Anti-b7h3 antibody, ligand-drug conjugate, and use thereof

Abstract

Provided are an anti-B7H3 antibody, a ligand-drug conjugate, and use thereof. The anti-B7H3 antibody or antigen-binding fragment can specifically bind to B7H3, has a high affinity, and can be used for the prevention and / or treatment of B7H3-mediated diseases or conditions.

Description

Anti-B7H3 antibodies, ligand-drug conjugates and their applications Technical Field

[0001] This invention belongs to the field of cellular immunology and biotechnology, specifically relating to antibodies, ligand-drug conjugates targeting B7H3 and their applications. Background Technology

[0002] B7 homologue 3 (B7H3, also known as CD276) is a type I transmembrane protein encoded by the CD276 gene on human chromosome 15. Due to exon replication, the extracellular domain in humans consists of two identical pairs of immunoglobulin variable and immunoglobulin constant domains (4IgB7-H3 isoform). B7H3 has a short intracellular tail and lacks known signal transduction motifs. B7H3 is universally expressed across species. A soluble form can also be detected in human serum, produced by surface cleavage by matrix metalloproteinases (MMPs) or by alternative splicing of introns.

[0003] B7H3 is induced on antigen-presenting cells and plays a crucial role in the suppression of T-cell function. Importantly, B7H3 is highly overexpressed in a wide range of human solid cancers and is generally associated with both negative prognosis and poor clinical outcomes in patients. Immunochemical assays show aberrant B7H3 expression in tumor tissues of over 60% and up to 93% of patients across the vast majority of cancer types, while limited expression is observed in normal healthy tissues. In most cases, high B7H3 expression is associated with poor prognosis and adverse clinical outcomes. Therefore, there is a need for improved therapies targeting B7H3.

[0004] Antibody-drug conjugates (ADCs) represent a relatively new class of therapeutic agents that comprise antibodies conjugated to cytotoxic drugs via chemical linkers. The therapeutic concept of ADCs combines the binding ability of antibodies and drugs, where the antibody is used to deliver the drug to tumor cells by binding to target surface antigens, including target surface antigens overexpressed in tumor cells.

[0005] There remains a need in the field for anti-B7H3 antibodies and anti-B7H3 ADCs that can be used for therapeutic purposes in cancer treatment. Summary of the Invention

[0006] The first aspect of the present invention provides an antibody or antigen-binding fragment thereof targeting B7H3.

[0007] A second aspect of the present invention provides a ligand-drug conjugate based on the above-mentioned antibody or its antigen-binding fragment, comprising an antibody or its antigen-binding fragment targeting B7H3, a chemical linker structure, and an effector molecule portion.

[0008] In another respect, the present invention relates to pharmaceutical compositions comprising antibody or ligand drug conjugates according to the present invention.

[0009] In another respect, the present invention relates to pharmaceutical compositions of antibody or ligand drug conjugates according to the invention, which are used as drugs.

[0010] In another respect, the present invention relates to the medical use of antibody or ligand drug conjugates according to the present invention.

[0011] definition

[0012] Unless otherwise specified, any polypeptide chain herein is described as having an amino acid sequence that begins at an N-terminus and terminates at a C-terminus.

[0013] The term “antibody and its antigen-binding portion” is used in the broadest sense herein, encompassing a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0014] As used in this article, "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains: two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions(s) (CDR(s)), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino acid terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, where the three CDRs of VH are HCDR1, HCDR2, and HCDR3, and the three CDRs of VL are LCDR1, LCDR2, and LCDR3. The amino acid assignment of each domain is generally consistent with the following definitions: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol., 196: 901-917 (1987); Chothia et al., Nature, 342: 878-883 (1989).

[0015] There are five main antibody types: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The C-terminal amino acid sequence of an antibody molecule is relatively stable; this region is called the constant region. The constant region is the same for the same antibody. The constant region of the antibody light chain consists of one Ig domain; the constant region of the heavy chain consists of 3-4 tandem Ig domains and a hinge region for increased flexibility. IgA, IgE, and IgG have three domains (CH1, CH2, CH3), while IgD and IgM have four domains (CH1, CH2, CH3, CH4).

[0016] The different types of heavy chain constant domains or regions corresponding to immunoglobulins are designated as α, δ, ε, γ, and μ, respectively. IgG molecules can be degraded into two Fab fragments and one Fc fragment by papain. The Fab fragment consists of the variable region of the antibody light chain, the constant region of the light chain, the variable region of the heavy chain, and the constant region of the heavy chain. The variable region is the site of antigen binding; therefore, the Fab fragment is also called the antigen-binding fragment. The Fc fragment contains the protein sequence common to all antibody molecules, as well as determinants unique to each class. The Fc fragment has various biological activities, including complement binding, Fc receptor binding, and placental cross-linking.

[0017] As used herein, the term "antigen-binding moiety" (or simply "antibody portion" or "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (such as the B7H3 protein). It has been demonstrated that the antigen-binding function of an antibody can be achieved by certain fragments of a full-length antibody. The binding fragments covered by the term "antigen-binding moiety" of an antibody include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL1, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments consisting of two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of VH domains; and (vi) CDR. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked together by a synthetic linker through recombination to become separate, linked chains, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). Such single-chain antibodies are also covered within the terminology of the "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as bispecific antibodies, are also included.

[0018] Different analyses can be used to determine or roughly estimate the CDR region. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition. The Kabat definition is a standard used to number residues in an antibody and is commonly used to determine the CDR region. See, for example, Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the location of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses an integrated set of computer programs mimicking the antibody structure, created by the Oxford Molecular Group. See, for example, Martin et al., Proc Natl Acad Sci (USA), 86: 9268-9272 (1989); "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies" Oxford, UK; Oxford Molecular, Ltd. AbM is defined as a model of the tertiary structure of an antibody built from a primary sequence using a known database and an ab initio method, such as the method described in, for example, Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198 (1999). Contact is defined based on the analysis of the effective complex crystal structure. See, for example, MacCallum et al., J.Mol.Biol., 5:732-45 (1996).

[0019] The antibodies of this invention are not limited in origin and can be antibodies from any animal, such as human antibodies, mouse antibodies, or rat antibodies. They can also be recombinant antibodies such as chimeric antibodies or humanized antibodies. Humanized antibodies are preferred.

[0020] The term "chimeric antibody" refers to an antibody that contains the variable regions of the heavy and light chains of antibodies from mammals other than humans, such as mice, and the constant regions of the heavy and light chains of human antibodies. Chimeric antibodies can be prepared using known methods. For example, chimeric antibodies can be prepared by inserting the gene of a hybridoma clone into a suitable vector and then introducing it into a host. Specifically, cDNA encoding the variable region (V) of an antibody is synthesized from the mRNA of the hybridoma using reverse transcriptase. When the DNA encoding the V region of the target antibody is obtained, it is ligated with DNA encoding the constant region (C region) of the desired human antibody and then inserted into an expression vector. Alternatively, the DNA encoding the V region of the antibody can be inserted into a DNA expression vector containing the C region of a human antibody. This insertion into the expression vector allows for expression under the regulation of the expression regulatory region. Subsequently, the chimeric antibody can be expressed by transforming host cells using this expression vector.

[0021] The term "humanized antibody" refers to an antibody containing at least one, usually two, nearly complete variable regions, in which all or almost all of the corresponding CDR regions are derived from a non-human antibody and all or almost all of the FR regions are derived from a human antibody.

[0022] The term "fully human antibody," also known as "fully human monoclonal antibody," refers to an antibody whose variable and constant regions are both human-derived, thus eliminating immunogenicity and toxic side effects.

[0023] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies. That is, each antibody that makes up the population is identical, except for a small number of mutations that may be naturally present. Monoclonal antibodies are highly specific and target a single antigen. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring antibodies to be produced by any particular method.

[0024] The term "bispecific antibody" in the context of this disclosure should be understood as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a single target. As used herein, the term "bispecific antibody" should be understood in its broadest sense, including full-length bispecific antibodies and their antigen-binding fragments. "Multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies, the former being antibodies with three different antigen-binding specificities and the latter being antibodies with four different antigen-binding specificities.

[0025] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D The affinity is represented by the dissociation constant (Kd) or binding constant (Ka). Affinity can be measured by general methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0026] The term "surface plasmon resonance" refers to an optical phenomenon that enables real-time interactive analysis by detecting changes in protein concentration in a biosensor matrix, for example, using the Biacore™ system.

[0027] The terms "neutralizing" or "blocking" antibodies refer to antibodies whose binding to the B7H3 protein results in inhibition of the biological activity of the B7H3 protein. This inhibition of B7H3 protein biological activity can be assessed by measuring one or more well-known indicators of B7H3 protein biological activity, such as B7H3 protein-induced cell activation and antibody binding to the B7H3 protein (see Examples below).

[0028] Synthesis sequence similarity, also known as sequence identity / origin, is typically measured by sequence analysis software. Protein analysis software uses similarity measures of various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG software includes programs such as Gap and Bestfit, which can determine the sequence homology or sequence identity of closely related peptides, such as homologous peptides from different species, using default parameters (see GCG version 6.1). The FASTA program in GCG version 6.1 can also be used to compare peptide sequences using default or suggested parameters. FASTA (such as FASTA2 and FASTA3) provides alignments of the best overlapping regions between queried and searched sequences and percentages of sequence identity (Pearson (2000) above). Another preferred computational program is the computer program BLAST, especially BLASTP or TBLASTN, when comparing sequences of the present invention with databases containing large numbers of sequences from different organisms, using default parameters during comparison.

[0029] The terms “substantially identical” or “substantially the same” when referring to a nucleic acid or a fragment thereof mean that, when optimally aligned with another nucleic acid (or its complementary strand) using appropriate nucleotide substitutions, insertions, or deletions, the nucleotide sequence is identical in at least about 80%, more preferably at at least about 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide bases.

[0030] When applied to peptides, the term "substantially similar" or "fundamentally similar" means that, when optimally aligned using procedures such as Gap or BESTFIT with default vacancy weights, two peptide sequences have at least 80% sequence identity, more preferably at least 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. The difference in the position of the dissimilar residue can be a substitution, deletion, or insertion of an amino acid; more preferably, the difference in the position of the dissimilar residue lies in a conserved amino acid substitution. A "conserved amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue containing a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ due to conserved substitution, the percentage of sequence identity or degree of similarity can be adjusted upwards to correct for the conservation of the substitution. Methods for making such adjustments are well known to those skilled in the art. Examples of amino acid groups containing side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxy side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions may be any changes with positive values ​​in the PAM250 log-likelihood matrix disclosed by Gonnet et al. (1992) Science 256:1443-1445. "Moderately conservative" substitution is any change in the PAM250 log-likelihood matrix that has a non-negative value.

[0031] The term "carrier" refers to any molecule or entity (such as nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information to a host cell.

[0032] The term "expression vector" refers to a vector suitable for transforming host cells and containing a nucleic acid sequence that directs and / or regulates the expression of one or more heterologous coding regions operatively linked thereto. Expression vectors may include, but are not limited to: sequences that affect or regulate transcription or translation; and, if introns are present, sequences that affect RNA splicing of coding regions operatively linked thereto.

[0033] The term "host cell" refers to the cell in which exogenous nucleic acids are introduced, including the progeny of said cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and their derived progeny (regardless of passage number). The nucleic acid content of the progeny may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as those screened or selected for in the original cells are included herein.

[0034] The term "transfection" refers to the absorption of foreign or exogenous DNA by a cell, which is "transfected" when the exogenous DNA is introduced into the cell membrane. Various transfection techniques are well known in the art. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. These techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0035] The term "treatment" includes therapeutic treatment, preventative treatment, and applications that reduce the risk of developing disease or other risk factors in a treated individual. Treatment does not require a complete cure for the disease, but rather includes implementation plans that alleviate symptoms or reduce underlying risk factors.

[0036] The term "prevention" does not require eliminating the possibility of an event 100%. More accurately, it means that the likelihood of an event occurring is reduced in the presence of the stated compound or method.

[0037] The term "secondary antibody" refers to a second antibody that binds to the primary antibody (antibody of the antibody). Its main function is to detect the presence of the primary antibody and amplify its signal. Secondary antibodies utilize the antigenic property of large protein molecules to immunize a foreign animal, producing immunoglobulins against that antibody in the animal's immune system. Secondary antibodies are reactive to all antibodies (such as IgG, IgM, or IgA) specific to a particular species (e.g., mice).

[0038] The term "drug" refers to a chemical substance that can alter or reveal the physiological functions and pathological states of an organism, and can be used to prevent, diagnose, and treat diseases. Drugs include cytotoxic drugs. There is no strict boundary between drugs and poisons; poisons are chemical substances that can cause harm to the body even in small doses, damaging human health. Excessive dosage of any drug can produce toxic reactions.

[0039] The term "effect molecule" is a molecule capable of exhibiting desired target activity. For example, the effect molecule is selected from radioactive isotopes, antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, and any combination thereof. In some embodiments, the effect molecule is a cytotoxic drug.

[0040] Cytotoxic drugs are substances that inhibit or prevent cellular function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also induce apoptosis in normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, radioactive isotopes, chemotherapeutic drugs, antibiotics, and ribolysins.

[0041] The term "linker unit" or "connecting segment" or "connecting unit" refers to a segment or bond in a chemical structure that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being connected to a drug.

[0042] The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker unit. In this disclosure, "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linker unit.

[0043] Drug loading, also known as drug-to-antibody ratio (DAR), is the average number of drugs conjugated to each antibody in an ADC. It can range from about 1 to about 12 drugs per antibody, and in some embodiments, from about 1 to about 8 drugs per antibody, preferably from the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. Exemplarily, the drug loading can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments of the invention, the drug loading can be expressed as m or m1, which can be a decimal or an integer. Drug loading can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC.

[0044] The term "hydrophilic unit" refers to -CH2-CH2-O-, hydrophilic amino acids, glucosamine, glycosyl group, phosphate group, sulfonate group, or combinations thereof; preferably, the hydrophilic unit is selected from -CH2-CH2-O-, sarcosine, alanine, serine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, glucosamine glutamate, glycosyl group, phosphate group, sulfonate group, or combinations thereof.

[0045] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. The term "C"... 1-6 "Alkyl" refers to a saturated straight-chain or branched hydrocarbon group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6 carbon atoms). For example, "C 1-6 "alkyl" can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl, etc.

[0046] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0047] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms are selected from nitrogen, oxygen, or sulfur, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring comprises 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0048] The term "aryl" refers to a 6- to 15-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, with phenyl being more preferred. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring.

[0049] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 15 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5- or 6-membered, such as furanyl, thiophene, pyridyl, pyrrole, N-alkylpyrrole, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring.

[0050] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens.

[0051] The term "deuterated alkyl" refers to an alkyl group that has been replaced by one or more deuterium atoms.

[0052] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0053] The terms “substitution” and “substituted” refer to the selective replacement of one or more (e.g., one, two, three, or four) hydrogen atoms on a specified atom by a designated group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and that the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound.

[0054] If a substituent is described as “optionally…substituted,” then the substituent may be (1) unsubstituted or (2) substituted. If an atom or group is described as being optionally substituted by one or more of the substituents in the list, then one or more hydrogen atoms on that atom or group may be substituted by independently selected, optional substituents. If a substituent is described as “independently selected” or “each independently is,” then each substituent is chosen independently of the others. Thus, each substituent may be the same as or different from another (other) substituent. For example, when a substituent or substitution position or different substituents or substitution positions have R groups (e.g., but not limited to R2, R3, Rh, Ri, Rx and / or Ry) that may have the same or different symbols, the individual Rs are chosen independently, i.e., they may be the same or different. The same applies to the selection of numerical values ​​such as d, g, m, n.

[0055] Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable location of the substituent.

[0056] When the bond of a substituent is such that it passes through the ring and connects two atoms, then such a substituent can be bonded to any cyclic atom in the substituted ring.

[0057] The terms “comprising,” “including,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other elements or method steps not listed. Those skilled in the art will understand that the foregoing term “comprising” encompasses the meaning of “consisting of.”

[0058] In this invention, "pharmaceuticalally acceptable carrier" refers to a diluent, excipient, formulation, or mediator administered co-administered with the active ingredient, and which, to the extent of reasonable medical judgment, is suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio. The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that can effectively treat one or more symptoms of a target disease or condition.

[0059] As used herein, the term "effective amount" (e.g., "therapeutic effective amount" or "preventive effective amount") refers to the amount of active ingredient that, when administered, will achieve the desired effect to a certain extent, such as relieving one or more symptoms of the treated condition or preventing the occurrence of the condition or its symptoms.

[0060] Unless otherwise stated, as used herein, the term “treatment” means to reverse, alleviate, or inhibit the progression of the condition or illness to which the term is applied, or one or more symptoms of the condition or illness, or to prevent the condition or illness, or one or more symptoms of the condition or illness. Attached Figure Description

[0061] Figure 1: Results of antibody binding activity in this invention

[0062] Figure 2: Plasma concentration variation curve of the small molecule toxic drug of the present invention in SD rats.

