 1.75, about 1.76, about 1.77, about 1.78, about 1.79, about 1.8, about 1.81, about 1.82, about 1.83, about 1.84, about 1.85, about 1.86, about 1.87, about 1.88, about 1.89, about 1.9, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99, about 2.0, about 2.01, about 2.02, about 2.03, about 2.04, about 2.05, about 2.06, about 2.07, about 2.08, about 2.09, about 2.1, about 2.11, about 2.12, about 2.13, about 2.14, about 2.15, about 2.16, about 2.17, about 2.18, about 2.19, about 2.2, about 2.21, about 2.22, about 2.23, about 2.24, about 2.25, about 2.26, about 2.27, about 2.28, about 2.29, about 2.3, about 2.31, about 2.32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, about 2.53, about 2.54, about 2.55, about 2.56, about 2.57, about 2.58, about 2.59, about 2.6, about 2.61, about 2.62, about 2.63, about 2.64, about 2.65, about 2.66, about 2.67, about 2.68, about 2.69, about 2.7, about 2.71, about 2.72, about 2.73, about 2.74, about 2.75, about 2.76, about 2.77, about 2.78, about 2.79, about 2.8, about 2.81, about 2.82, about 2.83, about 2.84, about 2.85, about 2.86, about 2.87, about 2.88, about 2.89, about 2.9, about 2.91, about 2.92, about 2.93, about 2.94, about 2.95, about 2.96, about 2.97, about 2.98, about 2.99, about 3.0, about 3.01, about 3.02, about 3.03, about 3.04, about 3.05, about 3.06, about 3.07, about 3.08, about 3.09, about 3.1, about 3.11, about 3.12, about 3.13, about 3.14, about 3.15, about 3.16, about 3.17, about 3.18, about 3.19, about 3.2, about 3.21, about 3.22, about 3.23, about 3.24, about 3.25, about 3.26, about 3.27, about 3.28, about 3.29, about 3.3, about 3.31, about 3.32, about 3.33, about 3.34, about 3.35, about 3.36, about 3.37, about 3.38, about 3.39, about 3.4, about 3.41, about 3.42, about 3.43, about 3.44, about 3.45, about 3.46, about 3.47, about 3.48, about 3.49, about 3.5, about 3.51, about 3.52, about 3.53, about 3.54, about 3.55, about 3.56, about 3.57, about 3.58, about 3.59, about 3.6, about 3.61, about 3.62, about 3.63, about 3.64, about 3.65, about 3.66, about 3.67, about 3.68, about 3.69, about 3.7, about 3.71, about 3.72, about 3.73, about 3.74, about 3.75, about 3.76, about 3.77, about 3.78, about 3.79, about 3.8, about 3.81, about 3.82, about 3.83, about 3.84, about 3.85, about 3.86, about 3.87, about 3.88, about 3.89, about 3.9, about 3.91, about 3.92, about 3.93, about 3.94, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4.0, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.06, about 4.07, about 4.08, about 4.09, about 4.1, about 4.11, about 4.12, about 4.13, about 4.14, about 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.2, about 4.21, about 4.22, about 4.23, about 4.24, about 4.25, about 4.26, about 4.27, about 4.28, about 4.29, about 4.3, about 4.31, about 4.32, about 4.33, about 4.34, about 4.35, about 4.36, about 4.37, about 4.38, about 4.39, about 4.4, about 4.41, about 4.42, about 4.43, about 4.44, about 4.45, about 4.46, about 4.47, about 4.48, about 4.49, about 4.5, about 4.51, about 4.52, about 4.53, about 4.54, about 4.55, about 4.56, about 4.57, about 4.58, about 4.59, about 4.6, about 4.61, about 4.62, about 4.63, about 4.64, about 4.65, about 4.66, about 4.67, about 4.68, about 4.69, about 4.7, about 4.71, about 4.72, about 4.73, about 4.74, about 4.75, about 4.76, about 4.77, about 4.78, about 4.79, about 4.8, about 4.81, about 4.82, about 4.83, about 4.84, about 4.85, about 4.86, about 4.87, about 4.88, about 4.89, about 4.9, about 4.91, about 4.92, about 4.93, about 4.94, about 4.95, about 4.96, about 4.97, about 4.98, about 4.99, or about 5.0.

