Anti-B7H3 Antibody-Drug Conjugate and Its Use

Abstract

The present invention provides an antibody-drug conjugate that specifically binds to B7H3 and a pharmaceutical composition containing the same. Also presented herein are methods of using the antibody-drug conjugate of the present invention and its uses.

Description

Technical Field

[0001] The present invention provides an antibody-drug conjugate that specifically binds to B7H3 and a composition containing the same. Also provided are methods of using the antibody-drug conjugate of the present invention and its uses.

Background Art

[0002] B7H3, also known as CD276, B7RP-2 and B7-H3, is a member of the B7 family and has 20% - 27% amino acid sequence homology to other members of the B7 family. B7H3 transcripts are widely expressed, but their expression is limited and maintained at low levels in normal tissues and immune cells, and is overexpressed in various human malignancies including melanoma, breast cancer, prostate cancer, etc. B7-H3 has been reported to be expressed in the cell membrane and cytoplasm of cancer cells or in the nucleus, and also in tumor-associated vasculature. B7H3 is not constitutively expressed on T cells and NK cells, but is constitutively expressed at low levels on some APCs such as DCs, and the expression of B7H3 on APCs can be induced by GM-CSF, IFNγ, etc.

[0003] B7-H3 is a very promising anti-cancer target. Although the exact function of B7-H3 is unknown, its diverse immune functions including stimulation, inhibition of T cell proliferation, and inhibition of NK cell function have been demonstrated. It is thought that different receptors exist on immune cells and these receptors can competitively bind to B7H3 on tumors. In addition to its immune checkpoint function, high levels of B7H3 expression have been reported to be associated with poor cancer prognosis and to enhance cell proliferation, migration, invasion, angiogenesis, metastatic ability and anti-cancer drug resistance.

[0004] Similar to other molecules of the B7 family, B7H3 is a type I transmembrane glycoprotein, and its extracellular domain contains an IgV-IgC domain. Different from the mouse B7H3 gene that has only one IgV-IgC domain, human B7H3 has two subtypes as a result of gene duplication and different splicing. One contains one copy of the IgV-IgC domain (referred to as 2IgB7H3), and the other contains two IgV-IgC domains (referred to as 4IgB7H3). 4IgB7H3, rather than 2IgB7H3, is the major subtype expressed on immune cells and malignant cells, suggesting that 4IgB7H3 may play a unique and important role in tumor development and tumor immunology.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Since B7H3 is overexpressed in many tumor cells, therapeutic molecules against B7H3 are currently being developed to treat related indications. However, there is still room for improvement and a clinical need for the development of new anti-B7H3 antibodies or antibody-drug conjugates.

Means for Solving the Problems

[0006] The present application provides an anti-B7H3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof that may have one or more effects selected from the group consisting of: (1) having inhibitory activity against the in vitro proliferation of tumor cells; (2) having better affinity for human B7H3; (3) having plasma stability; (4) having an in vivo anti-tumor effect; (5) having a bystander effect; (6) having the ability to inhibit transport by transporters; (7) having the ability to target tumors in vivo; and (8) having good in vivo safety.

[0007] In one aspect, the present application provides an antibody-drug conjugate comprising a B7H3 target antibody or an antigen-binding fragment thereof, a linker unit, and a cytotoxic drug, wherein the B7H3 target antibody or an antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4 or 7, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0008] In some embodiments, in the antibody-drug conjugates presented herein, the antibody or an antigen-binding fragment thereof is (I) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9, or a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 11, or (II) a heavy chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8 and a light chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9, or a heavy chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10 and a light chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11, or (III) a heavy chain variable region having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions in the framework region compared to SEQ ID NO: 8 or 10, and a light chain variable region having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions in the framework region compared to SEQ ID NO: 9 or 11 and comprises.

[0009] In some embodiments, in the antibody-drug conjugates presented herein, the antibody is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies.

[0010] In some embodiments, in the antibody-drug conjugates presented herein, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0011] In some embodiments, in the antibody-drug conjugates described herein, the antibody is a monoclonal antibody.

[0012] In some embodiments, in the antibody-drug conjugates presented herein, the antibody is a full-length antibody or its antigen-binding fragment, and the antigen-binding fragment is selected from the group consisting of, for example, Fab, Fab’, F(ab’)2, Fv, ScFv, Fab’-SH, sdAb, VHH, bispecific antibodies, and linear antibodies.

[0013] In some embodiments, in the antibody-drug conjugates presented herein, the antibody comprises an immunoglobulin constant region, and the immunoglobulin constant region is a human IgG constant region, such as a human IgG1 constant region.

[0014] In some embodiments, in the antibody-drug conjugates presented herein, the antibody is an antibody in the IgG1 form, an antibody in the IgG2 form, an antibody in the IgG3 form, or an antibody in the IgG4 form.

[0015] In some embodiments, in the antibody-drug conjugates presented herein, the antibody or its antigen-binding fragment is (I) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 12 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 13; or a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 14 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 15, or (II) A heavy chain having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12 and a light chain having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13; or a heavy chain having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14 and a light chain having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15 comprising.

[0016] In some embodiments, the antibody of the invention is the anti-B7H3 antibody WBP301088.

[0017] In certain preferred embodiments of the invention, in the antibody-drug conjugates described herein, certain groups in the compounds of formulas (A-1), (A-2), (A-1a) and (A-1b) or their pharmaceutically acceptable salts are defined as follows, and groups not described are as described in any one of the solutions of the present application (abbreviated as "in some embodiments").

[0018] In some embodiments, in the antibody-drug conjugates presented herein, the cytotoxic drug has the following structure: Formula (A-1) [Chemical formula] (wherein M is -L 2 -L 1 -C(O)-, L 2 is -O- or -S-, L 1 is -(C(R 1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkyl, or 3-6 membered saturated heterocyclyl, and C3-C6 saturated cycloalkyl and 3-6 membered saturated heterocyclyl are each independently optionally substituted with one or more R 2a s, m is selected from the group consisting of 1, 2, 3, and 4, and the 3- to 6-membered saturated heterocyclyl contains 1 to 3 heteroatoms selected from the group consisting of N, O, and S. Each R 1a is independently selected from the group consisting of hydrogen, halogen, hydroxy, amino, and C1-C6 alkyl, and the C1-C6 alkyl may be substituted with one or more R. R 1b and R 2a are each independently selected from the group consisting of halogen, hydroxy, amino, and C1-C6 alkyl, and the C1-C6 alkyl may be substituted with one or more R. Each R is independently hydrogen or halogen) selected from the group consisting of the structure shown in, and its tautomers, enantiomers, diastereoisomers and mixtures of isomers, and pharmaceutically acceptable salts and solvates thereof.

[0019] In some embodiments, in the antibody-drug conjugate presented herein, L 1 is -(C(R 1a )(R 1b )) m -CH2-, R 1a is selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl, and R 1b is selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl. In some embodiments, in the antibody-drug conjugate presented herein, L 1 is -(C(R 1a )(R 1b )) m -CH2-, R 1a is hydrogen or -CH3, and R 1b is selected from the group consisting of hydrogen and -CH3. For example, R 1a is -CH3, and R 1b is selected from the group consisting of hydrogen and -CH3.