[0063] Figure 3: Tumor volume change in the A375 model.

[0064] Figure 4: Tumor volume changes in the NCI-H358 model.

[0065] Figure 5: Tumor volume changes in the NCI-H526 model.

[0066] Figure 6: Tumor volume changes in the NCI-H526 model.

[0067] Figure 7: Tumor volume changes in the NCI-H526 model.

[0068] Figure 8: Tumor volume changes in the NCI-H358 model.

[0069] Figure 9: Tumor volume changes in the A375 model.

[0070] Figure 10: Bystander effect results of the ADC drug of the present invention

[0071] Figure 11: Tumor volume changes in the A375+MDA-MB-453 model.

[0072] Figure 12: Pharmacokinetic results of plasma ADC and total antibody in SD rats

[0073] Figure 13: Pharmacokinetic results of free small molecule loads in SD rats

[0074] Figure 14: Mouse survival curve

[0075] Figure 15: Mouse body weight change curve

[0076] Figure 16: In vivo antitumor activity of ADC drugs in the H526 CDX model Detailed Implementation

[0077] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0078] Abbreviations

[0079] PBS stands for Phosphate Buffer Solution.

[0080] PBST refers to the addition of Tween-20 to a PBS solution.

[0081] BSA stands for Bovine Serum Albumin. BSA blocking solution is prepared from bovine serum albumin.

[0082] TMB refers to 3,3',5,5'-tetramethylbenzidine.

[0083] Solution.

[0084] MEM medium refers to Minimum Essential Medium, which is a commonly used medium in animal cell culture.

[0085] FBS refers to Fetal Bovine Serum.

[0086] ND (Not Detected) means that the antibody did not exhibit neutralizing activity in vitro under experimental conditions.

[0087] Reagents and Instruments

[0088] biomaterial sources

[0089] The pCDNA3.1(+) expression vector was purchased from Invitrogen, catalog number V79020.

[0090] Mouse myeloma cells SP2 / 0 were purchased from the American Culture Collection Center, catalog number CRL-1581.

[0091] HEp-2 cells were purchased from ATCC, catalog number CCL-23.

[0092] 293F cells were purchased from ATCC, catalog number CRL-1573.

[0093] Part 1: B7H3 Antibody Section

[0094] Example 1. Screening of hybridoma cell lines

[0095] 1. Expression and purification of B7H3 recombinant protein

[0096] Based on the gene sequence of the B7H3 protein in the NCBI database, an expression gene with a His tag was chemically synthesized. This gene was then used to construct an expression plasmid for the B7H3 protein using the pCDNA3.1(+) expression vector. After transfection into 293F cells, the supernatant from cell expression was collected, concentrated, and purified using a nickel column (Cytiva) to obtain the B7H3 protein. Its amino acid sequence is as follows:

[0097] As shown in SEQ ID NO:1.

[0098] SEQ ID NO:1

[0099] 2. Construction of hybridoma cells

[0100] Female BALB / c mice aged 6-8 weeks were immunized after one week of rearing. The initial immunization used B7H3 protein. Before immunization, Sigma Adjuvant System adjuvant (Sigma) was resuspended in 1 ml of PBS, and then 1 ml of B7H3 protein (1 mg / ml) was added as an equal volume to emulsify the mixture. The mixture was injected intraperitoneally and subcutaneously, with each mouse receiving 100 μg per injection. Immunization was repeated every two weeks. Blood was collected after the third immunization to determine the titer, for a total of four immunizations. Spleens were harvested for fusion. A booster immunization was performed 72 hours before fusion, with a single intraperitoneal injection of 100 μL of B7H3 antigen protein per mouse.

[0101] Spleen cells from immunized mice were fused with SP2 / 0 mouse myeloma cells. The spleen was first ground to obtain a spleen cell suspension, which was then mixed 1:1 with SP2 / 0 mouse myeloma cells in logarithmic growth phase. The two cell types were fused together via electrofusion to obtain hybridoma cells. The fusion cell suspension was then diluted to 5000-10000 cells / ml and evenly seeded into 96-well plates. The fusion medium was DMEM complete selection medium (Gibco) containing HAT (Gibco) and 20% FBS. After incubation at 37°C and 5% CO2 for 7 days, positive hybridoma cell lines were selected using an indirect ELISA method. The medium was changed the day before selection.

[0102] 3. Production of monoclonal cell lines

[0103] Based on the hybridoma cell growth density, the cell supernatant was detected using an indirect ELISA method. Cells with strong binding ability to the B7H3 antigen protein were selected and promptly expanded and cryopreserved. Monoclonal cell lines were obtained through subcloning using a limiting dilution method. The supernatant of the monoclonal cell lines was collected and subjected to a Biacore assay to detect the binding activity of the monoclonal cell lines to the B7H3 antigen.

[0104] 4. Mouse-derived antibodies

[0105] The following procedures were followed to clone sequences from highly viable monoclonal hybridoma cell lines (12C12, 9H5, 9E6, 12B9, 10F3, 4G11, 18H6, 23G5, 10H5, and 12F12) in vitro. Hybridoma cells in logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) (following the kit instructions) and reverse transcribed (PrimeScript Reverse Transcriptase, Takara, cat#2680A). The resulting cDNA was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the obtained DNA sequences are shown in SEQ ID NO:2-21, and the CDR region sequences are shown in Table 1.

[0106] The murine antibody with variable region is shown below (underlined part is CDR region):

[0107] 12C12 heavy chain variable region sequence

[0108] 12C12 light chain variable region sequence

[0109] 9H5 heavy chain variable region sequence

[0110] 9H5 light chain variable region sequence

[0111] 9E6 heavy chain variable region sequence

[0112] 9E6 light chain variable region sequence

[0113] 12B9 heavy chain variable region sequence

[0114] 12B9 light chain variable region sequence

[0115] 10F3 heavy chain variable region sequence

[0116] 10F3 light chain variable region sequence

[0117] 4G11 heavy chain variable region sequence

[0118] 4G11 light chain variable region sequence

[0119] 18H6 heavy chain variable region sequence

[0120] 18H6 light chain variable region sequence

[0121] 23G5 heavy chain variable region sequence

[0122] 23G5 light chain variable region sequence

[0123] 10H5 heavy chain variable region sequence

[0124] 10H5 light chain variable region sequence

[0125] 12F12 heavy chain variable region sequence

[0126] 12F12 light chain variable region sequence

[0127] Table 1. Obtaining CDR Sequences via Kabat Numbering Rules

[0128] Example 2. Preparation of chimeric antibodies

[0129] 1. Construction and expression of chimeric antibodies

[0130] Chimeric antibodies were constructed using fusion PCR: The coding genes of the variable region of the murine antibody from Example 1 above, the heavy chain constant region of the human IgG1 antibody, and the human κ light chain constant region (amino acid sequence of the heavy chain constant region: SEQ ID NO: 82, amino acid sequence of the light chain constant region: SEQ ID NO: 83) were linked to construct a fusion gene. The fusion gene was amplified and cloned into the pCDNA3.1(+) expression vector to construct the expression plasmid of the chimeric antibody. After transfection into 293F cells, the cells were cultured and the supernatant of cell expression was collected. After concentration, the supernatant was purified by Protein A (Cytiva) to obtain the corresponding chimeric antibodies 12C12-Q1, 9H5-Q1, 9E6-Q1, 12B9-Q1, 10F3-Q1, 4G11-Q1, 18H6-Q1, 23G5-Q1, 10H5-Q1, and 12F12-Q1.

[0131] Heavy chain constant region of human IgG1 antibody (SEQ ID NO:82):

[0132] Human κ light chain constant region (SEQ ID NO:83):

[0133] 2. Chimeric antibody affinity detection

[0134] 1) The affinity of chimeric antibodies for B7H3 was detected using a capture antibody approach. A Sensor Chip Protein A biosensor chip (Cytiva, 29650263) was used to capture the antibody, and then the antigen B7H3-his was flowed over the chip surface. The reaction signal was detected in real-time using a Biacore K8 instrument (Cytiva) to obtain binding and dissociation curves. After dissociation in each experimental cycle, the chip was washed and regenerated with regeneration buffer. After the experiment, the data were fitted using a (1:1) Langmuir model with GE Biacore K8 Evaluation version 3.0 software to obtain the affinity values. The affinity of chimeric antibodies and humanized antibodies for the protein is shown in Table 2 below.

[0135] 2) The binding activity of the chimeric antibodies was detected by ELISA. The procedure is briefly described below: The purified B7H3-His antigen protein from Example 1 was coated at a concentration of 1 μg / mL. After overnight blocking with 2% BSA, serially diluted antibodies were added and incubated (Table 2 shows the initial antibody concentration as 10 μg / mL, 2-fold serial dilution). After incubation at 37°C for 1 hour, Mouse Anti-Human IgG Fab Antibody (12H3C4A6) [HRP] (Genscript, A01855-200) secondary antibody was added and incubated for 1 hour. Then, TMB (Solepro, RP1200-1000mL) was added for color development. The color development was stopped with stop solution (Beyotime, P0215). After reading the absorbance at OD450 using a microplate reader, the binding EC50 of each antibody was calculated using GraphPad Prism9. 50 The binding activities of each antibody were compared numerically, and the results are shown in Table 2 below.

[0136] Table 2. Chimeric antibody binding activity and affinity

[0137] As can be seen from Table 2, the exemplary antibody provided by this invention has good B7H3 binding activity.

[0138] Example 3. Antibody humanization

[0139] 1. Preparation of humanized antibodies

[0140] The mouse CDR regions of antibodies 9H5, 18H6, and 4G11 were retained, and their FR regions were humanized by transplantation. The steps for humanizing the FR regions are as follows:

[0141] (1) Homology modeling of the heavy and light chain sequences of the variable region of mouse antibody was performed using the Prediction function in Bioluminate software, and the reliability of the homology modeling was evaluated using the Reliable report function. Based on this evaluation, the model was further optimized to obtain the optimized antibody variable region model.

[0142] (2) The variable region of the antibody to be humanized was compared by NCBI Ig Blast, and the Human germline sequence with the highest homology was selected for CDR transplantation. In the first round of humanization, the FR sequence of the mouse antibody was completely replaced by humanization.

[0143] (3) Using the advanced option in CDR grafting, key sites such as the Canonical Structure Region and Vernier Zone in the frame region FR are predicted.

[0144] Selection and reversion mutations in the human FR region of antibody 9H5

[0145] (1) Selection of human-origin FR region

[0146] The humanized VH1 template for 9H5 is IGHV3-7*01, the humanized VH2 template is IGHV3-30*01, the humanized VL1 template is IGKV1-13*02, and the humanized VL2 template is IGKV1-9*01. The CDR of 9H5 was transplanted onto the humanized templates, and the resulting variable region sequence is as follows:

[0147] hu9H5VH1 - Heavy chain variable region:

[0148] hu9H5VH2 - Heavy chain variable region:

[0149] hu9H5VL1 - Light chain variable region:

[0150] hu9H5VL2 - Light chain variable region:

[0151] (2) Reversion mutation of humanized antibodies

[0152] The reversion mutation sites for the 9H5 humanized antibody are shown in the table below:

[0153] Table 3. Reversion mutations of the 9H5 humanized antibody Note: In the table, T28I indicates that the T-amino acid at position 28 of the humanized antibody has been reverse-mutated to I.

[0154] Single and multiple mutations were performed on the VH1, VH2, VL1, and VL2 sites of the humanized antibody hu9H5 according to the mutation sites in the table above. Different combinations of reversion mutant heavy and light chains were constructed, and the expressed proteins were subjected to affinity and cell activity assays. The humanized antibodies with high humanization and excellent activity, along with their corresponding reversion mutation sites, were finally determined as shown in the table below:

[0155] Table 4. 9H5 humanized antibodies and their corresponding reversion mutations

[0156] The variable region sequence is as follows:

[0157] 9H5-J1 / J2 heavy chain variable region VH sequence:

[0158] 9H5-J5 / J6 / J14 heavy chain variable region VH sequence:

[0159] 9H5-J1 / J5 / J17 / J29 light chain variable region VL sequence:

[0160] 9H5-J2 / J6 / J31 light chain variable region VL sequence:

[0161] 9H5-J3 / J7 light chain variable region VL sequence:

[0162] 9H5-J3 / J4 heavy chain variable region VH sequence:

[0163] 9H5-J4 / J8 / J14 light chain variable region VL sequence:

[0164] 9H5-J7 / J8 heavy chain variable region VH sequence:

[0165] 9H5-J17 heavy chain variable region VH sequence:

[0166] 9H5-J29 / J31 heavy chain variable region VH sequence:

[0167] The antibody sequence is as follows:

[0168] 9H5-J1 heavy chain sequence:

[0169] 9H5-J1 light chain sequence:

[0170] 9H5-J2 heavy chain sequence: (SEQ ID NO:94)

[0171] 9H5-J2 light chain sequence:

[0172] 9H5-J3 heavy chain sequence:

[0173] 9H5-J3 light chain sequence:

[0174] 9H5-J4 heavy chain sequence: (SEQ ID NO:97)

[0175] 9H5-J4 light chain sequence:

[0176] 9H5-J5 heavy chain sequence:

[0177] 9H5-J5 light chain sequence: SEQ ID NO:95

[0178] 9H5-J6 heavy chain sequence: (SEQ ID NO:100)

[0179] 9H5-J6 light chain sequence: (SEQ ID NO:96)

[0180] 9H5-J7 heavy chain sequence:

[0181] 9H5-J7 light chain sequence: (SEQ ID NO:98)

[0182] 9H5-J8 heavy chain sequence: (SEQ ID NO:101)

[0183] 9H5-J8 light chain sequence: (SEQ ID NO:99)

[0184] 9H5-J14 heavy chain sequence: (SEQ ID NO:100)

[0185] 9H5-J14 light chain sequence: (SEQ ID NO:99)

[0186] 9H5-J17 heavy chain sequence:

[0187] 9H5-J17 light chain sequence: (SEQ ID NO:95)

[0188] 9H5-J29 heavy chain sequence:

[0189] 9H5-J29 light chain sequence: (SEQ ID NO:95)

[0190] 9H5-J31 heavy chain sequence: (SEQ ID NO:103)

[0191] 9H5-J31 light chain sequence: (SEQ ID NO:96)

[0192] Selection and reversion mutations in the human FR region of antibody 4G11

[0193] (1) Selection of human-origin FR region

[0194] The humanized VH template for 4G11 is IGHV1-18*01, the template for humanized VL1 is IGKV1-13*02, and the template for humanized VL2 is IGKV3-15*01. The CDR of 4G11 was transplanted onto the human templates, and the resulting variable region sequence is as follows:

[0195] hu4G11VH - Heavy chain variable region:

[0196] hu4G11VL1 - Light chain variable region:

[0197] hu4G11VL2 - Light chain variable region:

[0198] (2) Reversion mutation of humanized antibodies

[0199] The reversion mutation sites of the 4G11 humanized antibody are shown in the table below:

[0200] Table 5. Reversion mutations of 4G11 humanized antibodies Note: In the table, M48I indicates that the 48th amino acid M of the humanized antibody has been reversed and mutated to I.

[0201] Single and multiple mutations were performed on the VH, VL1, and VL2 regions of the humanized antibody hu4G11 according to the mutation sites listed in the table above. Different combinations of the constructed reversion mutant heavy and light chains were then used to determine the affinity and cellular activity of the expressed proteins. The following table shows the humanized antibodies with high humanization and excellent activity, along with their corresponding reversion mutation sites:

[0202] Table 6. 4G11 humanized antibodies and their corresponding reversion mutations

[0203] The variable region sequence is as follows:

[0204] 4G11-J58 / J59 / J60 heavy chain variable region VH sequence:

[0205] 4G11-J58 light chain variable region VL sequence:

[0206] 4G11-J59 light chain variable region VL sequence:

[0207] 4G11-J60 light chain variable region VL sequence:

[0208] The antibody sequence is as follows:

[0209] 4G11-J58 heavy chain sequence:

[0210] sequence:

[0211] 4G11-J59 heavy chain sequence:

[0212] 4G11-J59 light chain sequence:

[0213] 4G11-J60 heavy chain sequence:

[0214] 4G11-J60 light chain sequence:

[0215] Selection and reversion mutations in the human FR region of 18H6

[0216] (1) Selection of human-origin FR region

[0217] The humanized VH template for 18H6 is IGHV1-18*01, the template for humanized VL1 is IGKV1-9*01, and the template for humanized VL2 is IGKV3-11*01. The CDR of 18H6 was transplanted onto the human templates, and the resulting variable region sequence is as follows:

[0218] hu18H6VH - Heavy chain variable region:

[0219] hu18H6VL1 - Light chain variable region:

[0220] hu18H6VL2 - Light chain variable region:

[0221] (2) Reversion mutation of humanized antibodies

[0222] The reversion mutation sites of the 18H6 humanized antibody are shown in the table below:

[0223] Table 7 Reversion Mutations of 18H6 Humanized Antibodies Note: In the table, M48I indicates that the 48th amino acid M of the humanized antibody has been reversed and mutated to I.