23. A compound represented by Formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:wherein,X, Aa, L1, L2, D1, and D2 are as defined in any one of claims 1 to 21;M1 is a precursor of the linking site that is attached to an antibody or antigen-binding fragment thereof;preferably, the antibody or antigen-binding fragment thereof is as defined in any one of claims 1 to 21;preferably, M1 is selected from the group consisting of the following structures:, , , , and ,more preferably, M1 is selected from the group consisting of the following structures:, , , , and ;more preferably, M1 is selected from the group consisting of the following structures: , , and ;wherein, each a is independently an integer ranging from 0 to 6, and b is an integer ranging from 1 to 10;LG represents a leaving group,preferably, each LG is independently selected from the group consisting of halogen (e.g., F, Cl, Br, I), halogenated C1-6 alkyl, C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated sulfonyl, C1-6 alkylsulfonate group, halogenated C1-6 alkylsulfonate group, C1-6 alkylsulfinate group, C1-6 alkylsulfoxide group, halogenated phenoxy, hydroxyl, sulfhydryl group, amino, nitro, azido, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragment, wherein the halogenated C1-6 alkyl, halogenated C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated sulfonyl, C1-6 alkylsulfonate group, halogenated C1-6 alkylsulfonate group, C1-6 alkylsulfinate group, C1-6 alkylsulfoxide group, halogenated phenoxy, alkenyl, alkynyl, and alkynyl-containing structural fragment are optionally substituted with one or more suitable substituents;preferably, LG is independently selected from the group consisting of halogen (e.g., F, Cl, Br, I), halogenated C1-6 alkyl, C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated sulfonyl, C1-6 alkylsulfonate group, halogenated C1-6 alkylsulfonate group, C1-6 alkylsulfinate group, C1-6 alkylsulfoxide group, halogenated phenoxy, hydroxyl, sulfhydryl group, amino, nitro, azido, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragment;more preferably, LG is C1-6 alkylsulfonyl, such as methylsulfonyl.

24. The compound or the pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof according to claim 23, wherein:M1 is , preferably , more preferably , a is an integer ranging from 0 to 6;L2 is , preferably , wherein g is an integer ranging from 0 to 6, h is an integer ranging from 1 to 10, and the position 10 of L2 is connected to Aa, and the position 11 is connected to D2;D2 is .

25. The compound or the pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof according to claim 23 or 24, wherein is selected from , preferably , more preferably , wherein, a is an integer ranging from 0 to 6; the position 6 is connected to L1, and the position 7 is connected to L2.

26. The compound or the pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof according to any one of claims 23 to 25, wherein is selected from ,preferably ,more preferably ,wherein, a is an integer ranging from 0 to 6, g is an integer ranging from 0 to 6, and h is an integer ranging from 1 to 10; the position 9 is connected to D1, and the position 11 is connected to D2.

27. The compound or the pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof according to any one of claims 23 to 26, wherein the compound is selected from the group consisting of the following structures: and .

28. An antibody or antigen-binding fragment thereof that specifically binds to B7H3, wherein the antibody or antigen-binding fragment thereof comprises:(1) the following heavy chain variable region (VH) and / or light chain variable region (VL): a heavy chain variable region (VH) comprising the following 3 CDRs: a CDR-H1 having a sequence as set forth in SEQ ID NO: 11 or a variant thereof, a CDR-H2 having a sequence as set forth in SEQ ID NO: 12 or a variant thereof, and a CDR-H3 having a sequence as set forth in SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: a CDR-L1 having a sequence as set forth in SEQ ID NO: 6 or a variant thereof, a CDR-L2 having a sequence as set forth in SEQ ID NO: 7 or a variant thereof, and a CDR-L3 having a sequence as set forth in SEQ ID NO: 5 or a variant thereof; (2) the following heavy chain variable region (VH) and / or light chain variable region (VL): a heavy chain variable region (VH) comprising the following 3 CDRs: a CDR-H1 having a sequence as set forth in SEQ ID NO: 14 or a variant thereof, a CDR-H2 having a sequence as set forth in SEQ ID NO: 15 or a variant thereof, and a CDR-H3 having a sequence as set forth in SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: a CDR-L1 having a sequence as set forth in SEQ ID NO: 6 or a variant thereof, a CDR-L2 having a sequence as set forth in SEQ ID NO: 7 or a variant thereof, and a CDR-L3 having a sequence as set forth in SEQ ID NO: 5 or a variant thereof;(3) the following heavy chain variable region (VH) and / or light chain variable region (VL):a heavy chain variable region (VH) comprising the following 3 CDRs: a CDR-H1 having a sequence as set forth in SEQ ID NO: 8 or a variant thereof, a CDR-H2 having a sequence as set forth in SEQ ID NO: 9 or a variant thereof, a CDR-H3 having a sequence as set forth in SEQ ID NO: 10 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: a CDR-L1 having a sequence as set forth in SEQ ID NO: 3 or a variant thereof, a CDR-L2 having a sequence as set forth in SEQ ID NO: 4 or a variant thereof, and a CDR-L3 having a sequence as set forth in SEQ ID NO: 5 or a variant thereof; or(4) the following heavy chain variable region (VH) and / or light chain variable region (VL):a heavy chain variable region (VH) comprising the following 3 CDRs: a CDR-H1 having a sequence as set forth in SEQ ID NO: 16 or a variant thereof, a CDR-H2 having a sequence as set forth in SEQ ID NO: 17 or a variant thereof, a CDR-H3 having a sequence as set forth in SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: a CDR-L1 having a sequence as set forth in SEQ ID NO: 6 or a variant thereof, a CDR-L2 having a sequence as set forth in SEQ ID NO: 7 or a variant thereof, and a CDR-L3 having a sequence as set forth in SEQ ID NO: 5 or a variant thereof. preferably, the antibody or antigen-binding fragment thereof comprises: a VH as set forth in SEQ ID NO: 23 or 1 or a variant thereof, and / or, a VL as set forth in SEQ ID NO: 2 or a variant thereof;preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain comprising a VH as set forth in SEQ ID NO: 23 or 1 or a variant thereof and a heavy chain constant region (CH) as set forth in SEQ ID NO: 18 or a variant thereof, and / or, a light chain comprising a VL as set forth in SEQ ID NO: 2 or a variant thereof and a light chain constant region (CL) as set forth in SEQ ID NO: 19 or a variant thereof;preferably, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 22 or 20, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 21;optionally, the N-terminal glutamine of the heavy chain or heavy chain variable region and / or the light chain or light chain variable region undergoes cyclization to form a pyroglutamic acid or pyroglutamate; and / or, the heavy chain or heavy chain constant region (CH) or variant thereof lacks a C-terminal lysine;preferably, the variant has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence from which it is derived, or has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) as compared with the sequence from which it is derived; preferably, the substitution is a conservative substitution.