[0020] In some embodiments, in the antibody-drug conjugate presented herein, L 1is -(C(R 1a )(R 1b )) m -CH2-, and m is 1 or 2.

[0021] In some embodiments, in the antibody-drug conjugates presented herein, L 1 is

Chemical formula

[0022] In some embodiments, in the antibody-drug conjugates presented herein, L 1 is C3-C6 saturated cycloalkyl or 3- to 6-membered saturated heterocyclyl, and C3-C6 saturated cycloalkyl and 3- to 6-membered saturated heterocyclyl are each independently optionally substituted with one or more R 2a , and each R 2a is independently selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl.

[0023] In some embodiments, in the antibody-drug conjugates presented herein, L 1 is C3-C6 saturated cycloalkyl optionally substituted with one or more R 2a , and each R 2a is independently selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl. In some embodiments, in the antibody-drug conjugates presented herein, L 1 is C3-C6 saturated cycloalkyl.

[0024] In some embodiments, in the antibody-drug conjugates presented herein, L 1 is optionally substituted with one, two, or three R 2a and

Chemical formula

[0025] In some embodiments, in the antibody-drug conjugates presented herein, L 1 is

Chemical formula

[0026] In some embodiments, in the antibody-drug conjugates presented herein, -M- is

Chemical formula

[0027] In some embodiments, in the anti-B7H3 antibody-drug conjugates, their isomers, their pharmaceutically acceptable salts, or mixtures thereof presented herein, -M- is

Chemical formula

[0028] In some embodiments, in the anti-B7H3 antibody-drug conjugates presented herein, the cytotoxic drug

Chemical formula

Chemical formula

[0029] In some embodiments, in the antibody-drug conjugates presented herein, the linker unit L is -L a -L b -L c -, and -L a - is

Chemical formula

[0030] In some embodiments, in the antibody-drug conjugates presented herein, linker unit L is L linked terminally to Ab a and L linked terminally to linker unit M c .

[0031] In some embodiments, in the antibody-drug conjugates presented herein, linker unit L is [Chem.] .

[0032] In some embodiments, in the antibody-drug conjugates presented herein, linker unit L is [Chem.] .

[0033] In some embodiments, the antibody-drug conjugates presented herein have the formula (A-2): [Chem.] (wherein p represents the average number of linkages, and p is selected from the group consisting of integers or decimals from 1 to 10, Ab, M, and L are as described in any one of the solutions of the present application) and have the structure shown.

[0034] In some embodiments, the antibody-drug conjugates presented herein have the formula (A-2):

Chemical formula

Chemical formula

[0035] In some embodiments, in the antibody-drug conjugates presented herein, p is an integer or a decimal from 2 to 8. For example, p is an integer or a decimal from 4 to 8. For example, p is an integer or a decimal from 4 to 6 or from 6 to 8.

[0036] In some embodiments, the antibody-drug conjugates presented herein have the formula (A-2a) or formula (A-2b):

Chemical formula

[0037] In some embodiments, the antibody-drug conjugates presented herein have the following structural formula:

Chemical formula

[0038] In some embodiments, in the antibody-drug conjugates presented herein or pharmaceutically acceptable salts thereof, the antibody-drug conjugate is

Chemical formula

[0039] In some embodiments, in the antibody-drug conjugates presented herein or pharmaceutically acceptable salts thereof, the antibody-drug conjugate is

Chemical formula

[0040] In yet another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0041] In yet another aspect, the present invention provides the use of the antibody-drug conjugate described herein, a pharmaceutically acceptable salt thereof or the pharmaceutical composition described herein in the preparation of a medicament for treating and / or preventing a B7H3-mediated disease or condition, preferably wherein the disease or condition is cancer. The disease or condition is preferably a disease or condition in which the expression of B7H3 is positive (positively express).

[0042] In yet another aspect, the present invention provides a method for treating and / or preventing a B7H3-mediated disease or condition, comprising administering to a subject in need thereof the antibody-drug conjugate described herein or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described herein, preferably wherein the disease or condition is cancer. The disease or condition is preferably a disease or condition in which the expression of B7H3 is positive.

[0043] In yet another aspect, the present invention provides an antibody-drug conjugate, an isomer thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof as described herein for treating and / or preventing a B7H3-mediated disease or condition, preferably, a disease or condition being cancer, an antibody-drug conjugate, an isomer thereof, or a pharmaceutically acceptable salt or pharmaceutical composition thereof as described herein. The disease or condition is preferably a disease or condition in which the expression of B7H3 is positive.

[0044] In some embodiments, the cancer of the present invention is selected from the group consisting of breast cancer, neurological tumors, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, urothelial cancer, lymphoma (e.g., non-Hodgkin lymphoma), chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma.

[0045] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0046] In yet another aspect, the present invention provides a kit comprising an antibody-drug conjugate as described herein or a pharmaceutical composition as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047]

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Figure 19A

Figure 19B

Figure 20A

Figure 20B

Mode for Carrying Out the Invention

[0048] Embodiments of the present invention will be described below with reference to specific examples. Also, other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of this specification.

[0049] Definition of Terms Unless otherwise specified, in the embodiments of the present invention, conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology are used, and all of them fall within the scope of the techniques in the relevant technical field.

[0050] To facilitate the understanding of the present invention, some scientific and technical terms are specifically defined as follows. Unless otherwise defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Regarding the definitions and terminology in the technical field, those skilled in the art can refer particularly to Current Protocols in Molecular Biology (Ausubel F. et al., John Wiley & Sons, New York, 2000). The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the technical field to indicate one of the 20 commonly used L-amino acids. The singular forms used herein (including the claims) also include their plural forms unless the context clearly indicates otherwise.

[0051] The term "about" generally means a variation of plus or minus 0.5% to 10% from the explicitly stated value, for example, a variation of plus or minus 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% from the explicitly stated value.