[0224] Single and multiple mutations were performed on the VH, VL1, and VL2 regions of the humanized antibody hu18H6 according to the mutation sites listed in the table above. Different combinations of the constructed reversion mutant heavy and light chains were then used to determine the affinity and cellular activity of the expressed proteins. The following table shows the humanized antibodies with high humanization and excellent activity, along with their corresponding reversion mutation sites:

[0225] Table 8. 18H6 humanized antibodies and their corresponding reversion mutations

[0226] The variable region sequence is as follows:

[0227] 18H6-J10 / J37 heavy chain variable region VH sequence:

[0228] 18H6-J10 light chain variable region VL sequence:

[0229] 18H6-J37 light chain variable region VL sequence:

[0230] The antibody sequence is as follows:

[0231] 18H6-J10 heavy chain sequence:

[0232] 18H6-J10 light chain sequence:

[0233] 18H6-J37 heavy chain sequence: (SEQ ID NO:117)

[0234] 18H6-J37 light chain sequence:

[0235] The above antibodies were cloned, expressed, and purified using conventional gene cloning and recombinant expression methods. 2. Indirect ELISA was used to detect the cross-binding of humanized antibodies with B7 family proteins.

[0236] 1. Instruments and reagents

[0237] Table 9

[0238] 2. Experimental Procedure

[0239] (1) Dilute B7 family recombinant proteins B7-1, B7-2, PD-L2, B7H1, B7H2, B7H3-his, B7H4, B7H5, and B7H6 to 1 μg / mL with coating buffer (CBS), coat ELISA plates with 100 μL / well, and incubate overnight at 4°C.

[0240] (2) Add 200 μL of 2% BSA to each well to block the microplate, and incubate at 37°C for 2 hours;

[0241] (3) Add 100 μL of antibody (10 μg / mL) and set up positive and negative controls. The concentration of the positive control antibody Ifinatamab (M30) and the novel fully humanized antibody 9H5J1 in the first well is 5 μg / mL. Dilute to 7 gradients at a ratio of 1:5. The last well is 0. Take 100 μg of the dilution solution from each well and add it to the microplate. Incubate at 37°C for 1 h.

[0242] (4) After washing twice with PBST, use HRP-labeled secondary antibody diluted 1:10000 to 10000, add 100 μL to each well, and incubate at 37℃ for 1 h;

[0243] (5) After washing with PBST 4 times, add 100 μL of single-component TMB colorimetric solution to each well and develop color in the dark for 5-10 min.

[0244] (6) Termination of reaction: Add 2M H2SO4 to terminate the reaction, 50μL per well;

[0245] (7) Reading: Place the microplate in the microplate reader and read the value. The detection wavelength is OD450. Use GraphPad Prism (version 5) to calculate EC based on nonlinear fitting of concentration and corresponding OD value. 50 The results are shown in Table 10-11 and Figure 1 below.

[0246] The positive control antibody of this invention is Ifinatamab (prepared using conventional antibody preparation methods):

[0247] Ifinatamab heavy chain (SEQ ID NO: 120)

[0248] Ifinatamab light chain (SEQ ID NO: 121)

[0249] Table 10 Cross-linking results between humanized antibodies and B7 family proteins.

[0250] Table 11 Results of antibody binding activity

[0251] Experimental results showed that, compared with the positive control antibody, 9H5-J1 had superior binding activity to B7H3 and did not cross-bind with other B7 family proteins such as B7-1 and B7-2.

[0252] Part Two: ADC Section

[0253] Example 2-1:

[0254] Compound 1

[0255] Synthesis route:

[0256] Synthesis of intermediates 1-3:

[0257] Dissolve 1-2 (1 g, 0.824 mmol) in DMF (4 mL), add 1-1 (280 mg, 0.824 mmol) and DIEA (150 μL, 0.906 mmol), and react at room temperature for 4 hours. After the reaction is complete, purify by reversed-phase column chromatography and concentrate under reduced pressure to give a colorless oily product (1.15 g, 97.0%). LC-MS (ESI): m / z found [M+1] + =1440.2.

[0258] Synthesis of intermediates 1-4:

[0259] Compounds 1-3 (1.15 g, 0.799 mmol) were dissolved in DMF (5 mL), and HOSU (124 mg, 1.079 mmol) and EDCI (206 mg, 1.079 mmol) were added. After reacting at room temperature for 2 hours, DIEA (132 μL, 0.799 mmol) and GGFG-AM (457 mg, 1.079 mmol) were added, and the reaction was carried out at room temperature for 2 hours. The reaction was monitored by LC-MS until complete, purified by reversed-phase column chromatography, and concentrated under reduced pressure to give a colorless oily product (1.1 g, 78.6%). LC-MS (ESI): m / z found [M / 2-36] + =885.0. Synthesis of intermediates 1-5:

[0260] Compounds 1-4 (500 mg, 0.271 mmol) were dissolved in DMF (5 mL), and 11 (138.5 mg, 0.326 mmol, synthesized according to WO2024230752A1), HATU (124 mg, 0.326 mmol), and DIEA (90 μL, 0.542 mmol) were added. The reaction was carried out at room temperature for 2.5 hours. After the reaction was monitored by LC-MS until complete, the product was purified by reversed-phase column chromatography and lyophilized to give a yellow solid product (341 mg, 55.9%). LC-MS (ESI): m / z found [M / 2+1] + =1127.35.

[0261] Synthesis of intermediates 1-6:

[0262] Compounds 1-5 (341 mg, 0.151 mmol) were dissolved in DMF (5 mL), and diethylamine (76 μL) was added. The mixture was reacted at room temperature for 2.5 hours. After the reaction was confirmed to be complete by LC-MS, the product was purified by reversed-phase column chromatography and concentrated under reduced pressure to give a yellow oily product (150 mg, 48.9%). LC-MS (ESI): m / z found [M / 2+1] + =1016.20.

[0263] Synthesis of intermediates 1-7:

[0264] Azideacetic acid (13.4 mg, 0.133 mmol) was dissolved in DMF (2 mL), followed by the addition of DMTMM (61 mg, 0.222 mmol), TEA (29 μL, 0.222 mmol), and compounds 1-6 (150 mg, 0.074 mmol). The reaction was carried out at room temperature for 3 hours. After the reaction was confirmed to be complete by LC-MS, the product was prepared by pre-HPLC and lyophilized to obtain a yellow solid product (32 mg, 20.5%). LC-MS (ESI): m / z found [M / 2+1] + =1057.75.

[0265] Synthesis of Compound 1:

[0266] Compounds 1-7 (30 mg, 0.014 mmol), 1-8 (15.7 mg, 0.014 mmol), copper sulfate pentahydrate (4.2 mg, 0.017 mmol), and ascorbic acid (4.8 mg, 0.029 mmol) were dissolved in DMSO (1.5 mL). The reaction was carried out at room temperature for 1.5 h under nitrogen protection. The reaction was confirmed to be complete by LCMS. The final product was prepared by HPLC and lyophilized to give compound 1 (0.93 mg, 2.2%) as a yellow solid. LCMS (ESI): m / z found [M / 2+H] + =1454.5.

[0267] Example 2-2:

[0268] Compound 2

[0269] Synthesis route:

[0270] Synthesis of intermediate 2-2:

[0271] Compound 2-1 (5.0 g, 5.24 mmol) was dissolved in DCM (50 mL), and ethylene glycol (8.4 g, 52.4 mmol) and PPTS (6.8 g, 10.86 mmol) were added sequentially. The reaction was allowed to proceed overnight at room temperature. The reaction was monitored by TLC until complete. The reaction solution was concentrated under reduced pressure, dissolved in a small amount of DMF, purified by reversed-phase column chromatography, and concentrated under reduced pressure to give a white solid (3.2 g, 61.3%).

[0272] Synthesis of intermediates 2-3:

[0273] Compound 2-2 (500 mg, 1.61 mmol) was dissolved in DCM (35 mL), and PNP2CO (1.96 g, 6.11 mmol) and DIEA (208.18 mg, 1.61 mmol) were added in portions. The mixture was reacted overnight at room temperature. The reaction proceeded to completion as monitored by TLC. The mixture was purified by normal-phase column chromatography and concentrated under reduced pressure to give a yellow oil (320 mg, 44.26%).

[0274] Synthesis of intermediates 2-4:

[0275] Compounds 2-3 (251.72 mg, 0.468 mmol) were dissolved in DMF (2 mL). HOBt (69.8 mg, 0.516 mmol) was added under ice bath conditions, and the mixture was reacted at room temperature for 15 min. Then, 11 (200 mg, 0.468 mmol) and DIEA (121.24 mmol, 0.94 mmol) were added sequentially, and the reaction was continued at room temperature for 1 h. The reaction was monitored by LC-MS to ensure complete reaction of the starting material. The mixture was purified by reversed-phase column chromatography and lyophilized to give a yellow solid (180 mg, 46.13%). LC-MS (ESI): m / z found [M+1] + =822.0.

[0276] Synthesis of intermediates 2-5:

[0277] Compounds 2-4 (50 mg, 0.06 mmol) were dissolved in DMF (1 mL), and piperidine (30.9 μL, 0.3 mmol) was added. The mixture was reacted at room temperature for 1 h. The reaction was monitored by LC-MS until complete. The mixture was purified by reverse-phase column chromatography and lyophilized to give a yellow solid (7 mg, 19.38%). LC-MS (ESI): m / z found [M+1] + =600.4.

[0278] Synthesis of intermediates 2-7:

[0279] At room temperature, 2-6 (1 g, 1.7 mmol) was dissolved in DMF (4 mL), and HOSU (215.2 mg, 1.87 mmol) and EDCI (358 mg, 1.87 mmol) were added. The mixture was reacted at room temperature for 6 hours, followed by the addition of 1-1 (578 mg, 1.7 mmol) and DIEA (444.3 μL, 2.55 mmol), and the reaction was continued at room temperature for 2 hours. The reaction mixture was checked by LC-MS to confirm complete reaction. The reaction solution was filtered, and the filtrate was purified by reversed-phase column chromatography and lyophilized to give a white oily substance (1.142 g, 73.77%). LC-MS (ESI): m / z found [M+H] + =911.8.

[0280] Synthesis of intermediates 2-8:

[0281] At room temperature, 2-7 (1.142 g, 1.25 mmol) was dissolved in ACN (10 mL), and HOSU (216.4 mg, 1.88 mmol) and DCC (388 mg, 1.88 mmol) were added. The reaction was carried out at room temperature for 2 hours, and then the reaction solution was filtered. The filtrate was collected and concentrated under reduced pressure. The crude product was dissolved in DMF (5 mL), and DIEA (327.5 μL, 1.88 mmol) and GGF (420 mg, 1.5 mmol) were added. The reaction was carried out at room temperature for 2 hours. The reaction mixture was monitored by LC-MS to ensure complete reaction of the starting material. The reaction solution was filtered, and the filtrate was purified by reversed-phase column chromatography and concentrated under reduced pressure to give a white oily substance (1.076 g, 73.22%). LC-MS (ESI): m / z found [M+H / 2] + =587.2.

[0282] Synthesis of intermediates 2-9:

[0283] At room temperature, 2-8 (400 mg, 0.34 mmol) was dissolved in DMF (2 mL), and piperidine (337 μL, 3.41 mmol) was added. The reaction was carried out at room temperature for 2 hours. LC-MS analysis showed that the reaction proceeded completely. The reaction solution was purified by automated pre-HPLC and concentrated under reduced pressure to obtain a white oil (159.3 mg, 49.14%). LC-MS (ESI): m / z found [M+H] + =950.7.

[0284] Synthesis of intermediate 2-10:

[0285] At room temperature, 2-9 (45 mg, 0.047 mmol) was dissolved in DMF (2.5 mL), followed by the addition of NHS azidoacetate (18.8 mg, 0.095 mmol) and DIEA (16.5 μL, 0.095 mmol). The reaction was allowed to proceed for 1.5 hours at room temperature. The reaction was monitored by LC-MS until complete. Pre-HPLC purification was performed, and the product was lyophilized to obtain a white oily substance (19.4 mg, 39.65%). LC-MS (ESI): m / z found [M+H] + =1033.75.

[0286] Synthesis of intermediate 2-11:

[0287] At room temperature, 2-10 (19.4 mg, 0.019 mmol) was dissolved in a mixed solution of EtOH (3 mL) and THF (1 mL), and 1-8 (13.54 mg, 0.017 mmol), CuSO4·5H2O (4.3 mg, 0.017 mmol), and ascorbic acid (3 mg, 0.017 mmol) were added. The mixture was purged with nitrogen three times and reacted at room temperature for 3 h. After the reaction was complete as monitored by LC-MS, the reaction solution was concentrated under reduced pressure, dissolved in a small amount of methanol, purified by pre-HPLC, and lyophilized to give a yellow solid compound (19.1 mg, 61.24%). LC-MS (ESI): m / z found [M+H / 2] + =914.45.

[0288] Synthesis of compound 2:

[0289] Compound 2-11 (10 mg, 0.005 mmol) was dissolved in DMF (2 mL) under ice bath conditions. HATU (2.5 mg, 0.007 mmol) was added, and the mixture was stirred under ice bath conditions for 20 min. Then, 2-5 (3.3 mg, 0.005 mmol) and DIEA (1.43 μL, 0.008 mmol) were added sequentially, and the mixture was reacted at room temperature for 2 h. The reaction was monitored by LC-MS until complete. The reaction solution was filtered, and the filtrate was purified by Pre-HPLC and lyophilized to give a yellow solid product (2.07 mg, 15.7%). LC-MS (ESI): m / z found [M+H / 2] + =1205.35. Examples 2-3:

[0290] Compound 3

[0291] Synthesis route:

[0292] Synthesis of intermediate 3-1:

[0293] At room temperature, 1-3 (488.5 mg, 0.339 mmol) was dissolved in ACN (5 mL), and HOSU (58.58 mg, 0.509 mmol) and DCC (105 mg, 0.509 mmol) were added. The mixture was reacted at room temperature for 2 hours. The reaction solution was filtered, and the filtrate was collected and concentrated under reduced pressure. The crude product was dissolved in DMF (3 mL), and DIEA (88.6 μL, 0.509 mmol) and GGF (113.7 mg, 0.407 mmol) were added. The mixture was reacted at room temperature for 2 hours. LC-MS analysis showed that the reaction was complete. The reaction solution was filtered, and the filtrate was purified by reversed-phase column chromatography and concentrated under reduced pressure to obtain a colorless oil (456.3 mg, 79.11%). LC-MS (ESI): m / z found [M+H / 2] + =851.85.

[0294] Synthesis of intermediate 3-2:

[0295] At room temperature, 3-1 (200 mg, 0.1176 mmol) was dissolved in DMF (2 mL), and piperidine (116.2 μL, 1.176 mmol) was added. The mixture was reacted at room temperature for 2 hours. LC-MS analysis showed that the reaction proceeded completely. The reaction solution was purified by Pre-HPLC and concentrated under reduced pressure to obtain a white oil (48.7 mg, 32.8%). LC-MS (ESI): m / z found [M+H] + =1479.25. Synthesis of intermediate 3-3:

[0296] At room temperature, 3-2 (48.7 mg, 0.033 mmol) was dissolved in DMF (2.5 mL), followed by the addition of NHS azidoacetate (13 mg, 0.066 mmol) and DIEA (11.5 μL, 0.066 mmol). The reaction was allowed to proceed for 1.5 hours at room temperature. LC-MS analysis confirmed complete reaction of the starting materials. The reaction solution was purified by Pre-HPLC and concentrated under reduced pressure to obtain a colorless oil (33.7 mg, 65.52%). LC-MS (ESI): m / z found [M+H] + =1562.25.

[0297] Synthesis of intermediates 3-4:

[0298] At room temperature, compound 3-3 (33.7 mg, 0.022 mmol) was dissolved in a mixed solution of EtOH (3 mL) and THF (1 mL), followed by the addition of compound 1-8 (15.56 mg, 0.020 mmol), copper sulfate pentahydrate (4.9 mg, 0.020 mmol), and ascorbic acid (3.5 mg, 0.020 mmol). The mixture was purged with nitrogen three times and reacted at room temperature for 2.5 h. After the reaction was complete, the reaction solution was dried under reduced pressure, dissolved in a small amount of methanol, purified by pre-HPLC, and lyophilized to give a yellow solid compound (28.8 mg, 56.66%). LC-MS (ESI): m / z found [M+H / 2] + =1178.85.

[0299] Synthesis of compound 3:

[0300] Under ice bath conditions, 3-4 (10 mg, 0.004 mmol) was dissolved in DMF (2 mL), and HATU (1.94 mg, 0.005 mmol) was added. After stirring in an ice bath for 30 min, 2-5 (2.55 mg, 0.004 mmol) and DIEA (1.11 μL, 0.006 mmol) were added sequentially, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by LC-MS to ensure complete reaction. The reaction solution was purified by pre-HPLC and lyophilized to give a yellow solid product (2.82 mg, 22.7%). LC-MS (ESI): m / z found [M+H / 2] + =1469.6.