29. A pharmaceutical composition, which comprises the antibody-drug conjugate according to any one of claims 1 to 21, the composition according to claim 22, the compound or the pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof according to any one of claims 23 to 27 or the antibody or antigen-binding fragment thereof according to claim 28, and one or more pharmaceutical excipients.

30. Use of the compound or the pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof according to any one of claims 23 to 27 or the antibody or antigen-binding fragment thereof according to claim 28, in the manufacture of an antibody-drug conjugate (e.g., the antibody-drug conjugate according to any one of claims 1 to 21).

31. Use of the antibody-drug conjugate according to any one of claims 1-21, the composition according to claim 22, the compound or the pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof according to any one of claims 23 to 27, the antibody or antigen-binding fragment thereof according to claim 28 or the pharmaceutical composition according to claim 29, in the manufacture of a medicament, particularly a medicament for treating and / or preventing a cancer (e.g., a cancer associated with B7H3 expression);preferably, the cancer is a B7H3-positive cancer;preferably, the cancer is selected from the group consisting of breast cancer (e.g., breast ductal carcinoma), gastric cancer, lung cancer (e.g., lung adenocarcinoma), and colon cancer.

32. A method for treating and / or preventing cancer (e.g., cancer associated with B7H3 expression), comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of the antibody-drug conjugate according to any one of claims 1-21, the composition according to claim 22, the compound or the pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof according to any one of claims 23 to 27, the antibody or antigen-binding fragment thereof according to claim 28 or the pharmaceutical composition according to claim 29;preferably, the cancer is a B7H3-positive cancer;preferably, the cancer is selected from the group consisting of breast cancer (e.g., breast ductal carcinoma), gastric cancer, lung cancer (e.g., lung adenocarcinoma), and colon cancer.

33. A compound as shown below, or a salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:wherein, PG1 is each independently H or an amino-protecting group, preferably an alkoxycarbonyl amino-protecting group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl(or ethyl)oxycarbonyl; an acyl amino-protecting group, such as phthaloyl (Pht), tosyl (Tos), trifluoroacetyl (Tfa), o-(p-)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; an alkyl amino-protecting group, such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), or benzyl (Bn).

34. A compound as shown below, or a salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof: or.

35. A compound represented by Formula (III), or a salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:,wherein,X, Aa, L1, L2, and M1 are as defined in any one of claims 23 to 27;G1 is p-nitrophenoxy or C1-6 alkanoyloxy;G2 is hydroxy or oxo;preferably, the compound is selected from the group consisting of: and .

36. A linker unit which is a compound having a structure represented by Formula (IV), or a salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:wherein, X, Aa, L1, a, g, and h are as defined in any one of claims 1 to 21.

37. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, which comprises the linker unit according to claim 36 and at least one of LG or D1, wherein the linker unit is connected to LG or D1, wherein D1 is connected to the position 9 of the linker unit; and LG is connected to the position 1 of the linker unit; wherein D1 is as defined in any one of claims 1 to 21, and LG is as defined in any one of claims 23 to 27.

38. An antibody-drug conjugate, which comprises the compound or the pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof according to any one of claims 23 to 27, and / or, the antibody or antigen-binding fragment thereof according to claim 28.

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