[0052] The term "antibody" or "Ab" is used herein in the broadest sense and encompasses various antibody structures including polyclonal antibodies, monospecific antibodies, and multispecific antibodies (e.g., bispecific antibodies). A native intact antibody is generally a Y-shaped tetrameric protein having two heavy (H) polypeptide chains and two light (L) polypeptide chains linked by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified as κ light chains and λ light chains. The heavy chains can be classified as μ, δ, γ, α, and ε, whereby the isotypes of the antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 amino acids or more, and the heavy chain further includes a "D" region of about 3 amino acids or more. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH region and the VL region can be further divided into hypervariable regions (referred to as complementary determining regions (CDRs)) and relatively conserved regions (referred to as framework regions (FRs)) that exist therebetween. Each VH and VL consists of three CDRs and four FRs in the following order: from the N-terminus to the C-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form an antigen-binding site, respectively. The ranges of the framework regions and CDRs can be precisely identified by using methods and systems known in the art, e.g., the Kabat definition, the Chothia definition, the AbM definition, the EU definition, and the contact definition, all of which are well-known in the art and are the definitions on Dr. Martin's website.See, for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Martin A. "Antibody bioinformatics website of Dr. Andrew Martin's lab at UCL," last updated on 31 July 2018; Chothia et al., (1989) Nature 342: 877; Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917, Al-lazikani et al (1997) J. Molec. Biol. 273: 927-948; Edelman et al., Proc Natl Acad Sci U S A. 1969 May;63 (1): 78-85; and Almagro, J. Mol. Recognit. 17: 132-143 (2004).hgmp.mrc.ac.uk and bioinf.org.uk / abs. Correspondence or alignments between numberings according to different definitions can for example be found at http: / / www.imgt.org / (see also Giudicelli V et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res. (1997) 25: 206-11; Lefranc MP et al., Unique database numbering system for immunogenetic analysis, Immunol Today (1997) 18: 509; and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Dev Comp Immunol. (2003) 27: 55-77). The antibody may be of different antibody isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies. The term "B7H3" is also known as the CD276 antigen, belongs to the B7 family, and refers to a type I transmembrane protein containing an extracellular domain consisting of a single IgV-IgC domain. Proteins of the B7 family contain extracellular IgV-like and IgC-like domains and have a short cytoplasmic tail. B7H3 is an immune checkpoint molecule that is abnormally overexpressed in various cancers. The amino acid sequence of the B7H3 protein includes the full-length B7H3 protein (e.g., human 4IgB7H3 protein or human 2IgB7H3 protein), or the extracellular domain of B7H3 (B7H3 ECD) or a fragment containing B7H3 ECD, or a B7H3-ECD fusion protein.Exemplary sequences of the B7H3 protein are shown below Uniprot ID: Q5ZPR3 (human 4IgB7H3) and Genebank accession numbers NP_001019907 (human), NP_001316557 (human), NP_001316558 (human), NP_079516 (human) and NP_598744 (mouse). Cynomolgus B7H3 has amino acid sequence identities of approximately 97% and 88% to human B7H3 and mouse B7H3, respectively.

[0053] The term "antibody that binds to B7H3" or "anti-B7H3 antibody" encompasses antibodies and antigen-binding fragments that specifically recognize the B7H3 protein, as well as antibodies and antigen-binding fragments that specifically bind to the B7H3 protein. As used herein, "anti-B7H3 antibody" encompasses monospecific monovalent antibodies, as well as bispecific antibodies that include a first antigen-binding site that binds to B7H3 and a second antigen-binding site that binds to a second antigen.

[0054] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., antibodies that constitute a population that is identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and target a single antigen epitope. In contrast, conventional (polyclonal) antibody preparations generally contain multiple antibodies that target (or are specific for) different epitopes. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as indicating that the antibody was produced by any particular method.

[0055] The term "full-length antibody" refers to a naturally occurring immunoglobulin molecule that contains four peptide chains, with two heavy (H) chains (approximately 50-70 kDa in full length) and two light (L) chains (approximately 25 kDa in full length) linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region (abbreviated as CH herein). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region (abbreviated as CL herein). The light chain constant region consists of one domain, CL. The VH region and the VL region can be further divided into more conserved regions called hypervariable complementarity determining regions (CDRs) and framework regions (FRs) separated by CDRs. Each VH or VL region consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including binding to the first component of the classical complement system (Clq) of various immune system cells (e.g., effector cells).

[0056] The term "CDR" refers to the complementarity-determining regions within the antibody variable sequences. There are three CDRs in each of the heavy-chain variable region and the light-chain variable region, which are designated as HCDR1, HCDR2, and HCDR3 for the heavy-chain variable region, or LCDR1, LCDR2, and LCDR3 for the light-chain variable region. The precise amino acid sequence boundaries of the CDRs of the variable regions of the antibodies of the present invention can be determined using any number of well-known schemes, including the Chothia (Chothia et al., (1989) Nature 342: 877-883; Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins, Journal of Molecular Biology, 273, 927-948 (1997)) based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), International ImMunoGenetics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a number of crystal structures. The CDR boundaries of the antibodies disclosed herein can be determined by those skilled in the art according to any scheme (e.g., different assignment methods or combinations) in the art.

[0057] The term "antigen-binding fragment" or "antibody fragment" of an antibody (the "parent antibody") encompasses fragments or derivatives of an antibody that retain at least a portion of the binding specificity of the parent antibody and generally contain at least one fragment (e.g., one or more CDRs) of the antigen-binding region or variable region of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab, Fab’, F(ab’)2 and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules, such as scFv; and nanobodies and multispecific antibodies formed by fragments of an antibody. An antigen-binding fragment or derivative generally retains at least 10% of the antigen-binding activity of the parent antibody when the binding activity to an antigen is expressed on a molar concentration basis. Preferably, the antigen-binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that antigen-antibody binding fragments may include conservative amino acid substitutions (referred to as "conservative variants" or "functionally conserved variants" of the antibody) or non-conservative amino acid substitutions that do not significantly alter their biological activity.

[0058] A "chimeric antibody" is an antibody having the variable domain of a first antibody and the constant domain of a second antibody, where the first antibody and the second antibody are derived from different species. Generally, the variable domain is obtained from an antibody of an experimental animal such as a rodent (the "parent antibody"), and the constant domain sequence is obtained from a human antibody, and thus the resulting chimeric antibody is less likely to induce a harmful immune response in a human subject compared to the parent rodent antibody.

[0059] A "humanized antibody" refers to an antibody form that contains both sequences derived from a human antibody and sequences derived from a non-human (e.g., mouse and rat, etc.) antibody. Generally, a humanized antibody contains substantially all of at least one, and generally two, variable domains, and all or substantially all of its hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework regions (FRs) are of human immunoglobulin sequences. A humanized antibody may include at least a portion of the human immunoglobulin constant region (Fc).

[0060] In the present application, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine, and may be, for example, fluorine or chlorine.

[0061] In the present application, the term "alkyl" generally refers to a residue resulting from the removal of a hydrogen atom from an alkane. The alkyl may or may not be substituted or replaced. The term "alkyl" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having a residue resulting from the removal of a hydrogen atom from the same carbon atom or two different carbon atoms of the parent alkane, and may be a straight-chain or branched group containing 1 to 20 carbon atoms, for example, 1 to 12 carbon atoms, for example, an alkyl chain containing 1 to 6 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. The alkyl may or may not be substituted or replaced. For example, when the alkyl is substituted, the substitution by the substituent may be made at any available linking site, and the substituent may be independently selected arbitrarily from the group consisting of one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, and for example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 may be an aliphatic group. For example, when substituted with cycloalkyl, the alkyl is cycloalkylalkyl.