[0301] Examples 2-4

[0302] Compound 4

[0303] Synthesis route:

[0304] Synthesis of intermediate 4-2:

[0305] At room temperature, 2-1 (100 mg, 0.272 mmol) was dissolved in DCM (2 mL), followed by the addition of 4-1 (42.38 mg, 0.543 mmol) and PPTS (136.44 mg, 0.543 mmol). The mixture was stirred at room temperature for 3 h under N2 protection. The reaction was monitored by LC-MS until complete. The reaction solution was concentrated under reduced pressure, dissolved in an appropriate amount of MeOH, purified by reversed-phase column chromatography, concentrated under reduced pressure, and lyophilized to obtain a white powder (45.9 mg, 43.7%). LC-MS (ESI): m / z found [M+H] + =617.40.

[0306] Synthesis of intermediate 4-3:

[0307] At room temperature, 4-2 (275.4 mg, 0.752 mmol) was dissolved in DMF (2 mL), followed by the addition of diethylamine (75 μL). The mixture was stirred at room temperature for 2 h under N2 protection. The reaction was monitored by LC-MS until complete. The product was purified by reversed-phase column chromatography, concentrated under reduced pressure, and lyophilized to obtain a yellow oil (127.2 mg, 100%). LC-MS (ESI): m / z found [M+H] + =163.2.

[0308] Synthesis of intermediates 4-6:

[0309] Under ice bath conditions, 4-4 (100 mg, 0.343 mmol) was dissolved in DMF (1 mL), followed by HOSU (47.41 mg, 0.412 mmol) and EDCI (79.09 mg, 0.412 mmol). The reaction was maintained on ice for 1 h. Then, 4-5 (95.88 mg, 0.343 mmol) and TEA (34.67 mg, 0.343 mmol) were added, and the reaction was continued at room temperature for 3 h. The reaction was monitored by LC-MS until complete. The reaction solution was purified by reversed-phase column chromatography, concentrated under reduced pressure, and lyophilized to give a yellow oily product (83.5 mg, 44%). LC-MS (ESI): m / z found [M+H] + =553.2.

[0310] Synthesis of intermediates 4-7:

[0311] At room temperature, 4-6 (168.3 mg, 0.305 mmol) was dissolved in THF (3 mL), followed by DCC (75.5 mg, 0.366 mmol) and HOSU (42.10 mg, 0.366 mmol), and stirred at room temperature for 2 h.

[0312] The reaction was confirmed to be complete by LC-MS. After stirring in an ice bath for 10 min, a large amount of white solid precipitated. The solid was filtered, washed with THF, and the filtrate was reserved. At room temperature, 4-3 (60 mg, 0.366 mmol) was dissolved in an aqueous solution of NaHCO3 (3 mL) and added dropwise to the filtrate. The mixture was stirred at room temperature for 2 h. The reaction was confirmed to be complete by LC-MS. The reaction solution was concentrated under reduced pressure, a small amount of water was added, and the pH was adjusted to 3 with a saturated sodium citrate solution. The solution was filtered, collected, concentrated under reduced pressure, and lyophilized to obtain a yellow oily substance (271.9 mg, 100%). LC-MS (ESI): m / z found [M+H] + =698.2.

[0313] Synthesis of intermediates 4-8:

[0314] At room temperature, 4-7 (100 mg, 0.143 mmol) was dissolved in DMSO (1 mL), followed by the addition of 1-8 (91 mg, 0.115 mmol), CuSO4·5H2O (35.82 mg, 0.143 mmol), and Vc (25.21 mg, 0.143 mmol). The mixture was stirred at room temperature for 5 min under N2 protection. The reaction was monitored by LC-MS until complete. The reaction solution was purified by reversed-phase column chromatography, concentrated under reduced pressure, and lyophilized to give a yellow solid product (83.5 mg, 29.8%). LC-MS (ESI): m / z found [M+H] + =1491.2.

[0315] Synthesis of compound 4:

[0316] At room temperature, 4-8 (108.2 mg, 0.073 mmol) was dissolved in DMF (1 mL), followed by the addition of HATU (33.10 mg, 0.087 mmol) and DIEA (25.27 μL, 0.145 mmol). The mixture was stirred at room temperature for 5 min under N2 protection, then 11 (30.87 mg, 0.0725 mmol) was added, and the mixture was stirred at room temperature for 2 h under N2 protection. The reaction was monitored by LC-MS until complete. Pre-HPLC was performed, and the product was lyophilized to obtain a yellow solid (7.3 mg, 5.2%). LC-MS (ESI): m / z found [M+H] + =950.2.

[0317] Examples 2-5

[0318] Compound 5

[0319] Synthesis route:

[0320] Compounds 5-1 and 5-2 were prepared according to WO2024230752A1;

[0321] Synthesis of compound 5:

[0322] 5-1 (316 mg, 0.211 mmol) was dissolved in DMAc (3 mL), and HATU (88 mg, 0.232 mmol) was added. After reacting at room temperature for 15 minutes, 5-2 (100 mg, 0.211 mmol) and DIEA (27.2 mg, 0.211 mmol) were added, and the reaction was continued at room temperature for 1 hour. After the reaction of the starting materials was complete as monitored by LC-MS, the mixture was prepared by Pre-HPLC and lyophilized to obtain a yellow solid (139 mg, 33.8%). LC-MS (ESI): m / z found [M+H] + =973.2

[0323] Examples 2-6

[0324] Compound 6

[0325] Prepared according to patent WO2024230752A1.

[0326] Examples 2-7

[0327] Compound 7

[0328] Prepared according to patent WO2024230752A1.

[0329] Compound 8-48 in Examples 2-8 to 2-48 below was prepared with reference to patent WO2024230752A1.

[0330] Examples 2-8

[0331] Compound 8:

[0332] Examples 2-9

[0333] Compound 9:

[0334] Example 2-10

[0335] Compound 10:

[0336] Example 2-11

[0337] Compound 11:

[0338] Example 2-12

[0339] Compound 12:

[0340] Example 2-13

[0341] Compound 13:

[0342] Example 2-14

[0343] Compound 14:

[0344] Example 2-15

[0345] Compound 15:

[0346] Example 2-16

[0347] Compound 16:

[0348] Example 2-17

[0349] Compound 17

[0350] Example 2-18

[0351] Compound 18

[0352] Example 2-19

[0353] Compound 19

[0354] Example 2-20

[0355] Compound 20

[0356] Example 2-21

[0357] Compound 21

[0358] Example 2-22

[0359] Compound 22

[0360] Example 2-23

[0361] Compound 23

[0362] Example 2-24

[0363] Compound 24

[0364] Example 2-25

[0365] Compound 25

[0366] Example 2-26

[0367] Compound 26

[0368] Example 2-27

[0369] Compound 27

[0370] Example 2-28

[0371] Compound 28

[0372] Example 2-29

[0373] Compound 29

[0374] Examples 2-30

[0375] Compound 30

[0376] Example 2-31

[0377] Compound 31

[0378] Example 2-32

[0379] Compound 32

[0380] Example 2-33

[0381] Compound 33

[0382] Examples 2-34

[0383] Compound 34

[0384] Example 2-35

[0385] Compound 35

[0386] Examples 2-36

[0387] Compound 36

[0388] Example 2-37

[0389] Compound 37

[0390] Example 2-38

[0391] Compound 38

[0392] Example 2-39

[0393] Compound 39

[0394] Example 2-40

[0395] Compound 40

[0396] Example 2-41

[0397] Compound 41

[0398] Example 2-42

[0399] Compound 42

[0400] Examples 2-43

[0401] Compound 43

[0402] Example 2-44

[0403] Compound 44

[0404] Example 2-45

[0405] Compound 45

[0406] Examples 2-46 Compound 46

[0407] Synthesis route:

[0408] Synthesis of compound 46:

[0409] At room temperature, 27-1 (16 mg, 0.011 mmol) and HATU (5.1 mg, 0.013 mmol) were dissolved in DMF (2 mL), and compound 2a (10 mg, 0.011 mmol) and DIEA (3.8 μL, 0.022 mmol) were added. The product was purified by HPLC to give a yellow solid, 46 (2.02 mg, 30%). LC-MS (ESI): m / z found [M / 2+H]+ = 948.32.

[0410] Example 2-47

[0411] Compound 47

[0412] Synthesis route:

[0413] Synthesis of compound 47:

[0414] Compound 27-1 (17 mg, 0.0114 mmol) and HATU (5.2 mg, 0.0137 mmol) were dissolved in DMF (3 mL) under ice bath conditions and reacted for 15 min. Then, compound 3a (5 mg, 0.0114 mmol) and DIPEA (3.9 μL) were added, followed by slow heating to room temperature and stirring for 2 h. The reaction was monitored by LC-MS until complete. The reaction solution was purified by pre-HPLC and lyophilized to give a yellow solid, compound 47 (7.01 mg, 32%). LC-MS (ESI): m / z found [M / 2+H]+=955.2.

[0415] Example 2-48

[0416] Compound 48

[0417] Synthesis route:

[0418] Synthesis of intermediate 29-2:

[0419] 29-1 (500 mg, 1.61 mmol) was dissolved in DCM (35 mL), and (PNP)₂CO (1.96 g, 6.44 mmol) and DIEA (208 mg, 1.61 mmol) were added. The mixture was reacted at room temperature for 16 h. TLC showed that the reaction proceeded substantially to completion. After concentration under reduced pressure, the mixture was purified by normal-phase column chromatography and concentrated under reduced pressure to give a yellow solid compound (550 mg, 63.7%).

[0420] Synthesis of intermediate 29-3:

[0421] 29-2 (61.2 mg, 0.114 mmol) was dissolved in DMF (2 mL), HOBt (17 mg, 0.126 mmol) was added, and the mixture was stirred for 30 min. Then, DIEA (30 mg, 0.228 mmol) and compound 3a (50 mg, 0.114 mmol) were added, and the mixture was reacted at room temperature for 2 h. The mixture was purified by reversed-phase column chromatography, concentrated under reduced pressure, and lyophilized to give a yellow solid compound (76 mg, 79%). LC-MS (ESI): m / z found [M+H] + =835.4.

[0422] Synthesis of intermediate 29-4:

[0423] 29-3 (76 mg, 0.091 mmol) was dissolved in DMF (3 mL), and piperidine (78 mg, 0.91 mmol) was added. The reaction was carried out at room temperature for 2 h. The reaction was monitored by LC-MS until complete. The compound was purified by pre-HPLC and lyophilized to give a yellow solid compound (20 mg, 30%). LC-MS (ESI): m / z found [M+H] + =612.35.

[0424] Synthesis of compound 48:

[0425] 29-5 (11 mg, 8.17 μmol) was dissolved in DMF (1 mL), and HATU (5 mg, 9.8 μmol) was added. The mixture was reacted at room temperature for 2 h. Then, DIEA (1 mg, 8.17 μmol) and 29-4 (3.73 mg, 8.17 μmol) were added sequentially, and the mixture was reacted at room temperature for 6 h. The reaction was monitored to be complete by LC-MS. The mixture was prepared by pre-HPLC and lyophilized to give a yellow solid compound 48 (2.01 mg, 12.6%). LC-MS (ESI): m / z found [M / 2+H] + =970.85.

[0426] Table 12 Structure and LC-MS of Compound 49

[0427] Example 2-50

[0428] Compound 50

[0429] Synthesis route:

[0430] Synthesis of intermediate 31-2:

[0431] Compound 31-1 (200 mg, 0.112 mmol) was dissolved in DCM (5 mL) at room temperature, and TFA (2 mL) was added. The reaction was carried out at room temperature for 2 h. After dilution with water, one drop of the free product DIEA was added, followed by extraction with EA, concentration under reduced pressure, and purification by reversed-phase column chromatography to obtain a yellow solid compound (140 mg, 86%). LC-MS (ESI): m / z found [M+H] + =469.20. Synthesis of intermediate 31-3:

[0432] At room temperature, Boc-L-Val (50 mg, 0.088 mmol) and HATU (136 mg, 0.112 mmol) were dissolved in DMF (1.5 mL), followed by the addition of compound 31-2 (50 mg, 0.088 mmol) and DIEA (116 mg, 0.120 mmol). The reaction was carried out at room temperature for 2 h. The reaction was monitored by LC-MS until complete. The compound was purified by reversed-phase column chromatography and concentrated under reduced pressure to obtain the compound (150 mg, 71%). LC-MS (ESI): m / z found [M+H] + =668.2.

[0433] Synthesis of intermediate 31-4:

[0434] At room temperature, 31-3 (75 mg, 0.112 mmol) was dissolved in dichloromethane (5 mL), and TFA (2 mL) was added. The reaction was allowed to proceed for 2 h at room temperature. The reaction was monitored by LC-MS until complete, and the solid was purified by reversed-phase column chromatography to obtain a yellow solid (60 mg, 86%). LC-MS (ESI): m / z found [M+H] + =568.20.

[0435] Synthesis of intermediate 31-5:

[0436] At room temperature, Azido-dPEG 4-acid (28 mg, 0.088 mmol) was dissolved in DMF (1.5 mL), followed by the sequential addition of HATU (55 mg, 0.112 mmol), 31-4 (55 mg, 0.088 mmol), and DIEA (25 mg, 0.120 mmol). The reaction was carried out at room temperature for 2 h. The reaction was monitored by LC-MS until complete. The product was purified by reversed-phase column chromatography and lyophilized to obtain the target product (37 mg, 56%). LC-MS (ESI): m / z found [M+H]+ =840.30.

[0437] Synthesis of Compound 50:

[0438] At room temperature, 31-6 (16 mg, 0.088 mmol) was dissolved in DMSO (1.5 mL), followed by the addition of ascorbic acid (8.5 mg, 0.112 mmol), anhydrous copper sulfate (6 mg), and 31-5 (55 mg, 0.088 mmol). The mixture was reacted at room temperature for 2 h. LC-MS monitoring showed that the reaction was essentially complete. The mixture was purified by pre-HPLC and lyophilized to give 50 (2.3 mg) of a yellow solid. LC-MS (ESI): m / z found [M / 2+H] + =818.70.

[0439] Example 2-51

[0440] Compound 51

[0441] Synthesis route:

[0442] Synthesis of compound 51:

[0443] Compound 10a (5 mg, 0.0114 mmol) was dissolved in N,N-dimethylaniline (3 mL) under ice bath conditions, followed by the addition of N,N-diisopropylethylamine (2 μL, 0.0114 mmol). After reacting for 15 min, compound 31-1 (16 mg, 0.0114 mmol) and N,N-diisopropylethylamine (2 μL, 0.0114 mmol) were added, followed by incubation at room temperature for 2 h. The reaction was monitored by LC-MS to ensure complete reaction. The reaction solution was purified by pre-HPLC to obtain compound 51 (4.84 mg, 25%). LC-MS (ESI): m / z found [M+H] + =1721.0.

[0444] Example 2-52

[0445] Compound 52

[0446] Synthesis route:

[0447] Synthesis of compound 52:

[0448] Under ice bath conditions, 27-1 (11.19 mg, 0.0075 mmol) was dissolved in DMA (2 mL), and HATU (3.14 mg, 0.0083 mmol) was added. After stirring in an ice bath for 10 min, compound 2a (3.18 mg, 0.0075 mmol) and DIEA (1.31 μL, 0.0075 mmol) were added sequentially, and the reaction was carried out at room temperature for 1 h. LC-MS analysis confirmed complete reaction of the starting material. The reaction solution was filtered, and the filtrate was purified manually by pre-HPLC to obtain a yellow solid compound 52 (3.54 mg, 24.86%). LC-MS (ESI): m / z found [M+H / 2] + =949.5.

[0449] Examples 2-53

[0450] Compound 53

[0451] Synthesis route:

[0452] Synthesis of intermediate 34-2:

[0453] Compound 34-1 (1 g, 0.0063 mmol) was dissolved in methanol (60 mL) at room temperature. MeSNa (884 mg, 0.0126 mmol) and K₂CO₃ (1.74 g, 0.0126 mmol) were added, and the reaction was allowed to proceed overnight at room temperature. LCMS (5-100, 3 min) was used to determine if the reaction was complete. The reaction solution was dried under reduced pressure. The crude product was dissolved in water, and the aqueous phase was washed three times with ethyl acetate. The collected aqueous phase was placed in an ice bath, and 2N hydrochloric acid aqueous solution was slowly added repeatedly until a large amount of solid precipitated. The mixture was filtered, and the filter cake was washed with pure water. The filter cake was collected and lyophilized to give a white solid, compound 34-2 (707 mg, 65.93%). LCMS (ESI): m / z found [M+H] + =171.0.

[0454] Synthesis of intermediate 34-3:

[0455] Compound 34-2 (300 mg, 1.765 mmol) was dissolved in 10 mL of DCM under ice bath conditions. HOSU (245 mg, 2.118 mmol) and EDCI (406 mg, 2.118 mmol) were added sequentially. After stirring in an ice bath for 2 h, amino-hexaethylene glycol-carboxylic acid (624 mg, 1.765 mmol) and TEA (245 μL, 1.176 mmol) were added, and the reaction was allowed to proceed at room temperature for 2 h. LC-MS analysis confirmed complete reaction of the starting material. The reaction solution was dried under reduced pressure, dissolved in a small amount of methanol, purified by reverse column chromatography, and lyophilized to obtain a pale yellow oily solid, compound 34-3 (285.9 mg, 32.04%). LC-MS (ESI): m / z found [M+H] + =506.2.

[0456] Synthesis of intermediate 34-4:

[0457] Compound 34-3 (285.9 mg, 0.565 mmol) was dissolved in 10 mL of DCM under ice bath conditions. M-chloroperoxybenzoic acid (243.96 g, 1.414 mmol) was added, and the mixture was in an ice bath for 10 min, then the reaction was carried out at room temperature for 3 h. LC-MS (100-1000, 4 min) was used to determine if the reaction was complete. The reaction solution was dried under reduced pressure, dissolved in a small amount of DMF, and purified by reverse-phase column chromatography and lyophilized to give compound 34-4 (173 mg, 56.9%) as a pale yellow oily solid. LC-MS (ESI): m / z found [M+H] + =538.4.

[0458] Synthesis of intermediate 34-6:

[0459] Compound 34-4 (50 mg, 0.093 mmol) was dissolved in DMF (2.5 mL) under ice bath conditions. HATU (40.32 mg, 0.106 mmol) was added, and the mixture was reacted under ice bath conditions for 10 min. Then, DIEA (30.78 μL, 0.176 mmol) and compound 34-5 (31.3 mg, 0.093 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction was monitored for completeness by LC-MS (5-100, 3 min). The reaction solution was filtered and purified manually by pre-HPLC to obtain a white solid 34-6 (10 mg, 12.56%). LC-MS (ESI): m / z found [M+H] + =856.2.

[0460] Synthesis of compound 53:

[0461] Under ice bath conditions, 34-6 (6.1 mg, 0.0071 mmol) was dissolved in DMF (2.5 mL), and HATU (3.2 mg, 0.0085 mmol) was added. The mixture was reacted under ice bath conditions for 10 min, followed by the addition of DIEA (2.5 μL, 0.0142 mmol) and compound 2a (3.0 mg, 0.0071 mmol). The mixture was stirred at room temperature for 2 h. The reaction was monitored for completeness by LC-MS (5-100, 3 min). The reaction solution was filtered and purified manually by pre-HPLC to obtain a white solid compound 53 (2.45 mg, 27.35%). LC-MS (ESI): m / z found [M+H] + =1261.34.

[0462] Example 2-54

[0463] Compound 54

[0464] Synthesis route:

[0465] Synthesis of intermediate 35-2:

[0466] Compound 11a (18.3 mg, 0.043 mmol) was dissolved in DCM (3 mL) at room temperature, followed by the addition of compound 35-1 (78.66 mg, 0.21 mmol) and TFA (25 μL). The reaction was allowed to proceed at room temperature for 4 h. LCMS (5-100, 3 min) was used to determine the completeness of the reaction. The reaction solution was dried under reduced pressure, dissolved in a small amount of DMF, and purified by pre-HPLC to obtain a white solid compound 35-2 (13.5 mg, 40.17%). LCMS (ESI): m / z found [M+H]+=787.2.

[0467] Synthesis of intermediate 35-3:

[0468] Compound 35-2 (13.5 mg, 0.017 mmol) was dissolved in DCM (4 mL) at room temperature, and diethylamine (1 mL) was added. The reaction was carried out at room temperature for 1.5 h. LC-MS (5-100, 3 min) showed that the reaction proceeds were completely reacted. The reaction solution was dried under reduced pressure, dissolved in a small amount of DMF, and purified by pre-HPLC to obtain a white solid compound 35-3 (9.7 mg, 100%). LCMS (ESI): m / z found [M+H]+=565.2.

[0469] Synthesis of intermediate 35-5:

[0470] Compound 35-4 (11.68 mg, 0.023 mmol) was dissolved in DMF (2 mL) under ice bath conditions. HATU (10.48 mg, 0.028 mmol) was added, and the mixture was reacted under ice bath conditions for 10 min. Then, DIEA (6 μL, 0.034 mmol) and compound 35-3 (13 mg, 0.023 mmol) were added, and the mixture was stirred at room temperature for 1.5 h. The reaction was monitored for completeness by LC-MS. The reaction solution was filtered and purified by pre-HPLC to obtain a yellow solid compound 35-5 (4 mg, 19.86%). LCMS (ESI): m / z found [M+H]+ = 1056.2. Synthesis of compound 54:

[0471] Compound 35-5 (4 mg, 0.001 mmol) was dissolved in a mixed solution of EtOH (1.5 mL) and THF (0.5 mL) at room temperature. Compound 31-6 (3 mg, 0.001 mmol), copper sulfate pentahydrate (1.14 mg, 0.0012 mmol), and vitamin C (0.8 mg, 0.0012 mmol) were added. The mixture was purged with nitrogen three times and reacted at room temperature for 2 h. The reaction was monitored by LC-MS to ensure complete reaction. The reaction solution was dried under reduced pressure, dissolved in a small amount of DMF, and purified by pre-HPLC to obtain a yellow solid compound 54 (0.3 mg, 4.28%). LC-MS (ESI): m / z found [M+H / 2]+=925.0.

[0472] Example 2-55

[0473] Compound 55

[0474] Synthesis route:

[0475] Synthesis of intermediate 36-1:

[0476] Compound 25a (135 mg, 0.331 mmol) was dissolved in a mixed solvent of DMF (1 mL) and DMSO (1 mL), and bis(p-nitrobenzene) carbonate (301 mg, 0.993 mmol) and DIEA (59 μL, 0.331 mmol) were added. The reaction was carried out at room temperature for 3 h. The reaction was monitored by LCMS until complete. The product was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by normal-phase column chromatography to give a yellow solid product (100 mg, 52.9%). LCMS (ESI): m / z found [M+H] + =574.0. Synthesis of intermediate 36-3:

[0477] Compound 36-2 (150 mg, 0.239 mmol) was dissolved in DMA (4 mL), and N,N'-dimethylethylenediamine (26 μL, 0.239 mmol) was added under ice bath conditions. The reaction was carried out at room temperature for 1.5 h. LCMS analysis showed the reaction was complete. The product was purified by reversed-phase column chromatography, concentrated under reduced pressure by distillation, and lyophilized to give a yellow solid (100 mg, 41.3%). LCMS (ESI): m / z found [M+H] + =577.2. Synthesis of intermediate 36-4:

[0478] Compounds 36-3 (100 mg, 0.174 mmol) and 36-4 (100 mg, 0.174 mmol) were dissolved in DMA (2.5 mL) and reacted in an ice bath for 1.5 h. The reaction was confirmed to be complete by LCMS. The product was purified by reversed-phase column chromatography, concentrated under reduced pressure by distillation, and lyophilized to give a yellow solid product (55 mg, 31.4%). LCMS (ESI): m / z found [M+H] + =1011.2.

[0479] Synthesis of compound 55:

[0480] Compounds 36-4 (20 mg, 0.020 mmol), 31-6 (15.7 mg, 0.020 mmol), copper sulfate pentahydrate (5.9 mg, 0.024 mmol), and ascorbic acid (4.2 mg, 0.024 mmol) were dissolved in a mixed solvent of anhydrous ethanol (1.5 mL) and tetrahydrofuran (0.5 mL). The reaction was carried out under nitrogen protection at room temperature for 2.5 h. The reaction was confirmed to be complete by LCMS. The solvent was evaporated under reduced pressure, and the solution was prepared by HPLC and lyophilized to give a yellow solid compound 55 (7.04 mg, 19.7%). LCMS (ESI): m / z found [M / 2+H] + =902.8.

[0481] Example 2-56

[0482] Compound 56

[0483] Synthesis route:

[0484] Synthesis of intermediate 37-1:

[0485] Compound 36-1 (40 mg, 0.070 mmol) was dissolved in DMA (2.5 mL), and N-BOC-N,N'-dimethylethylenediamine (13.6 μL, 0.070 mmol) was added. The reaction was allowed to proceed at room temperature for 1.5 h. The reaction was monitored by LCMS until complete. The product was purified by reversed-phase column chromatography, concentrated under reduced pressure by distillation, and lyophilized to give a yellow solid product (40 mg, 92.0%). LCMS (ESI): m / z found [M+H] + =623.2. Synthesis of intermediate 37-2:

[0486] Compound 37-1 (40 mg, 0.064 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. The reaction was carried out at room temperature for 0.5 h. LCMS analysis showed that the reaction was complete. The system was concentrated under reduced pressure to obtain the crude product, which was then directly added to the next reaction (35 mg). LCMS (ESI): m / z found [M+H] + =523.2.

[0487] Synthesis of compound 56:

[0488] Compound 37-3 (40 mg, 0.028 mmol) was dissolved in DMA (3 mL), and HATU (12.8 mg, 0.034 mmol) was added. After reacting at room temperature for 15 min, compound 37-2 (14.8 mg, 0.028 mmol) and DIEA (7.6 μL, 0.042 mmol) were added, and the reaction was carried out at room temperature for 1 h. The reaction was monitored by LCMS until complete. The product was prepared by HPLC and lyophilized to give a yellow solid product 56 (8.6 mg, 16.1%). LCMS (ESI): m / z found [M / 2+H] + =954.2.

[0489] Example 2-57

[0490] Compound 57

[0491] Synthesis route:

[0492] Synthesis of intermediate 38-1:

[0493] Compound 24a (80 mg, 0.172 mmol) was dissolved in toluene (2.5 mL), and compound 35-1 (316 mg, 0.858 mmol) and zinc acetate (63 mg, 0.343 mmol) were added. The reaction was carried out at room temperature for 3 h. The reaction was monitored by LCMS until it was complete. The product was purified by pre-HPLC and lyophilized to give a yellow solid (25 mg, 18.8%). LCMS (ESI): m / z found [M+H]+=775.2.

[0494] Synthesis of intermediate 38-2:

[0495] Compound 38-2 (25 mg, 0.032 mmol) was dissolved in DMF (1 mL), and diethylamine (0.2 mL) was added. The reaction was carried out at room temperature for 2 h. The reaction was monitored by LCMS until it was complete. The product was purified by pre-HPLC and lyophilized to give a yellow solid product (5 mg, 28.1%). LCMS (ESI): m / z found [M+H]+=553.0.

[0496] Synthesis of compound 57:

[0497] Compound 29-5 (5 mg, 0.009 mmol) was dissolved in DMF (0.5 mL), and HATU (4 mg, 0.011 mmol) was added. After stirring at room temperature for 15 min, compound 38-2 (316 mg, 0.858 mmol) and DIEA (1.16 mg, 0.343 mmol) were added. The reaction was allowed to proceed at room temperature for 2 h. The reaction was confirmed to be complete by LCMS. The product was purified by HPLC and lyophilized to give a yellow solid product 57 (1.95 mg, 11.5%). LCMS (ESI): m / z found [M / 2+H]+=940.8.

[0498] Example 2-58

[0499] Compound 58

[0500] Synthesis route:

[0501] Synthesis of intermediate 39-1:

[0502] Compound 24a (64 mg, 0.137 mmol) was dissolved in DMF (0.6 mL), and DIEA (48 μL) and (PNP)₂CO (167 mg, 0.5494 mmol) were added. The mixture was stirred at room temperature for 12 h. The reaction was confirmed to be complete by LCMS. The product was purified by reversed-phase column chromatography to give a yellow solid (40 mg, 46.2%). LCMS (ESI): m / z found [M+H] +=632.0.

[0503] Synthesis of intermediate 39-2:

[0504] Compound 39-1 (20 mg, 0.137 mmol) was dissolved in DMA (0.6 mL), and DIEA (11 μL), HOBT (5 mg, 0.035 mmol), and N,N'-dimethylethylenediamine (3.5 μL) were added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until complete. The product was purified by reversed-phase column chromatography (42% ACN in H2O) to give a yellow solid product (22 mg, 27.6%). LCMS (ESI): m / z found [M+H] + =581.2.

[0505] Synthesis of compound 58:

[0506] Compound 37-3 (24 mg, 0.017 mmol) was dissolved in DMA (0.6 mL), and DIEA (4.5 μL), HATU (7.9 mg, 0.021 mmol), and 39-2 (10 mg, 0.017 mmol) were added. The mixture was stirred at room temperature for 2 h. The reaction was confirmed to be complete by LC-MS. The reaction solution was filtered, concentrated, and purified by HPLC to give a yellow solid product 58 (0.71 mg). LC-MS (ESI): m / z found [M / 2+H] + =983.9.

[0507] Except for replacing the corresponding raw materials, Examples 2-59 were synthesized according to the synthesis method of Examples 2-54. The structure and LC-MS information are shown in Table 13.

[0508] Table 13 Structure and LC-MS of Examples 2-59

[0509] Example 2-60

[0510] Compound 60

[0511] Except for replacing the corresponding reaction raw materials, the synthesis methods of Examples 2-51 were used to synthesize Examples 2-60. The structures and LC-MS information are shown in Table 14.

[0512] Table 14 Structure and LC-MS of Examples 2-60

[0513] Example 2-61

[0514] Compound 61

[0515] Except for replacing the corresponding raw materials, Examples 2-61 were synthesized according to the synthesis method of Examples 2-58. The structure and LC-MS information are shown in Table 15 below.

[0516] Table 15. Structural Examples and LC-MS

[0517] Example 2-62

[0518] Compound 62

[0519] Synthesis route:

[0520] Synthesis of compound 62:

[0521] 5-1 (316 mg, 0.211 mmol) was dissolved in DMAc (3 mL), and HATU (88 mg, 0.232 mmol) was added. After reacting at room temperature for 15 minutes, 5-2 (100 mg, 0.211 mmol) and DIEA (27.2 mg, 0.211 mmol) were added, and the reaction was carried out at room temperature for 1 hour. After the reaction of the starting materials was complete as monitored by LC-MS, the solution was prepared by Pre-HPLC and lyophilized to obtain a yellow solid 62 (139 mg, 33.8%). LC-MS (ESI): m / z found [M+H] + =973.2.

[0522] Example 2-63

[0523] Compound 63

[0524] Synthesized according to WO2024230752A1, the structure and LC-MS are shown in Table 16 below:

[0525] Table 16. Embodiment Structure and LC-MS

[0526] Example 2-64

[0527] Compound 64

[0528] Except for replacing the corresponding raw materials, the synthesis was carried out according to Examples 2-55. The structure and LC-MS results are shown in Table 17 below:

[0529] Table 17. Structural Examples and LC-MS

[0530] Example 2-65

[0531] Compound 65

[0532] Synthesized according to WO2024230752A1, the structure and LC-MS are shown in Table 18 below:

[0533] Table 18. Structural Examples and LC-MS

[0534] Example 2-66

[0535] Compound 66

[0536] Synthesis route:

[0537] Synthesis of compound 66:

[0538] Linker 3 (10 mg, 8.33 μmol) was dissolved in DMF (1 mL), and HATU (5 mg, 9.16 μmol) was added. The mixture was reacted at room temperature for 2 h, followed by the addition of DIEA (1 mg, 16.67 μmol) and 9-4 (3.73 mg, 8.33 μmol), and then reacted again at room temperature for 2 h. The reaction was monitored by LC-MS until complete. After pre-HPLC purification, the product was lyophilized to give a yellow solid, 66 (3.69 mg, 25.2%). LC-MS (ESI): m / z found [M / 2+H] + =897.85.

[0539] Experimental Example 1: In vitro tumor-suppressive activity of camptothecin-based small molecules

[0540] The small molecule used in this ADC was prepared according to WO2024230752A1, and the positive control drug was DXD.

[0541] The small molecule structures used in the ADC disclosed herein include compounds P-1, P-2, and P-3:

[0542] 1. Experimental Methods

[0543] 1. Prepare cell suspensions using fresh cell culture medium containing 10% FBS. Add 2000 cells per well of NCI-H358, LNCAP, DU145, and MDA-MB-453, and 4000 cells per well of NCI-H526, to 96-well cell culture plates. Add PBS to the outermost wells of the cell plate and incubate at 37°C and 5% CO2 for 24 hours.

[0544] 2. Prepare culture medium solutions containing small molecules. The initial concentration of NCI-H358, LNCAP, DU145, and MDA-MB-453 in each well is 1200 nM. These solutions are then serially diluted 5-fold with fresh culture medium, replacing the old medium with 100 μL per well. The initial concentration of NCI-H526 in each well is 2400 nM. This solution is also serially diluted 5-fold with fresh culture medium, adding 50 μL to each well. There are 10 concentration points (the 10th point being the zero concentration point). The cells are then incubated at 37°C and 5% CO2 for 5 days.

[0545] 3. On the last day, discard the old culture medium and add 100 μL of culture medium containing CCK8 detection solution to each well (blank medium: CCK8 ratio of 9:1). Continue to incubate at 37℃ and 5% CO2 for 4 hours.

[0546] 4. Measure the absorbance at 450 nm using an ELISA reader.