[0062] In the present application, the term "alkylene" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues resulting from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, and may be a straight-chain or branched group containing 1 to 20 carbon atoms. For example, the term "methylene" may refer to a residue resulting from the removal of two hydrogen atoms from a one-carbon atom group. Methylene may or may not be substituted, and may or may not be replaced. For example, alkylene contains 1 to 12 carbon atoms, for example, an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylidene (-CH(CH3)-), 1,2-ethylidene (-CH2CH2-), 1,1-propylidene (-CH(CH2CH3)-), 1,2-propylidene (-CH2CH(CH3)-), 1,3-propylidene (-CH2CH2CH2-), 1,4-butylidene (-CH2CH2CH2CH2-), 1,5-pentylidene (-CH2CH2CH2CH2CH2-), and the like. Alkylene may or may not be substituted, and may or may not be replaced. For example, when alkylene is substituted, the substitution by the substituent may be made at any available linkage point, and the substituent is preferably independently selected from the group consisting of one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo. For example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6It may be an aliphatic group. Methylene or alkylene may or may not be substituted.

[0063] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where alkyl or cycloalkyl is as defined herein. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. Alkoxy may or may not be substituted. When alkoxy is substituted, the substituents are preferably independently one or more of the groups selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0064] In the present application, the term "alkenyl" generally refers to a linear or branched hydrocarbon group containing one or more double bonds. Exemplary examples of alkenyl include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, hexenyl, and the like. Exemplary examples of C2-6 alkenyl containing more than one double bond include butadienyl, pentadienyl, hexadienyl, and hexatrienyl, and their branched forms. The position of the unsaturated bond (double bond) may be at any position within the carbon chain. The alkenyl may or may not be substituted. For example, when the alkenyl is substituted, the substitution by the substituent may be made at any available linkage point, and the substituent is preferably independently selected from the group consisting of one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo. For example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 may be an aliphatic group.

[0065] In the present application, the term "alkenylene" generally refers to a group having a residue resulting from the removal of two hydrogen atoms from a carbon atom from an alkene. For example, alkenylene may be acrol, vinylene, butenylene, pentenylene, hexenylene, etc. Alkenylene may or may not be substituted. For example, when alkenylene is substituted, the substitution by a substituent may be made at any available linkage point, and the substituent is preferably independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, one or more of which, for example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 may be an aliphatic group.

[0066] In this application, the term "alkynyl" generally refers to a straight-chain or branched hydrocarbon group containing one or more triple bonds. The alkynyl may be an unsaturated straight-chain or branched alkynyl, such as ethynyl, 1-propynyl, propargyl, or butynyl. The alkynyl may or may not be substituted. For example, when the alkynyl is substituted, the substitution by the substituent may be made at any available linking point, and the substituent is preferably independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, and may consist of one or more of them. For example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 may be an aliphatic group.

[0067] In the present application, the term "alkynylene" generally refers to a group having a residue resulting from the removal of two hydrogen atoms from a carbon atom from an alkyne. For example, alkynylene may be ethynylene, propynylene, propargylene, butynylene, etc. Alkynylene may or may not be substituted. For example, when alkynylene is substituted, the substitution by a substituent may be made at any available linking point, and the substituent is preferably independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, one or more of which may be selected. For example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 may be an aliphatic group.

[0068] In the present application, the term "aryl" generally refers to a group having a residue resulting from the removal of a hydrogen atom from an aromatic ring. The term "aromatic ring" can refer to a monocyclic or fused polycyclic ring (i.e., rings sharing adjacent pairs of carbon atoms) having a conjugated π-electron system and consisting entirely of carbon atoms with 6 to 14 members, and can be 6 to 10 members, for example, benzene and naphthalene. The aromatic ring can be fused with a heteroaryl, heterocyclyl or cycloalkyl ring, in which case the ring linked to the parent structure is an aryl ring. Aryl may or may not be substituted. When aryl is substituted, the substituents may independently be one or more of the groups selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio. Aryl may or may not be substituted.

[0069] In the present application, the term "heteroaryl" generally refers to a group having a residue resulting from a hydrogen atom from a carbon atom from a heteroaromatic ring. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms can be selected from the group consisting of oxygen, sulfur, and nitrogen. Heteroaryl may be 5 to 10 members, and may be 5-membered or 6-membered, for example, furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, and tetrazolyl. The heteroaromatic ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, in which case the ring linked to the parent structure is a heteroaromatic ring. Heteroaryl may or may not be substituted. When heteroaryl is substituted, the substituents may independently be one or more of the groups selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. Heteroaryl may or may not be substituted.

[0070] In the present application, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, and the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Examples of polycyclic cycloalkyl include spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl. The cycloalkyl may or may not be substituted. When the cycloalkyl is substituted, the substitution by the substituent may be made at any available linking site, and the substituent is preferably independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0071] In the present application, the term "partially unsaturated" generally means that the cyclic structure contains at least one double bond or triple bond between the ring molecules. The term "partially unsaturated" encompasses cyclic structures having multiple unsaturated sites, but does not encompass aromatic or heteroaromatic rings as defined herein. The term "unsaturated" means that the moiety has one or more degrees of unsaturation.

[0072] In the present application, the term "heterocyclyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and the remaining ring atoms are carbon atoms. Preferably, the heterocyclyl contains 3 to 12 ring atoms, 1 to 4 of which are heteroatoms; more preferably, the heterocyclyl contains 3 to 8 ring atoms, 1 to 3 of which are heteroatoms; still more preferably, the heterocyclyl contains 3 to 6 ring atoms, 1 to 3 of which are heteroatoms; most preferably, the heterocyclyl contains 5 or 6 ring atoms, 1 to 3 of which are heteroatoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Non-limiting examples of polycyclic heterocyclyls include spiroheterocyclyl, fused heterocyclyl, and bridged heterocyclyl. The heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, and the ring attached to the parent structure is heterocyclyl. The heterocyclyl may or may not be substituted. When the heterocyclyl is substituted, the substitution by the substituent may be made at any available linking site, and the substituent is preferably independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0073] In the present application, the term "ring-forming atom" generally refers to an atom contained in a cyclic structure. For example, the ring-forming atom can be a carbon atom in a benzene ring or a nitrogen atom in a pyridine ring. When a hydrogen atom is linked to the ring-forming atom, the ring-forming atom may or may not be substituted.

[0074] In the present application, the term "independently of each other" generally means that a variable applies in any case, regardless of whether there are variables having the same or different definitions in the same compound. For example, a variable may refer to the type or number of substituents in a compound, the type of atoms in a compound, etc. For example, if R appears twice in a compound and R is defined as "independently, carbon or nitrogen", both Rs may be carbon, both Rs may be nitrogen, or one R may be carbon and the other R may be nitrogen.

[0075] In the present application, the term "may be (optional)" or "may be (optionally)" generally means that the event or situation described before it may occur but is not necessarily so, and that the description includes the case where the event or situation occurs or does not occur. For example, "a heterocyclic group optionally substituted with alkyl" means that alkyl may or may not be present, and that this description includes the case where the heterocyclic group is substituted with alkyl or not substituted with alkyl.

[0076] In the present application, the term "substituted" generally means that one or more hydrogen atoms in a group, for example, up to 5 (e.g., 1 to 3) hydrogen atoms, are each independently substituted with the corresponding number of substituents. Substituents are present only at possible chemical positions, and those skilled in the art can determine possible or impossible substitutions without undue experimentation (either experimentally or theoretically). For example, an amino or hydroxy having a free hydrogen bonded to a carbon atom having an unsaturated (such as olefin) bond may be unstable.