[0547] 5. Process and analyze the results using Graphpad Prism. The results are shown in Table 19 below.

[0548] Table 19. Activity data of some small molecules in the ADC compounds disclosed herein. "-" indicates that no test was performed.

[0549] Experimental results show that the small molecule disclosed herein has comparable, superior or significantly superior proliferation inhibitory activity against cell lines NCI-H358, NCI-H526, LNCaP, DU145, MDA-MB-453, T47D and HT1376 compared with DXD.

[0550] Example 2: Pharmacokinetic Study of Small Molecule Camptothecin in Rats

[0551] I. Grouping, dosage, and administration volume are shown in the table below:

[0552] II. Dosage frequency and method: Single tail vein administration, with a dosage volume of 5 mL / kg;

[0553] III. Blood collection points: 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h;

[0554] IV. Blood collection method: Whole blood was collected through the orbital plexus, and 300 μL of blood was collected.

[0555] V. Blood Sample Processing and Detection: After serum collection, the sample was allowed to stand for 1 hour until clear stratification occurred. The sample was then centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected. The obtained serum samples were stored at -80℃. After the experiment, the samples were tested for free small molecules. The results are shown in Figure 2 and Table 20 below.

[0556] Table 20 Pharmacokinetic Results in SD Rats

[0557] Experimental results show that, compared with DXD, the small molecule disclosed herein has a longer half-life and higher exposure in rats.

[0558] Experimental Example 3: ADC Coupling

[0559] The positive control antibody of this invention is Ifinatamab (also known as M30, prepared using conventional antibody preparation methods) and 2E3-02 as described in WO2023231988A1;

[0560] Ifinatamab heavy chain (SEQ ID NO: 120)

[0561] Ifinatamab light chain (SEQ ID NO: 121)

[0562] 2E3-02 Heavy Chain (SEQ ID NO: 122)

[0563] 2E3-02 Light Chain (SEQ ID NO: 123)

[0564] The exemplary B7H3 antibody sequences 9H5-J1 and 18H6-J37 selected in this invention are as shown above. Compound A of this invention was synthesized according to Example 72 of WO2023138635A1:

[0565] The antibody-drug conjugates disclosed herein were prepared using the following methods. The names and specific structures of the antibody-drug conjugates are shown in Table 21.

[0566] 1. Prepare the solution:

[0567] 1) Prepare a 1mM EDTA-PBS buffer solution using 0.5M EDTA: 20μL EDTA + 10mL PBS.

[0568] 2) Dilute 0.5M TCEP to 5mM: 10Ml TCEP + 990μL EDTA-PBS.

[0569] 3) Prepare a 10% DMSO solution: 100 μL DMSO + 900 μL EDTA-PBS.

[0570] 4) The disclosed antibody or positive reference antibody Ifinatamab or antibody 2E3-02 is diluted to 10 mg / mL.

[0571] 2. TCEP reduction and cleavage of disulfide bonds: Antibody:TCEP = 1:5 (molar ratio);

[0572] 3. Incubate at 37℃ on a shaker for 2 hours (normal shaking speed is 210-230 rpm), and place on ice after shaking.

[0573] 4. Dissolve the Linker-payload (referring to the linker and small molecule drug portions in the ligand-drug conjugate, excluding the ligand portion): Dissolve in pure DMSO to prepare a 5 mg / mL solution.

[0574] 5. Add the Linker-payload to the antibody at a molar ratio of 1:5.

[0575] 6. Mix thoroughly using a rotary mixer for 2 hours.

[0576] 7. After mixing, desalinate using a desalination column and ultrafilter 3 times.

[0577] 8. The concentration of ADC coupling products was determined using the BCA method for the samples obtained after ultrafiltration.

[0578] 9. Determine whether the coupling was successful by HPLC-HIC analysis.

[0579] 10. Antibody purity was determined by SEC-HPLC.

[0580] 11. Detect DAR value using LC-MS.

[0581] Table 21 Names and structural formulas of antibody-drug conjugates

[0582] After ultrafiltration, the concentration of the ADC conjugation product was determined using absorbance measurements at 280 nm and 365 nm; successful conjugation was confirmed by HPLC-HIC analysis; antibody purity was determined by SEC-HPLC; and the DAR value was determined by LC-MS. The conjugation results are shown in Table 22.

[0583] Table 22 B7H3-ADC Coupling Results

[0584] Experimental Example 4: In vitro proliferation inhibition test of the antibody-drug conjugate of the present invention on tumor cells targeting B7H3.

[0585] The cell lines used in this experiment were human A375 (ATCC, catalog number: CRL-1619), NCI-H358 (ATCC, catalog number: CRL-5807), NCI-H345 (BIOBW Beijing Bio-Tech Biotechnology, catalog number: bio-133296), and NCI-H526 cell lines with high B7H3 expression. Cell suspensions were prepared using fresh cell culture medium containing 10% FBS and then diluted to a density of 2 × 10⁻⁶ cells / mL. 4 cells / mL, 4×10 4 cells / mL and 2×10 4 cells / mL, 5×10 3 Cells / mL, A375, NCI-H358, and NCI-H345 were added at 100 μL per well to a 96-well cell culture plate (Thermo catalog number: 167425), and NCI-H526 was added at 50 μL per well to a 96-well cell culture plate (Thermo catalog number: 167425). The plates were incubated at 37°C for 24 h with 5% carbon dioxide.

[0586] The ADC sample was prepared at 10 μM using PBS. This was used as the initial concentration, and the samples were serially diluted five-fold with PBS to obtain nine different concentrations. 50 μL of culture medium was aspirated from each well, and then 50 μL of the above ADC solution was added to each well, resulting in an initial ADC concentration of 5 μM and a final volume of 100 μL per well. The samples were incubated at 37°C with 5% CO2 for 3 days. 100 μL of CTG was added to each well. The Luminescent Cell Viability Assay (Promega, catalog number: G7573) was mixed on a decolorizing shaker at room temperature for 30 min, incubated for 10 min, and the chemiluminescence was read on a microplate reader (TECAN, Spark). Data analysis was performed using Graphpad Prism 5 software. The results are shown in Table 23.

[0587] Table 23 IC50 of the antibody-drug conjugate of the present invention against in vitro proliferation of cancer cells. 50 value "-" indicates that no detection was performed.

[0588] Conclusion: The antibody-drug conjugate targeting B7H3 in this invention has significant inhibitory activity against the proliferation of A375, NCI-H358, NCI-H345, NCI-H526, LNCaP, and DU145 cell lines.

[0589] Experimental Example 5: In vitro and in vivo activity of chimeric antibody-drug conjugates

[0590] Prepare the antibody-drug conjugates listed in Table 24 according to the preparation method in Experimental Example 3.

[0591] I. In vitro activity

[0592] a. Plating & Culture: A375 cells were seeded into 96-well plates at a density of 103 cells / 100 μL / well, and 200 μL of PBS was added to the outermost well (to reduce the evaporation of the culture medium). The plates were then incubated at 37°C and 5% CO2 for 48 h.

[0593] b. Adding the drug and incubating: Take out the 96-well plate and add 100 μL of the diluted compound (the concentration of the first well is 10 μm, and it is serially diluted 5 times. A total of 9 dilutions are performed, and the concentration of the last point is 0). Treat at 37°C and 5% CO2 for 120 h.

[0594] Detection and data processing

[0595] 1. Remove the 96-well plate, remove the culture medium, and add 100 μL of detection solution (CCK8: culture medium = 1:9) to each well; incubate at 37°C in a 5% CO2 incubator for 4 hours;

[0596] 2. Use an ELISA reader (CEY0017) to read the values ​​at 450nm and record them using Excel;

[0597] 3. Use Graphpad Prism 9.0 to analyze and process data.

[0598] Table 24 Cell killing results

[0599] Experimental Example 6. Intra-pharmaceutical efficacy of chimeric antibody-drug conjugates

[0600] Experimental Example 6.1 A375 Efficacy Experiment

[0601] Human melanoma cells A375 (5 × 10⁵) were subcutaneously injected into the right rib area of ​​BALB / c-Nude nude mice. 6 / 200μL / with only 50% low-growth-factor artificial basement membrane. After cell seeding, the tumor grew to a volume of 130mm after 7 days. 3 Animals were randomly divided into eight groups (D0) of five animals each. Administered via tail vein injection (M30-compound A, 12C12-Q1-compound A, 9H5-Q1-compound A) at a single dose of 5 mg / kg, once daily. Tumor volume and body weight were measured twice weekly, and the data were recorded. The results are shown in Figure 3.

[0602] Conclusion: The ADC drug of this invention has excellent inhibitory effect on the proliferation of human melanoma cells (A375 cells).

[0603] Experimental Example 6.2 NCI-H358 Efficacy Test

[0604] BALB / c-Nude nude mice were subcutaneously inoculated with 3 × 10⁶ human non-small cell lung cancer cells NCI-H358 in the right rib area. 6 / 200μL / cell, with an artificial basement membrane containing 50% low growth factors. After cell seeding, tumors grew to a volume of 130mm in 7 days. 3 After resection, the animals were randomly divided into eight groups (D0), with five animals in each group. Medications (M30-compound A, 4G11-Q1-compound A, 18H6-Q1-compound A) were administered via tail vein injection at a single dose of 1.5 mg / kg, once daily. Tumor volume and body weight were measured twice weekly, and the data were recorded. The results are shown in Figure 4.

[0605] Tumor volume (V): V = 1 / 2L major diameter * L minor diameter * L minor diameter

[0606] Conclusion: The ADC drug of this invention has excellent inhibitory effect on the proliferation of human non-small cell lung cancer cells (NCI-H358 cells).

[0607] Experimental Example 6.3: In vitro assay of the humanized antibody-drug conjugate of the present invention to inhibit tumor cell proliferation.

[0608] Following the preparation method in Example 3, antibody-drug conjugates listed in Table 25-27 were prepared (the corresponding numbered antibodies were conjugated with compound A). The results were tested according to the method in Example 6.1. The results are shown in Table 25-27.

[0609] Table 25 Results of Activity Assay for 9H5 Humanized Antibody-Drug Conjugate

[0610] Table 26 Results of Activity Assay for 4G11 Humanized Antibody-Drug Conjugate

[0611] Table 27 Results of activity assay of 18H6 humanized antibody-drug conjugate

[0612] Conclusion: The ADC drug of this invention has excellent inhibitory effect on the proliferation of human melanoma cells (A375 cells).

[0613] Experimental Example 7: In vivo efficacy of humanized antibody ADC

[0614] NCI-H526 model

[0615] Following the method described in Example 3, the humanized antibody was conjugated to the small molecule (compound 7) with a DAR of 4. The resulting ADCs were 4G11-J58-compound 7, 4G11-J60-compound 7, 18H6-J37-compound 7, 9H5-J1-compound 7, and Ifinatamab-compound 7.

[0616] Female BALB / c nude mice aged 6-8 weeks were selected and subcutaneously injected with 4×10⁴ mg of a solution dissolved in 200 μL (containing 100 μL of low growth factor matrix gel) in the right posterior region. 6 Human non-small cell lung cancer cells (NCI-H526) were used until the tumor grew to an average volume of 120 mm. 3 At approximately 10:00 AM, mice were randomly divided into 7 groups of 5 mice each, based on their body weight and tumor size. One group served as the solvent control group, and the other as the single-dose control group. The mice received a single dose of 1.5 mg / kg. Body weight and tumor volume were measured twice weekly, and the animals' survival status was observed throughout the experiment. The results are shown in Table 28 and Figure 5.

[0617] Table 28. In vivo antitumor activity of the ADC drug of the present invention in the NCI-H526 model.

[0618] Conclusion: The ADC drug of the present invention exhibits excellent tumor suppressor activity in the small cell lung cancer NCI-H526 CDX model.

[0619] Example 8: Animal efficacy of different linker-payload ADCs in the NCI-H526 CDX model. 9H5-J1 was conjugated with compounds 4, 5, 6, 7, and 2, with a DAR of 4. Female 6-8 week old BALB / c nude mice were subcutaneously injected with 4 × 10⁻⁶ ADCs dissolved in 200 μL (containing 100 μL of low growth factor matrix gel) on the right posterior side. 6 Human non-small cell lung cancer cells (NCI-H526) were used until the tumor grew to an average volume of 120 mm. 3 Around 10:00 AM, mice were randomly divided into 11 groups of 5 mice each, based on their body weight and tumor size. These groups served as a solvent-based blank control group and a drug-treated group, respectively, with a single dose administered. Body weight and tumor volume were measured twice weekly, and the animals' survival status was observed throughout the experiment. The results are shown in Tables 29 and 30 and Figures 6 and 7.

[0620] Table 29. In vivo antitumor activity of the ADC drug of the present invention in the NCI-H526 model.

[0621] Table 30. In vivo tumor activity of the ADC of the present invention in the NCI-H526 model.

[0622] Conclusion: The ADC drug of this invention showed excellent antitumor activity in the NCI-H526 CDX model of small cell lung cancer, and had a three-fold dose advantage compared with the positive control drug DS7300.

[0623] Experimental Example 9: Animal efficacy of the ADC of the present invention in the NCI-H358 CDX model

[0624] Female BALB / c nude mice aged 6-8 weeks were selected and subcutaneously injected into the right posterior region with a solution of 5×10⁶ mg / L of a solution dissolved in 200 μL (containing 100 μL of low growth factor matrix gel). 6 Human non-small cell lung cancer cells (NCI-H358) were used until the tumor grew to an average volume of 120 mm. 3 At approximately 10:00 AM, mice were randomly divided into 7 groups of 5 mice each, based on their body weight and tumor size. The groups were: a solvent-free control group; and groups receiving DS7300, 9H5-J1-compound 4, 9H5-J1-compound 5, 9H5-J1-compound 6, 9H5-J1-compound 7, and 9H5-J1-compound 2, administered as a single dose of 1 mg / kg. Body weight and tumor volume were measured twice weekly, and the animals' survival status was observed throughout the experiment. The results are shown in Table 31 and Figure 8.

[0625] Table 31. In vivo antitumor activity of the ADC drug of the present invention in the NCI-H358 model.

[0626] Conclusion: The ADC drug of the present invention exhibits excellent tumor suppressive activity in the NCI-H358 CDX model of non-small cell lung cancer, and is significantly superior to the positive control drug DS7300.

[0627] Experimental Example 10: Animal efficacy of the ADC of the present invention in the A375 CDX model.

[0628] (1) Human melanoma cells A375 (5 × 10⁶) were subcutaneously inoculated into the right rib area of ​​BALB / c-Nude nude mice. 6 / 200μL / cell (artificial basement membrane with 50% low growth factor). After cell seeding, tumors grew for 7 days, reaching a volume of 120mm. 3 The animals were then randomly grouped into three groups (D0), with five animals in each group.

[0629] (2) The drug was administered via tail vein injection at doses of 1 mg / kg and 2 mg / kg, as a single injection. Tumor volume and body weight were measured twice weekly, and the data were recorded. Data were statistically analyzed using Excel 2016 software: average values ​​were calculated as average; SD values ​​were calculated as STDEV; and SEM values ​​were calculated as STDEV / SQRT. Tumor growth curves were generated using GrapHPad Prism 9 software.

[0630] Tumor volume (V): V = 1 / 2L major diameter * L minor diameter * L minor diameter

[0631] Relative tumor volume (RTV): RTV = VT / V0 Relative tumor proliferation rate T / C (%) = TRTV / CRTV × 100% Tumor inhibition rate (%) = (CRTV - TRTV) / CRTV (%) Tumor weight inhibition rate (%) = (1 - average tumor weight in the treatment group / average tumor weight in the control group) * 100%

[0632] V0 and VT represent the tumor volumes at the start and end of the experiment, respectively. CRTV and TRTV represent the relative tumor volumes of the blank control group (PBS) and the experimental group at the end of the experiment, respectively. The results are shown in Table 32 and Figure 9.

[0633] Table 32. In vivo antitumor activity of the ADC drug of the present invention in the A375 CDX model.

[0634] Conclusion: The ADC drug of the present invention exhibits excellent antitumor activity in melanoma A375 CDX and is significantly superior to the positive control drug DS-7300.

[0635] Experimental Example 11: Bystander Effect of the In Vivo and In Vivo ADC of the Present Invention

[0636] 1. External bystander effect

[0637] Experimental steps:

[0638] a. Cell seeding (mixed wells: NCI-H358 (positive cells): MDA-MB-453 (negative cells) = 1:1, 1×10⁻⁶ cells of each type) 5 In 6-well plates, the single cell per well is 1×10⁶. 5 Each well contains 4 mL of 10% FBS 1640 medium.

[0639] b. On day 2, the ADC drug was added to the 6-well plate at concentrations of 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, 0.05 nM, and 0 nM.

[0640] c. On the 4th day after drug administration and incubation, discard the supernatant, digest the cells in the well plate, collect them in centrifuge tubes, and collect the cells at 300xg for 5 min.

[0641] d. Wash the cells once with 2% FACS, and seed 100 μL into each well of a 96-well plate, making 3 wells for each concentration.