[0077] In the present application, the term "substituted with 0 or more (e.g., 0 or 1 or more, 0 or 1, or 0) methylene units" generally means that when the structure contains one or more methylene units, one or more of those methylene units may not be substituted, and may be substituted with one or more groups other than methylene (e.g., -NHC(O)-, -C(O)NH-, -C(O)-, -OC(O)-, -C(O)O-, -NH-, -O-, -S-, -SO-, -SO2-, -PH-, -P(=O)H-, -NHSO2-, -SO2NH-, -C(=S)-, -C(=NH)-, -N=N-, -C=N-, -N=C- or -C(=N2)-).

[0078] In the present application, the "linkage" between the X group and the Y group may generally be in any direction, and generally means that when the X group is used as a linker between the Y group and the Z group, two or more linking sites of the X group may be optionally linked to either the Y group or the Z group.

[0079] In the present application, the term "compound" generally refers to a substance having two or more different elements. For example, the compounds disclosed herein may be organic compounds. For example, the compounds disclosed herein may be compounds having a molecular weight of 500 Da or less, compounds having a molecular weight of 1000 Da or less, compounds having a molecular weight of 1000 Da or more, or compounds having a molecular weight of 10,000 Da or more or 100,000 Da or more. In the present application, a compound may refer to a compound involved in linkage by chemical bonds, for example, one or more molecules having a molecular weight of 1000 Da or less are linked to a biological macromolecule by chemical bonds, and the biological macromolecule may be a polysaccharide, a protein, a nucleic acid, a polypeptide, etc. For example, the compounds disclosed herein may include compounds in which a protein is linked to one or more molecules having a molecular weight of 1000 Da or less, may include compounds in which a protein is linked to one or more molecules having a molecular weight of 10,000 Da or less, and may include compounds in which a protein is linked to one or more molecules having a molecular weight of 100,000 Da or less.

[0080] In the present application, terms such as "alkyl", "alkenyl", and "cycloalkyl" may, as is known to those skilled in the art, be preceded by a designation indicating the number of atoms present in the group under certain circumstances, such as C1-C4 alkyl, C3-C7 cycloalkoxy, and C1-C4 alkylcarbonylamino, and the subscript number following "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group containing 3 carbon atoms (e.g., n-propyl, or isopropyl), and C 1-10 in which the members of the group may contain any number of carbon atoms in the range of 1 to 10.

[0081] One or more hydrogen atoms of the group, for example, up to 5 (e.g., 1 to 3) hydrogen atoms, are each independently substituted with the corresponding number of substituents. The substituents are present only at their possible chemical positions, and those skilled in the art can determine possible or impossible substitutions without undue experimentation (either experimentally or theoretically). For example, an amino or hydroxy having a free hydrogen bonded to a carbon atom having an unsaturated (such as olefinic) bond may be unstable.

[0082] In the present application, the compounds or ligand-drug conjugates of the present application include their tautomers, mesomers, racemates, enantiomers, and / or diastereoisomers. In the present application, the term "diastereoisomer" generally refers to stereoisomers that have two or more chiral centers and the molecules are not mirror images of each other. Diastereoisomers can have different physical properties, such as melting point, boiling point, spectral characteristics, and reactivity. In the present application, the terms "tautomer" or "tautomeric form" are used interchangeably and generally refer to structural isomers that have different energies and can be converted into each other by crossing a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions by proton transfer such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions by recombination of some bonding electrons. In the present application, the term "mesomer" generally means that the molecule contains asymmetric atoms but the overall optical rotation is zero due to the presence of a symmetry factor. The term "racemate" or "racemic mixture" refers to a composition of two enantiomeric substances in equimolar amounts.

[0083] In the present application, the term "isomer" of a compound or ligand-drug conjugate generally includes the tautomers, mesomers, racemates, enantiomers, and diastereoisomers of that compound.

[0084] In the present application, the term "ligand-drug conjugate" generally means that the ligand is linked to a bioactive cytotoxic drug via a stable linking unit. In the present application, the "ligand-drug conjugate" can be an antibody-drug conjugate (ADC), which means that an antibody or antibody fragment is linked to a bioactive drug (e.g., a cytotoxic drug) via a stable linking unit.

[0085] In this application, the term "ligand" generally refers to a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell. The role of the ligand can be to deliver a drug to a population of target cells to which the ligand binds, and ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules capable of binding to cells, receptors, and / or antigens. In this application, the ligand may be denoted as Ab, and the linkage bond between the ligand antigen and the linking unit is formed via a heteroatom of the ligand. The ligand may be an antibody or an antigen-binding fragment thereof (Ab), where the antibody may be selected from the group consisting of chimeric antibodies, humanized antibodies, fully human antibodies, and mouse antibodies, and the antibody may be a monoclonal antibody.

[0086] The term "cytotoxic drug" generally refers to a toxic drug, and the cytotoxic drug may be a chemical molecule sufficiently potent to interfere with the normal growth of tumor cells within the tumor cells. The cytotoxic drug can kill tumor cells at a sufficiently high concentration. "Cytotoxic drugs" can include low molecular weight toxins or enzymatically active toxins such as those of bacterial origin, fungal origin, plant origin, or animal origin, radioisotopes (e.g., radioisotopes of At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 or Lu), toxic drugs, chemotherapeutic agents, antibiotics, and nucleic acid degrading enzymes. For example, the cytotoxic drug may be a toxic drug including, but not limited to, a camptothecin derivative such as exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3’,4’:6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione).

[0087] The term "linker unit" or "linker structure" generally refers to a chemical structural fragment or bond that links to a ligand at one end and to a cytotoxic drug at the other end, or that links to another linker and then to a cytotoxic drug. Direct or indirect linkage of a ligand can mean that a group is directly linked to the ligand via a covalent bond and may also be linked to the ligand via a linker structure. For example, chemical structural fragments or bonds including acid-labile linker structures (e.g., hydrazone), protease-sensitive (e.g., peptidase-sensitive) linker structures, photosensitive linker structures, dimethyl linker structures or disulfide-containing linker structures can be used as linker structures.

[0088] The term "structure that may be linked to other molecular moieties" generally means that the structure is not linked to any other chemical structure, or that the structure is linked to one or more other chemical structures different from the structure (e.g., ligands described herein) (e.g., via a chemical bond or a linker structure).

[0089] The term "drug loading" generally refers to the average amount of cytotoxic drug loaded per ligand and can also be expressed as the ratio of cytotoxic drug to antibody. The cytotoxic drug loading can range from 0 to 12 (e.g., 1 to 10) cytotoxic drugs per ligand (Ab). In embodiments of the present application, the drug loading is indicated as DAR, and exemplary values can be 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 on average. The drug loading per ADC molecule after the coupling reaction can be characterized by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC.

[0090] In the present application, certain atoms of the compounds of the present application may exist in more than one isotopic form. For example, hydrogen may exist as protium ( 1 H), deuterium ( 2 H), and tritium ( 3 H), and carbon may exist as three different isotopes ( 12 C,13 C, and 14 (C) may occur naturally. Examples of isotopes that can be incorporated into the compounds of the present application include, but are not limited to, 15 N, 18 O, 17 O, 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125 I, or similar isotopes. Thus, the compounds of the present application may be enriched in one or more of these isotopes compared to their natural abundances. Such isotope-enriched compounds can be used for various purposes known to those skilled in the art. For example, substitution with heavy isotopes such as deuterium ( 2 H) may provide certain therapeutic advantages, perhaps due to higher metabolic stability. For example, the natural abundance of deuterium ( 2 H) is about 0.015%. Thus, one out of about 6500 hydrogen atoms is a deuterium atom. Accordingly, the abundance of deuterium at one or more sites (where applicable) in the deuterium-containing compounds of the present application exceeds 0.015%. Unless otherwise specified, the structures described herein may include compounds that differ only in the presence or absence of one or more isotope-enriched atoms. For example, compounds having the same structure as those disclosed herein, except that a hydrogen atom is replaced with deuterium or tritium or a carbon atom is replaced with carbon-13 or carbon-14, are within the scope of the present application.

[0091] The term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism, ease the absorption of the active ingredient, and thereby exert biological activity. Regarding the preparation of conventional pharmaceutical compositions, reference can be made to the Chinese Pharmacopoeia. Pharmaceutical compositions may be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. The suspensions can be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable or suspension prepared in a non-parenterally acceptable non-toxic diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, sterile non-volatile oils can be customarily used as solvents or suspending media. For example, any miscible non-volatile oil containing synthetic monoglycerides or diglycerides can be used. Additionally, fatty acids such as oleic acid can also be used in the preparation of injectables.

[0092] The term "pharmaceutically acceptable salt" generally refers to salts of the compounds or ligand-drug conjugates disclosed herein, or salts of the compounds described herein. Such salts may be safe and / or effective when used in mammals, may have the required biological activity, and the antibody-drug conjugates disclosed herein can form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0093] The term "pharmaceutically acceptable carrier" generally refers to a carrier or vehicle that provides a therapeutic agent, such as an antibody or polypeptide, a gene, and other therapeutic agents. This term refers to any pharmaceutical carrier that, by itself, can be administered without inducing the production of antibodies harmful to the individual receiving the composition and without causing undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, poly(amino acids), amino acid copolymers, lipid aggregates, and inactivated virus particles. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids such as water, physiological saline, glycerol, and ethanol. Auxiliary substances such as wetting or emulsifying agents, or pH buffering substances may also be present in these carriers.

[0094] The terms "treatment" and "treating" generally refer to methods of achieving a beneficial or desired effect that includes, but is not limited to, a therapeutic benefit. Therapeutic benefits include, but are not limited to, eradication, inhibition, alleviation, or amelioration of the underlying disease being treated. Further, a therapeutic benefit is achieved by eradicating, inhibiting, alleviating, or ameliorating one or more physiological symptoms associated with the underlying disease, and thus an improvement is observed in the patient, although the patient may still be afflicted with the underlying disease.

[0095] The term "B7H3-related disease" refers to any disease or condition caused by or exacerbated by, or otherwise associated with, an increase or decrease (generally an increase) in the expression or activity of B7H3 (such as human B7H3).

[0096] The terms "prevention" and "preventing" generally refer to methods of achieving beneficial or desired effects, including but not limited to prophylactic benefits. For prophylactic benefits, a pharmaceutical composition can be administered to a patient at risk of developing a particular disease or to a patient who has reported having one or more physiological symptoms of a disease even though the disease has not yet been diagnosed.

[0097] The term "subject" or "patient" generally refers to a human (i.e., a male or female of any age group, such as a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young adult, middle-aged adult, or elderly adult) and / or other primates (e.g., cynomolgus monkeys or rhesus monkeys); mammals including commercially relevant mammals, such as cows, pigs, horses, sheep, goats, cats, and / or dogs; and / or poultry including commercially relevant poultry, such as chickens, ducks, geese, quails, and / or turkeys.

[0098] The terms "therapeutically effective amount", "therapeutically effective dose", and "effective amount" refer to an amount of a ligand-drug conjugate of the present invention that, when administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents, is effective in preventing or ameliorating one or more symptoms of a disease or condition or the occurrence of a disease or condition. A therapeutically effective dose also refers to an amount sufficient to cause an improvement in symptoms, e.g., an amount for treating, curing, preventing, or ameliorating a related condition or for promoting the treatment, cure, prevention, or amelioration of such a condition. When the active ingredient is administered alone to an individual, the therapeutically effective dose refers to the amount of the ingredient only. When administered in combination, the therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether these active ingredients are administered in combination, sequentially, or simultaneously. The diagnostic index or parameter is increased by at least 10%, generally at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% by the effective amount of the therapeutic agent.

[0099] As used herein, the term "cancer" refers to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancer can be benign (also referred to as a benign tumor), pre-cancerous, or malignant. Cancer cells can be solid cancer cells or blood cancer cells. As used herein, the term "tumor" refers to one or more cells that include cancer. As used herein, the term "tumor growth" refers to the proliferation or growth of one or more cells that include cancer, corresponding to an increase in the size or spread of the cancer.

[0100] Anti-B7H3 antibody The present invention provides antibodies that specifically bind to B7H3, for example, antibodies that specifically bind to human B7H3, mouse B7H3, cynomolgus monkey B7H3, and its ECD domain. As used herein, the term "antibody" is as defined above and includes full-length immunoglobulins and antibody portions thereof that bind to the same antigen. Antibodies can be, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, or single-chain antibodies. In some embodiments, the antibody portion is a Fab fragment or an F(ab')2 fragment. In some embodiments, the antibody portion retains the ability to specifically bind to B7H3.

[0101] Recombinant anti-B7H3 antibodies, such as chimeric antibodies and humanized monoclonal antibodies that contain human and non-human portions, can be prepared using standard recombinant DNA techniques and are within the scope of the present invention. Such chimeric monoclonal antibodies and humanized monoclonal antibodies can be produced by recombinant DNA techniques such as those described in U.S. Patent No. 7,112,421; Better et al., (1988) Science, 240: 1041-1043; or Liu et al., (1987) Proc. Natl. Acad. Sci. USA, 84: 3439-3443. The present invention also includes humanized monoclonal antibodies that have been further modified / optimized, for example, by affinity maturation, back mutations, and removal of post-translational modification sites.

[0102] The antibodies or antigen-binding sites thereof disclosed herein preferably can bind to human 4IgB7H3 with high affinity. The antibodies or antigen-binding portions thereof disclosed herein can bind to human 2IgB7H3 with much lower affinity. The antibodies or antigen-binding portions thereof disclosed herein can bind to human 4IgB7H3 with high affinity, but bind to human 2IgB7H3 with much lower affinity.