[0642] e. Add primary antibody M30 (1 μg / 100 μL / well), mix well, and incubate at 4°C for 1 hour.

[0643] f. Wash twice with 2% FACS, add secondary antibody anti-human FC (1:200) and NIR staining solution (1:1000), 100 μL / well, and incubate at 4 degrees Celsius in the dark for 30 min.

[0644] g. Wash 3 times with 2% FACS, wash once with PBS, and then perform flow cytometry detection after resuspending in PBS.

[0645] The experimental results are shown in Table 33 and Figure 10.

[0646] Table 33. The lethality of the bystander effect.

[0647] Conclusion: The ADC drug of the present invention has excellent in vitro bystander effect and is significantly superior to the positive control drug DS-7300.

[0648] 2. In vivo bystander effect in the A375(B7H3 Positive)+MDA-MB-453(B7H3 Negative) model

[0649] Select 6-8 week old female BALB / c nude mice and subcutaneously inject 200 μL (containing 100 μL of low growth factor matrix gel) into the right posterior region with 5×10 6 Human malignant melanoma cells (A375) and 6×10 6 A mixture of human small cell lung cancer cells (MDA-MB-453) was used until the tumor grew to an average volume of 210 mm. 3 At approximately 10:00 AM, mice were randomly divided into 8 groups (n=5 per group) based on their body weight and tumor size. The groups were: a blank control group (solvent-free); groups receiving DS7300, 9H5-J1-compound 6, 9H5-J1-compound 7, and 9H5-J1-compound 8 at 2 mg / kg each; and groups receiving 9H5-J1-compound 9, 9H5-J1-compound 10, and 9H5-J1-compound 11 at 1 mg / kg each. All mice received a single dose. Body weight and tumor volume were measured twice weekly, and the animals' survival status was observed throughout the experiment. The results are shown in Table 34 and Figure 11.

[0650] Table 34. In vivo antitumor activity and positive / negative cell ratio of the ADC drug of the present invention in A375+MDA-MB-453.

[0651] Experimental results show that the ADC of the present invention has excellent in vivo bystander effect and is significantly better than the positive control drug DS-7300.

[0652] Pharmacokinetics of SD Rats (Example 12)

[0653] After administering candidate and control drugs via a single intravenous injection into the tail vein of rats, the basic pharmacokinetic characteristics of the ADC drugs at different doses were observed in rats.

[0654] The grouping, dosage, and administration volume are shown in Table 35 below:

[0655] Table 35

[0656] (2) Dosage frequency and method: Single tail vein administration, with a dosage volume of 5 mL / kg;

[0657] (3) Blood collection points: 15min, 3.5h, 24h, 48h, 72h (Day 3), 168h (Day 7), 240h (Day 10), 336h (Day 14), 504h (Day 21);

[0658] (4) Blood collection method: Whole blood was collected via the jugular vein, and 300 μL of blood was drawn;

[0659] (5) Blood sample processing and detection: After the serum was collected, it was left to stand. After 1 hour, the serum was clearly separated into layers. The supernatant was collected by centrifugation at 4000 rpm for 10 min. The obtained serum samples were divided into two portions and frozen in a -80℃ freezer. After the experiment, the free small molecules, ADC and total antibody were detected. The experimental results are shown in Figures 12 and 13.

[0660] Experimental results show that the ADC in this invention has a lower release of cytotoxic small molecule payloads compared to the positive control drug DS7300, which is expected to reduce off-target toxicity.

[0661] Experimental Example 13: Toxicological Experiment in BALB / c Mice

[0662] (I) Experimental Grouping

[0663] (II) Experimental Methods

[0664] Route of administration: Intravenous injection, once a week for a total of 4 weeks, Day 0, Day 7, Day 14, and Day 21.

[0665] Dosage frequency: Weekly administration for 4 consecutive weeks;

[0666] Experiment duration: 4 weeks

[0667] Dosage volume: 15 mL / kg

[0668] (III) Detection Indicators:

[0669] Clinical pathology: blood cell count, blood biochemistry, coagulation function and urine analysis, once on the day of autopsy, and pharmacological and toxicological tests were commissioned;

[0670] Observe the cage twice a day (including observation of death and near-death experiences), and draw a survival curve based on the survival status;

[0671] Detailed clinical observation: once a week. If abnormalities are observed at the cage edge, the observation frequency can be increased to once a day until the symptoms disappear (twice a week in this experiment).

[0672] Weight: 1-2 times per week, once before administration. Twice per week.

[0673] The experimental results are shown in Figures 14 and 15.

[0674] Experimental results show that the ADC of the present invention has good safety in mice. Example 14: Animal efficacy of the ADC of the present invention in the H526 CDX model.

[0675] Select 6-8 week old female BALB / c nude mice and subcutaneously inject them with 4×10⁴ mg of a solution dissolved in 200 μL (containing 100 μL of low growth factor matrix gel) into the right posterior region of the mouse. 6 Human small cell lung cancer cells (NCI-H526) were used until the tumor grew to an average volume of 125 mm. 3 At approximately 10:00 AM, mice were randomly divided into 6 groups (n=6 per group) based on their body weight and tumor size. These groups consisted of a solvent blank control group, a low-dose YL-201 group, a high-dose YL-201 group, and groups receiving 9H5-J1-DXD, 9H5-J1-ADC-7, and DS7300. Except for the low-dose YL-201 group (1 mg / kg), all other groups received 2 mg / kg of YL-201, with each group receiving a single dose. Body weight and tumor volume were measured twice weekly, and the animals' survival status was observed throughout the experiment. The results are shown in Table 36 and Figure 16.

[0676] Table 36. In vivo antitumor activity of ADC drugs in the H526 CDX model.

[0677] Conclusion: Experimental results show that the ADC of the present invention has excellent tumor inhibition effect in the H526 CDX model, and is significantly better than the control drugs YL-201 and DS7300.

[0678] The present invention has been described through the above embodiments. However, it should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the invention to the scope of the described embodiments. Furthermore, those skilled in the art will understand that the present invention is not limited to the above embodiments, and many variations and modifications can be made based on the teachings of the present invention, all of which fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antibody or its antigen-binding fragment, comprising: The three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained within the peptide with the amino acid sequence SEQ ID NO: 94, 97, 100, 101, 102, 103, 108, or 117; and / or The three light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide with amino acid sequences of SEQ ID NO: 95, 96, 98, 99, 109, 110, 111, 118 or 119; Preferably, the antibody or its antigen-binding fragment specifically binds to B7H3.

2. An antibody or its antigen-binding fragment, comprising: 1) HCDR3, wherein HCDR3 comprises any amino acid sequence from the group consisting of the following amino acid sequences: SEQ ID NO: 24, 30, 36, 42, 48, 54, 60, 66, 72, and 78; and 2) LCDR3, wherein the LCDR3 comprises any amino acid sequence from the group consisting of the following amino acid sequences or any amino acid sequence from the group consisting of the following amino acid sequences: SEQ ID NO: 27, 33, 39, 45, 51, 57, 63, 69, 75, and 81; Preferably, the antibody or its antigen-binding fragment specifically binds to B7H3.

3. The antibody or antigen-binding fragment thereof as described in claim 2, further comprising: 1) HCDR1, wherein HCDR1 comprises any amino acid sequence from the group consisting of the following amino acid sequences or any amino acid sequence from the group consisting of the following amino acid sequences: SEQ ID NO: 22, 28, 34, 40, 46, 52, 58, 64, 70, and 76; and 2) LCDR1, wherein LCDR1 comprises any amino acid sequence from the group consisting of the following amino acid sequences or any amino acid sequence from the group consisting of the following amino acid sequences: SEQ ID NO: 25, 31, 37, 43, 49, 55, 61, 67, 73, and 79; Preferably, the antibody or its antigen-binding fragment further comprises: 3) HCDR2, wherein HCDR2 comprises any amino acid sequence from the group consisting of the following amino acid sequences or any amino acid sequence from the group consisting of the following amino acid sequences: SEQ ID NO: 23, 29, 35, 41, 47, 53, 59, 65, 71, and 77; and 4) LCDR2, wherein the LCDR2 comprises any amino acid sequence from the group consisting of the following amino acid sequences or any amino acid sequence from the group consisting of the following amino acid sequences: SEQ ID NO: 26, 32, 38, 44, 50, 56, 62, 68, 74, and 80.

4. The antibody or antigen-binding fragment thereof as described in any one of claims 1-3, comprising: 1) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:2, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the peptide segment shown in SEQ ID NO:3; or 2) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:4, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the peptide segment shown in SEQ ID NO:5; or 3) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:6, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the peptide segment shown in SEQ ID NO:7; or 4) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:8, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the peptide segment shown in SEQ ID NO:9; or 5) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:10, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the peptide segment shown in SEQ ID NO:11; or 6) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:12, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the peptide segment shown in SEQ ID NO:13; or 7) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:14, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the peptide segment shown in SEQ ID NO:15; or 8) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:16, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the peptide segment shown in SEQ ID NO:17; or 9) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:18, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the peptide segment shown in SEQ ID NO:19; or 10) The amino acid sequence is the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the peptide segment shown in SEQ ID NO:20, and The amino acid sequence is the three light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:

21.

5. The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, comprising: 1) Contains or consists of the sequence shown in SEQ ID NO:22, HCDR1 HCDR2 contains the sequence shown in SEQ ID NO:23 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:24 or is composed of it. LCDR1, which contains the sequence shown in SEQ ID NO:25 or is composed of it, LCDR2, which contains the sequence shown in SEQ ID NO:26 or is composed of therein, and LCDR3 comprising the sequence shown in SEQ ID NO:27 or thereof; or 2) Contains or consists of the sequence shown in SEQ ID NO:28, HCDR1 HCDR2 contains the sequence shown in SEQ ID NO:29 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:30 or is composed of it. LCDR1 contains the sequence shown in SEQ ID NO:31 or is composed of it. LCDR2, which contains the sequence shown in SEQ ID NO:32 or is composed of therein, and LCDR3 comprising or consisting of the sequence shown in SEQ ID NO:33; or 3) Contains or consists of the sequence shown in SEQ ID NO:34, HCDR1 HCDR2 contains the sequence shown in SEQ ID NO:35 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:36 or is composed of it. LCDR1 contains the sequence shown in SEQ ID NO:37 or is composed of it. LCDR2, which contains the sequence shown in SEQ ID NO:38 or is composed of therein, and LCDR3 comprising the sequence shown in SEQ ID NO:39 or thereof; or 4) Contains or consists of the sequence shown in SEQ ID NO:40, HCDR1 HCDR2 contains the sequence shown in SEQ ID NO:41 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:42 or is composed of it. LCDR1, which contains the sequence shown in SEQ ID NO:43 or is composed of it, LCDR2, which contains the sequence shown in SEQ ID NO:44 or is composed of therein, and LCDR3 comprising the sequence shown in SEQ ID NO:45 or thereof; or 5) Contains or consists of the sequence shown in SEQ ID NO:46, HCDR1 HCDR2 contains the sequence shown in SEQ ID NO:47 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:48 or is composed of it. LCDR1, which contains the sequence shown in SEQ ID NO:49 or is composed of it, LCDR2, which contains the sequence shown in SEQ ID NO:50 or is composed of therein, and LCDR3 comprising the sequence shown in SEQ ID NO:51 or thereof; or 6) Contains or consists of the sequence shown in SEQ ID NO:52, HCDR1 HCDR2 contains the sequence shown in SEQ ID NO:53 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:54 or is composed of it. LCDR1, which contains the sequence shown in SEQ ID NO:55 or is composed of it, LCDR2, which contains the sequence shown in SEQ ID NO:56 or is composed of therein, and LCDR3 comprising or consisting of the sequence shown in SEQ ID NO:57; or 7) HCDR1 containing or consisting of the sequence shown in SEQ ID NO:58, HCDR2 contains the sequence shown in SEQ ID NO:59 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:60 or is composed of it. LCDR1 contains the sequence shown in SEQ ID NO:61 or is composed of it. LCDR2, which contains the sequence shown in SEQ ID NO:62 or is composed of therein, and LCDR3 comprising the sequence shown in SEQ ID NO:63 or thereof; or 8) HCDR1 containing or consisting of the sequence shown in SEQ ID NO:64, HCDR2 contains the sequence shown in SEQ ID NO:65 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:66 or is composed of it. LCDR1 contains the sequence shown in SEQ ID NO:67 or is composed of it. LCDR2, which contains the sequence shown in SEQ ID NO:68 or is composed of therein, and LCDR3 comprising the sequence shown in SEQ ID NO:69 or thereof; or 9) HCDR1 containing or consisting of the sequence shown in SEQ ID NO:70, HCDR2 contains the sequence shown in SEQ ID NO:71 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:72 or is composed of it. LCDR1, which contains the sequence shown in SEQ ID NO:73 or is composed of it, LCDR2, which contains the sequence shown in SEQ ID NO:74 or is composed of therein, and LCDR3 comprising the sequence shown in SEQ ID NO:75 or thereof; or 10) HCDR1 containing or consisting of the sequence shown in SEQ ID NO:76, HCDR2 contains the sequence shown in SEQ ID NO:77 or is composed of it. HCDR3 contains the sequence shown in SEQ ID NO:78 or is composed of it. LCDR1 contains the sequence shown in SEQ ID NO:79 or is composed of it. LCDR2, which contains the sequence shown in SEQ ID NO:80 or is composed of therein, and LCDR3 contains the sequence shown in SEQ ID NO:81 or is composed of it.

6. An antibody or its antigen-binding fragment, comprising: The heavy chain variable region has an amino acid sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 84, 85, 89, 91, 92, 93, 104, and 112; preferably, the heavy chain variable region has a CDR region amino acid sequence that is identical to the amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 84, 85, 89, 91, 92, 93, 104, or 112, and the amino acid sequence of the frame region of the heavy chain variable region is identical to the amino acid sequence .... The frame regions of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 84, 85, 89, 91, 92, 93, 104, or 112 have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity; preferably, the heavy chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 84, 85, 89, 91, 92, 93, 104, and 112; and / or The light chain variable region has an amino acid sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 86, 87, 88, 90, 105, 106, 107, 113, 114, 115, and 116; preferably, the amino acid sequence of the light chain variable region has the same CDR region as the amino acid sequences SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 86, 87, 88, 90, 105, 106, 107, 113, 114, 115, or 116, and the frame region of the light chain variable region is identical to the sequence .... The frame regions of SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 86, 87, 88, 90, 105, 106, 107, 113, 114, 115, or 116 have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity; preferably, the light chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 86, 87, 88, 90, 105, 106, 107, 113, 114, 115, and 116; Preferably, the antibody or its antigen-binding fragment specifically binds to B7H3.

7. The antibody or its antigen-binding fragment as described in claim 6, It comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence pairs of the heavy chain variable region and the light chain variable region are selected from any one of the following groups of amino acid sequence pairs: SEQ ID NO:2 and SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, SEQ ID NO:84 and SEQ ID NO:86, SEQ ID NO:84 and SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89, SEQ ID NO:89 and SEQ ID NO:90, SEQ ID NO:85 and SEQ ID NO:86, SEQ ID NO:85 and SEQ ID NO:87, SEQ ID NO:91 and SEQ ID NO:88, SEQ ID NO:91 and SEQ ID NO:90, SEQ SEQ ID NO:85 and SEQ ID NO:90, SEQ ID NO:92 and SEQ ID NO:86, SEQ ID NO:93 and SEQ ID NO:86, SEQ ID NO:93 and SEQ ID NO:87, SEQ ID NO:104 and SEQ ID NO:105, SEQ ID NO:104 and SEQ ID NO:106, SEQ ID NO:104 and SEQ ID NO:107, SEQ ID NO:112 and SEQ ID NO:115, and SEQ ID NO:112 and SEQ ID NO:

116.

8. The antibody or antigen-binding fragment thereof as described in any one of claims 1-7, The antibody or its antigen-binding fragment is a chimeric antibody, a humanized antibody, or a fully human antibody; preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region and / or a light chain constant region, more preferably comprising a mouse-derived or humanized heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region is an IgG heavy chain constant region, and / or the light chain constant region is a κ or λ light chain constant region; preferably, the heavy chain constant region is an IgG1 heavy chain constant region, and / or the light chain constant region is a κ light chain constant region; preferably, the amino acid sequence of the humanized heavy chain constant region is as shown in SEQ ID NO:82, and / or the amino acid sequence of the humanized light chain constant region is as shown in SEQ ID NO:

83.

9. The antibody or antigen-binding fragment thereof as described in any one of claims 1-8, The antibody or its antigen-binding fragment is selected from Fab, Fab', Fab'-SH, F v ,scF v And F(ab')2.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, comprising a heavy chain and / or a light chain, wherein, The amino acid sequence of the heavy chain has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 94, 97, 100, 101, 102, 103, 108, or 117; preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 94, SEQ ID NO: 94, 97, 100, 101, 102, 103, 108, or 117; and / or The amino acid sequence of the light chain has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 95, 96, 98, 99, 109, 110, 111, 118, or 119; preferably, the amino acid sequence of the light chain is as shown in SEQ ID NO: 95, 96, 98, 99, 109, 110, 111, 118, or 119.

11. The antibody or antigen-binding fragment thereof as described in any one of claims 1-10, The amino acid sequences of its heavy and light chains are selected from any group consisting of the following amino acid sequence pairs: SEQ ID NO:94 and SEQ ID NO:95, SEQ ID NO:94 and SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98, SEQ ID NO:97 and SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:95, SEQ ID NO:100 and SEQ ID NO:96, SEQ ID NO:101 and SEQ ID NO:98, SEQ ID NO:101 and SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:99, SEQ ID NO:102 and SEQ ID NO:95, SEQ ID NO:103 and SEQ ID NO:95, SEQ ID NO:103 and SEQ ID NO:96, SEQ ID NO:108 and SEQ ID NO:109, SEQ ID NO:108 and SEQ ID NO:110, SEQ ID NO:108 ...8 and SEQ ID NO:96, SEQ ID NO:108 and SEQ ID NO:109, SEQ ID NO:108 and SEQ ID NO:110, SEQ ID NO:108 and SEQ ID NO:109, SEQ ID NO:108 and SEQ ID NO:109, SEQ ID NO: SEQ ID NO:111, SEQ ID NO:117 and SEQ ID NO:118, and SEQ ID NO:117 and SEQ ID NO:

119.

12. The antibody or antigen-binding fragment thereof as described in any one of claims 1-11, The HCDR and LCDR are defined according to the Kabat definition scheme, the Chothia definition scheme, the Abm definition scheme, the IMGT definition scheme, and / or the Contact definition scheme; preferably, the HCDR and LCDR are defined according to the Kabat or Chothia definition scheme.

13. The antibody or antigen-binding fragment thereof as described in any one of claims 1-12, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

14. A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-13.

15. A nucleic acid encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1-13 or a bispecific or multispecific antibody as described in claim 14.

16. An expression vector comprising the nucleic acid as described in claim 15.

17. A host cell comprising the expression vector as described in claim 16.

18. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell as described in claim 17 and recovering the antibody or antigen-binding fragment thereof expressed therefrom from the culture.

19. The method of claim 18, wherein the host cell is a prokaryotic cell or a eukaryotic cell; Preferably, the host cell is an Escherichia coli cell, yeast cell, insect cell, plant cell, or mammalian cell; preferably, the host cell is a Chinese hamster ovary cell (CHO), a CHO cell variant, a 293 cell, or an NSO cell.

20. A ligand-drug conjugate as shown in Formula 1 or a pharmaceutically acceptable salt thereof, in, Ab is the antibody or antigen-binding fragment of any one of claims 1-13 or the bispecific or multispecific antibody of claim 14; n is selected from any integer or decimal from 1 to 20; preferably, n is selected from any integer or decimal from 1 to 12. L represents the chemical bonding structure; D represents the effector molecule.

21. The ligand-drug conjugate of claim 20 or a pharmaceutically acceptable salt thereof, wherein... D is selected from the cytotoxic drug fraction, the drug fraction for treating autoimmune diseases, and the anti-inflammatory drug fraction; preferably, D is the cytotoxic drug fraction; preferably, D is selected from the topoisomerase I (TOP1) inhibitor fraction, the tubulin polymerization inhibitor fraction, the topoisomerase II (TOP2) inhibitor fraction, the dihydrofolate reductase inhibitor fraction, the DNA alkylating agent fraction, the thymidine synthase inhibitor fraction, the purine nucleoside synthase inhibitor fraction, the nucleotide reductase inhibitor fraction, the DNA topoisomerase inhibitor fraction, the RNA polymerase II inhibitor fraction, and other compound fractions capable of inhibiting cell proliferation; preferably, D is selected from the DNA topoisomerase inhibitor fraction; preferably, D is selected from camptothecin or its derivatives; preferably, D is selected from... in, q1 is selected from 0 and 1; A1 is selected from O and S; R 1a R 1a1 R 1a2 R 2a R 3a and R 5a Each occurrence is independently selected from H, deuterium, F, Cl, Br, I, C1-C6 alkyl, C1-C6 deuterated alkyl, C2-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkoxy, C1-C6 alkylamino, hydroxyl, nitro, cyano, amino, and carboxyl; preferably, R 1a R 1a1 R 1a2 R 2a R 3a and R 5a Each occurrence is independently selected from H, cyano, F, Cl, Br, I, carboxyl, hydroxyl, amino, C1-C3 alkyl, and C1-C3 alkoxy groups; or R 1a1 and R 1a2 Together with the carbon atoms attached thereto, they form 4-7 member heterocycles or 4-7 member carbon rings containing 1, 2, or 3 heteroatoms each independently selected from N, O, and S, wherein the heterocycles or carbon rings may optionally be separated by 0, 1, 2, 3, 4, or 5 R atoms. 1a replace; or R 2a and R 3a Together with the carbon atoms attached thereto, they form 4-7 member heterocycles containing 1, 2, or 3 heteroatoms independently selected from N, O, and S, or 5-7 member carbon rings, wherein the heterocycles or carbon rings may optionally be connected by 0, 1, 2, 3, 4, or 5 R atoms. 1a replace; R 4a and R 6a Each is independently selected from single bonds, NR 1a A combination of one or more of -CO-, C1-C6 alkylene, -O-, C3-C6 cycloalkylene, and 4-6 heterocyclic groups; wherein the alkylene, cycloalkylene, and heterocyclic groups may optionally be surrounded by 0, 1, 2, or 3 R groups. 1a Replacement; preferably, R 4a Selected from single bonds, -O-, -NH-, -NH-CO-5-membered heterocyclic group-, -NH-CO-CH2-O-, -NH-CO-O-CH2-CH2-O-, -NH-CO-CD2-O-, -CH2-CH2-CH2-O-, -CH2-CH2-CH2-CH2-O-, -NH-CO-C4-C6 cycloalkyl-O-; R 7a Each occurrence is independently selected from H and Preferably, D is selected from the following structures: The above structure may optionally be substituted with 1, 2, 3, 4, 5, 6 or more R1s, wherein R1 is selected from deuterium, F, Cl, Br, I, C1-C6 alkyl, C1-C6 deuterated alkyl, C2-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkoxy, C1-C6 alkylamino, hydroxyl, nitro, cyano, amino, and carboxyl; preferably, R1 is selected from cyano, F, Cl, Br, I, carboxyl, hydroxyl, amino, C1-C3 alkylamino, C1-C3 alkyl and C1-C3 alkoxy. Preferably, D is selected from the following structures:

22. The ligand-drug conjugate as described in claim 20 or 21, or a pharmaceutically acceptable salt thereof. Where L is the structure shown in Equation 1-1 Where *Ab indicates that the side is linked to the antibody or antigen-binding fragment of the ligand-drug conjugate, and *D indicates that the side is linked to the D fragment of the ligand-drug conjugate. T is selected from The T can be optionally replaced by 1, 2, 3 or more Ra; wherein the left side of the T structure The right side of the T structure represents the connection point with the antibody or its antigen-binding fragment. Indicates the connection point with M; M is selected from single bonds, 6-10 arylene groups, 5-13 heteroarylene groups, 3-15 cycloalkyl groups, and 3-15 heteroalkyl groups, wherein the arylene group, heteroarylene group, cycloalkyl group, and heteroalkyl group are not substituted or optionally surrounded by 1, 2, 3, or more R groups. a replace; L1 is selected from single bond, alkyne group, alkenyl group, and -NR group. a -、-O-、-C(O)-、-NR a -C(O)-, a combination of one, two, three or more of alkylene and heteroalkylene groups, wherein the ynylene, alkenylene, alkylene, and heteroalkylene groups are not substituted or optionally further substituted by one or more R groups. a replace; L2 is selected from single bond, -(CH2) n7 -(OCH2CH2) n8 -O-(CH2) n9 -Cy3-(CH2) n10 -C(O)-, -(CH2) n7 -(CH2CH2O) n8 -(CH2) n9 -Cy3-(CH2) n10 -C(O)-, -(CH2) n7 -Cy3-(CH2CH2O) n8 -(CH2) n9 -C(O)-, -(CH2) n11 -(OCH2CH2) n12 -O-(CH2) n13 -Cy3-(CH2) n14 -(OCH2CH2) n15 -C(O)-, -(CH2) n11 -(CH2CH2O) n12 -(CH2) n13 -Cy3-(CH2CH2O) n15 -(CH2) n14 -C(O)-, -(CH2) n7 -(OCH2CH2) n8 -(CH2) n10 -C(O)-、 One, two, three or more combinations; n7, n9, n10, n11, n13 and n14 are each independently selected from 0, 1, 2, 3, 4, 5 and 6 when they appear; Each time n8 and n17 appear, they are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28; Each time n12 and n15 appear, they are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, and n12 and n15 are not both 0 at the same time; Each time j appears, it is independently selected from 0, 1, 2, 3, 4, 5, and 6; Cy3 is selected from 6-10 arylene, 5-13 heteroarylene, 3-15 cycloalkylene, and 3-15 heteroalkylene, wherein the arylene, heteroarylene, cycloalkylene, and heteroalkylene are unsubstituted or optionally surrounded by 1, 2, 3, 4, 5, or 6 R's. a replace; M, L1, and L2 are not all single bonds at the same time; L4 is a peptide residue consisting of 2-8 amino acids, wherein the amino acids are not substituted or optionally further substituted by one or more Ra. L5 is selected from single bonds, -N(R) b )-(CH2) g -OC 3-6 Alkylene-C(O)-, -N(R) b )-(CH2) g -O-(C(R b )2) g -C(O)-、-N(R b )-(CH2) g -O-(CH2) g -OC(O)-、-N(R b )-(CH2) g -OC 3-6 Cycloalkylene-C(O)-, -N(R) b )-(CH2) g -O-(CH2CH2O) g -C(O)-、-N(R b )-(CH2) g -、 The C 3-6 The alkylene group is not substituted or is represented by 1, 2, 3 or more R groups. a replace; Each time n20 appears, it is independently selected from any integer between 1 and 30; preferably, each time n20 appears, it is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28; Each time g appears, it is independently selected from 1, 2, 3, 4, 5, and 6; and R a and R b Each time it appears, it is independently selected from H, deuterium, F, Cl, Br, I, C1-C6 alkyl, C1-C6 deuterated alkyl, C2-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkoxy, hydroxyl, nitro, cyano, amino, carboxyl, C3-C6 cycloalkyl, 3-6 membered heterocyclic, C1-C6 alkylamino, C1-C6 alkyl-C(O)-, C1-C6 alkyl-OC(O)-, C1-C6 alkyl-NHC(O)-, -C 1-6 Alkylene-C(O)-NH2, C 1-6 Alkyl OC(O)NH-, -C 1-6 Alkylene-NHC(O)-NH2, C1-C6 alkyl-NHC(O)-NH-, C6-C 15 aryl and 5-13-membered heteroaryl, wherein the C1-C6 alkyl, C2-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3-6-membered heterocyclic, C1-C6 alkylamino, C6-C 15 The aryl and 5-13-membered heteroaryl groups are each independently and optionally selected from F, Cl, Br, I, C1-C6 alkyl, C2-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkoxy, hydroxyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, cyano, amino, nitro, C3-C6 cycloalkyl, 3-6-membered heterocyclic, C1-C6 alkylamino, C6-C 15 It is substituted by one, two, three or more substituents in aryl and 5-13 heteroaryl groups.

23. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 20-22, wherein, T is selected from and / or M is selected from single bonds, phenylene, 5-7-membered heteroaryl, 3-7-membered cycloalkylene, and 3-7-membered heteroalkylene, wherein the phenylene, heteroaryl, cycloalkylene, and heteroalkylene are optionally surrounded by one, two, three, or four Rs. a Replace; and / or L1 is selected from single bonds, C 2-6 Ethyne group, C 2-6 imidene group, -NR a -、-O-、-C(O)-、-NR a -C(O)-、C 1-6 Alkylene and C 2-6 A combination of one, two, three, or more heteroalkyl groups, wherein the ynylene, alkenylene, alkylene, and heteroalkyl groups are optionally represented by one, two, three, four, five, or six R groups. a Replacement; preferably, L1 is selected from single bonds, C 2-3 Ethyne group, C 2-3 imidene group, -NR a -、-O-、-C(O)-、-NR a -C(O)-、C 1-3 Alkylene and C 2-3 A combination of one, two, three, or more heteroalkyl groups, wherein the ynylene, alkenylene, alkylene, and heteroalkyl groups are optionally represented by one, two, three, four, five, or six R groups. a Replace; and / or Cy3 is selected from Preferably, Cy3 is selected from and / or L4 is a peptide residue consisting of 2, 3, 4, 5, 6, or 7 amino acids, wherein the amino acids are selected from D-alanine, L-alanine, phenylalanine, glycine, valine, lysine, leucine, citrulline, serine, glutamic acid, aspartic acid, arginine, and asparagine, and the amino acids are not substituted or optionally surrounded by 1, 2, 3, 4, 5, or 6 R's. a Replace; and / or R a and R b Each time it appears, it is independently selected from H, cyano, deuterium, F, Cl, Br, I, carboxyl, hydroxyl, amino, C1-C3 alkylamino, C1-C3 alkyl and C1-C3 alkoxy.

24. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 20-23, wherein, T is selected from and / or M is selected from single bond, Preferably, M is selected from single bonds, and and / or L1 is selected from single bonds, -O-, -(CH2)5-CO-, -(CH2)5-CO-NH-, -CH(COOH)-CH2-NH-CO-, -CH(CH2NH2)-CO-NH-, -CO-NH-, -CH2-CO-, and / or L2 is selected from single bond, Wherein, n17 is independently selected from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 each time it appears; preferably, n17 is independently selected from 6, 8, 12, and 24 each time it appears; preferably, L2 is selected from and / or L4 is selected from Preferably, L4 is selected from and / or L5 is selected from: single key, Preferably, L5 is selected from and / or R a and R b Each time it appears, it is independently selected from H, cyano, deuterium, F, Cl, Br, I, carboxyl, hydroxyl, amino, C1-C3 alkylamino, C1-C3 alkyl and C1-C3 alkoxy.

25. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 20-24, wherein, L is selected from 26. The ligand-drug conjugate of any one of claims 20-25 or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate is selected from: in, Ab is the antibody or antigen-binding fragment thereof as described in any one of claims 1-14; Each time n appears, it is independently selected from an integer or decimal number from 1 to 10; preferably, each time n appears, it is independently selected from an integer or decimal number from 2 to 8; preferably, each time n appears, it is independently selected from an integer or decimal number from 2 to 6; preferably, each time n appears, it is independently selected from about 4; preferably, each time n appears, it is independently selected from 1, 2, 3, 4, 5, 6, 7, and 8. Preferably, the ligand-drug conjugate is selected from the following structures: Wherein, Ab is the antibody or antigen-binding fragment thereof as described in any one of claims 1-14; Each time n appears, it is independently selected from an integer or decimal number from 1 to 10; preferably, each time n appears, it is independently selected from an integer or decimal number from 2 to 8; preferably, each time n appears, it is independently selected from an integer or decimal number from 2 to 6; preferably, each time n appears, it is independently selected from about 4; preferably, each time n appears, it is independently selected from 1, 2, 3, 4, 5, 6, 7, and 8. Preferably, the ligand drug conjugate is selected from the antibody drug conjugates in Table 21, and is not DS7300, YL-201 and compound M30-ADC-1.

27. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-13, a bispecific or multispecific antibody as described in claim 14, or a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 20-26, and a pharmaceutically acceptable carrier.

28. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-13, the bispecific or multispecific antibody of claim 14, the ligand-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 20-26, or the pharmaceutical composition of claim 27 in the preparation of a medicament for treating and / or preventing diseases or conditions related to B7H3 expression; preferably, wherein the disease or condition is a cancer related to B7H3 expression; preferably, the cancer is melanoma, lung cancer, or prostate cancer; preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

29. A kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-13, a bispecific or multispecific antibody as claimed in claim 14, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as claimed in any one of claims 20-26, or a pharmaceutical composition as claimed in claim 27.

30. A method for treating and / or preventing a disease or condition associated with B7H3 expression, comprising administering to a subject in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-13, or a bispecific or multispecific antibody as described in claim 14, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 20-26, or a pharmaceutical composition as described in claim 27; preferably, wherein the disease or condition is a cancer associated with B7H3 expression; preferably, the cancer is melanoma, lung cancer, or prostate cancer; preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

31. The compound of any one of claims 1-13, the bispecific or multispecific antibody of claim 14, or the ligand-drug conjugate of any one of claims 20-26, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 27, for the treatment and / or prevention of diseases or conditions related to B7H3 expression; preferably, wherein the disease or condition is a cancer related to B7H3 expression; preferably, the cancer is melanoma, non-small cell lung cancer, small cell lung cancer, or prostate cancer; preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

32. Use of a compound in the preparation of a medicament for the treatment or prevention of tumors or cancer, wherein the tumor or cancer is prostate cancer, lung cancer, breast cancer, or bladder cancer, and said compound is selected from the following structures Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

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