[0103] The binding of the antibodies of the present invention to B7H3 can be evaluated using one or more techniques recognized in the art, such as ELISA or flow cytometry. In some embodiments, the antibodies can be analyzed and tested by flow cytometry, in which case the antibodies are reacted with human B7H3-expressing cell lines, such as MCF-7 cancer cells or CHOK1 cells transfected to express B7H3 on the cell surface. Further, the antibodies can also be tested by binuclear binding analysis for binding including binding kinetics (e.g., K D value). In some other embodiments, the antibodies are tested by ELISA, in which case the antibodies are reacted with soluble B7H3 protein.

[0104] The present invention provides antibodies that bind to human 4IgB7H3 protein with a K 8 of 1×10− D M or less, a K 9 of 1×10− D M or less, a K 10 of 5×10− D M or less, a K 10 of 1×10− D M or less, a K 11 of 9×10− D M or less, a K 11 of 8×10− D M or less, or a K 11 of 7×10− D M or less as measured by surface plasmon resonance (SPR). The antibodies disclosed herein also have a K 9 of 5×10−D 、 1×10- 8 M or more of K D 、 2×10- 8 M or more of K D 、 3×10- 8 M or more of K D 、 or 4×10 -8 M or more of K D and can be combined. In some embodiments, the antibodies disclosed herein bind to human 4IgB7H3 with a K D value less than 1 / 500, less than 1 / 100, or less than 1 / 50 of the K D value for binding to human 2IgB7H3. These K D values for comparison can be measured by surface plasmon resonance (SPR).

[0105] In some embodiments, the antibodies of the invention bind to human or cynomolgus B7H3-expressing cell lines with an EC 50 less than 5 nM, less than 4 nM, less than 3 nM, or less than 2 nM as determined by FACS

[0106] In some embodiments, the antibody or its antigen-binding site is a chimeric antibody or a murine antibody that specifically binds to B7H3, preferably human 4IgB7H3. In some further embodiments, the antibody or its antigen-binding site is a humanized antibody that specifically binds to B7H3, preferably human 4IgB7H3. In some further embodiments, the humanized antibody or its antigen-binding site contains one or more revertant mutations within the framework region. In some further embodiments, the humanized antibody or its antigen-binding site contains one or more modifications at potential post-translational modification (PTM) sites, such as removing any amino acids except NG, NS, and DG in the CDR, and NXS and NXT (where X can be any amino acid other than P) throughout the length.

[0107] In some embodiments, the antibodies or their antigen-binding sites disclosed herein are as follows: (i) HCDR1 comprising SEQ ID NO: 1, (ii) An HCDR2 comprising SEQ ID NO: 2, and (iii) An HCDR3 comprising SEQ ID NO: 3 One or more heavy chain CDRs (HCDRs) selected from the group consisting of at least one of the following, and / or The following: (i) An LCDR1 comprising SEQ ID NO: 4, 7, or 18, (ii) An LCDR2 comprising SEQ ID NO: 5, and (iii) An LCDR3 comprising SEQ ID NO: 6 One or more light chain CDRs (LCDRs) selected from the group consisting of at least one of the following are included.

[0108] In some embodiments, the antibody or antigen-binding site thereof described above may comprise an HCDR1 comprising or consisting of SEQ ID NO: 1, an HCDR2 comprising or consisting of SEQ ID NO: 2, an HCDR3 comprising or consisting of SEQ ID NO: 3, an LCDR1 comprising or consisting of SEQ ID NO: 7, an LCDR2 comprising or consisting of SEQ ID NO: 5, and an LCDR3 comprising or consisting of SEQ ID NO: 6. In particular, the antibody may further comprise one or more modifications in which potential PTM sites in the CDRs are removed. In some embodiments, the antibody comprises one or more modifications in which the potential PTM site "NS" within the sequence is removed in LCDR1 as compared to the LCDR1 shown in SEQ ID NO: 7 ("KSSQSLL NS SNQKNYLA"). In some preferred embodiments, the antibody comprises a substitution at position 8 or 9 of the amino acid sequence of LCDR1 as compared to the LCDR1 shown in SEQ ID NO: 7 ("KSSQSLL NS SNQKNYLA"). In some more preferred embodiments, the antibody comprises a substitution from N to Q at position 8 of the amino acid sequence of LCDR1 (i.e., SEQ ID NO: 4, "KSSQSLL QS SNQKNYLA"). In some other embodiments, the antibody comprises a substitution from S to P at position 9 of the amino acid sequence of LCDR1 (i.e., SEQ ID NO: 18, "KSSQSLL NP") SNQKNYLA. Those skilled in the art will understand that other types of substitutions may be selected as long as the binding affinity for B7H3 is substantially retained.

[0109] In some embodiments, the antibody or antigen-binding portion thereof comprises an HCDR1 comprising or consisting of SEQ ID NO: 1, an HCDR2 comprising or consisting of SEQ ID NO: 2, an HCDR3 comprising or consisting of SEQ ID NO: 3, an LCDR1 comprising or consisting of SEQ ID NO: 4, an LCDR2 comprising or consisting of SEQ ID NO: 5, and an LCDR3 comprising or consisting of SEQ ID NO: 6.

[0110] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region each comprise the above HCDR1 to HCDR3 and LCDR1 to LCDR3.

[0111] In some embodiments, the heavy chain variable region of the antibody or antigen-binding portion thereof (i) the amino acid sequence of SEQ ID NO: 8 or 10; (ii) an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 8 or 10, or (iii) an amino acid sequence having an addition, deletion and / or substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids as compared to SEQ ID NO: 8 or 10.

[0112] In some embodiments, the amino acid substitution(s) may be conservative substitutions.

[0113] In some embodiments, the light chain variable region of the antibody or antigen-binding portion thereof (i) The amino acid sequence of SEQ ID NO: 9 or 11; (ii) an amino acid sequence having at least 85%, at least 90% or at least 95% identity to SEQ ID NO: 9 or 11, or (iii) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 9 or 11.

[0114] In some embodiments, the amino acid substitution(s) may be conservative substitutions.

[0115] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH (i) The amino acid sequence of SEQ ID NO: 8 or 10, or (ii) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid substitutions within the framework region(s) compared to SEQ ID NO: 8 or 10. and / or VL (i) The amino acid sequence of SEQ ID NO: 9 or 11, or (ii) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid substitutions within the framework region(s) compared to SEQ ID NO: 9 or 11.

[0116] In some embodiments, the amino acid substitution(s) may be conservative substitutions.

[0117] In some embodiments, the antibody or its antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the VH region shown in SEQ ID NO: 8 or 10, and LCDR1, LCDR2 and LCDR3 of the VL region shown in SEQ ID NO: 9 or 11.

[0118] As will be understood by those skilled in the art, the exact numbering and placement of CDRs will vary with different numbering schemes. However, it should be understood that the disclosure of a variable heavy chain sequence and / or variable light chain sequence encompasses the disclosure of the relevant (endogenous) CDRs. Thus, the disclosure of each heavy chain variable region is the disclosure of the heavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR3), and the disclosure of each light chain variable region is the disclosure of the light chain CDRs (e.g., LCDR1, LCDR2, and LCDR3). Amino acids can be assigned to each CDR according to one or a combination of all numbering schemes well known in the art. A comparison of CDR numbers is shown below. See Lafranc et al., Dev. Comp. Immunol. 27 (1): 55-77 (2003). [Table 1]

[0119] In some embodiments, the amino acid sequence of a CDR can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the above sequences. In some embodiments, the amino acid sequence of a variable region can have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the above sequences.

[0120] As noted above, additions, deletions, and / or substitutions of at least one amino acid within a VH or VL region can be in the framework (FR) sequences and not in any of the CDR sequences. For example, an isolated antibody or its antigen-binding site can include one or more amino acid substitutions in the framework sequences of the VH or VL region, e.g., FR1, FR2, FR3, and / or FR4. In some embodiments, the framework region of the VH region includes one or more of the following substitutions: S025T, V071R, Y091F, V089T.

[0121] In some embodiments, the changes in the six CDRs can be a total of 0, 1, 2, 3, 4, or 5 amino acid modifications (preferably amino acid substitutions), and changes in the framework regions (excluding the CDRs) of the heavy and light chain variable regions may be included as long as the framework (excluding the CDRs) maintains at least about 80%, 85%, or 90% identity to the parental antibody (e.g., W301088-1.145.16). Thus, the same CDRs described herein can be combined with different framework sequences derived from the human germline sequence as long as the framework region maintains at least 80%, 85%, or 90% identity to the human germline sequence.

[0122] In certain embodiments, the isolated antibody or antigen-binding portion thereof presented herein can include any suitable framework region (FR) sequence as long as the antigen-binding domain specifically binds to B7H3, preferably human Ig4B7H3.

[0123] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies presented herein, and thus, Fc region variants can be generated. The Fc region variants can include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0124] In some embodiments, it may be necessary to generate antibodies modified by cysteine modification, such as "sulfono-MAb" in which one or more residues of the antibody are replaced by cysteine residues.

[0125] In some embodiments, the antibodies presented herein can be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and glucan or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0126] The antibodies of the present invention can be produced using any suitable method for producing antibodies. Any suitable form of B7H3 can be used as an immunogen (antigen) for antibody production. By way of example, and not limitation, any B7H3 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells that produce mouse monoclonal anti-human B7H3 antibodies can be generated by methods well known in the art. Antibodies derived from rodents (e.g., mice) can induce unwanted immunogenicity of the antibody when used as therapeutic agents in vivo. Repeated use of these antibodies induces an immune response to the therapeutic antibody in the human body. Such an immune response results in at least a loss of therapeutic efficacy and, in severe cases, potentially lethal allergic reactions. One method of reducing the immunogenicity of rodent antibodies is the production of chimeric antibodies in which the mouse variable regions are fused to human constant regions (Liu et al. (1987), Proc. Natl. Acad. Sci. USA 84: 3439-3443). However, the presence of intact rodent variable regions in chimeric antibodies still has the potential to induce harmful immunogenicity in patients. Transplantation of the complementarity determining region (CDR) loops of the rodent variable domain into the human framework (i.e., humanization) has been used to further minimize rodent sequences (Jones et al., (1986) Nature 321: 522; Verhoeyen et al., (1988) Science 239: 1534).

[0127] In some embodiments, the chimeric or humanized antibodies of the present invention can be prepared based on the sequences of the prepared mouse monoclonal hybridoma antibodies. DNA encoding the immunoglobulin heavy and light chains can be obtained from the desired mouse hybridoma and modified using standard molecular biology techniques to include non-mouse (e.g., human) immunoglobulin sequences.

[0128] In some embodiments, the chimeric B7H3 antibodies described herein can be prepared by operably linking the immunoglobulin heavy and light chain variable regions of hybridoma origin to human IgG constant regions, respectively, using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). In some embodiments, the chimeric antibodies of the invention can include a constant region selected from any human IgG subtype, such as IgG1, IgG2, IgG3, and IgG4, preferably IgG1.

[0129] In some embodiments, the chimeric B7H3 antibodies of the invention can be obtained by "mixing and matching" a chimeric light chain expression plasmid and a chimeric heavy chain expression plasmid and transfecting the expression cells therewith. The binding of such "mixed and matched" antibodies to B7H3 can be tested using the binding assays described above and other conventional binding assays (e.g., ELISA).

[0130] With respect to the humanized antibodies described herein, the mouse CDR regions can be inserted into the human germline framework regions using methods known in the art (see U.S. Patent No. 5,225,539 to Winter et al. and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.).

[0131] In some embodiments, the CDR sequences of the antibodies used in the drug conjugates, compositions, uses or methods of the present invention comprise the CDR sequences of antibody W301088-1.145.16-z3-p1-uIgG1KV320 (abbreviated as WBP301088). In some embodiments, the CDR sequences of the antibodies used in the drug conjugates, compositions, uses or methods of the present invention comprise the CDR sequences of antibody W301088-1.145.16-xIgG1KV320. In some embodiments, the variable region sequences of the antibodies used in the drug conjugates, compositions or uses of the present invention comprise variable region sequences derived from antibody W301088-1.145.16-z3-p1-uIgG1KV320 or W301088-1.145.16-xIgG1KV320. In some embodiments, the amino acid sequences of the antibodies used in the drug conjugates, compositions, uses or methods of the present invention comprise the full-length amino acid sequences derived from antibody W301088-1.145.16-z3-p1-uIgG1KV320 or W301088-1.145.16-xIgG1KV320.

[0132] The anti-B7H3 antibody W301088-1.145.16-xIgG1KV320 or an antigen-binding fragment thereof described herein is prepared as described in the Examples. In some embodiments, the CDR sequences of the antibodies used in the drug conjugates, compositions, uses or methods of the present invention comprise HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In some embodiments, the variable region sequences of the antibodies used in the drug conjugates, compositions or uses of the present invention comprise a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the antibodies used in the drug conjugates, compositions, uses or methods of the present invention comprise a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 14 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0133] The anti-B7H3 antibody W301088-1.145.16-z3-p1-uIgG1KV320 (abbreviated as WBP301088) described in this specification or its antigen-binding fragment is prepared as described in the examples. In some embodiments, the CDR sequences of the antibodies used in the drug conjugates, compositions, uses or methods of the present invention include HCDR1, HCDR2 and HCDR3 containing the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 containing the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In some embodiments, the variable region sequences of the antibodies used in the drug conjugates, compositions or uses of the present invention include a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the antibodies used in the drug conjugates, compositions, uses or methods of the present invention include a heavy chain containing the amino acid sequence shown in SEQ ID NO: 12 and a light chain containing the amino acid sequence shown in SEQ ID NO: 13.

[0134] The amino acid sequences of the chimeric antibody W301088-1.145.16-xIgG1KV320 and the humanized antibody W301088-1.145.16-z3-pl-ulgGlKV320 of the present invention are shown below. In some embodiments, the CDRs of the antibodies of the present invention are numbered using a combined Kabat and Chothia numbering scheme.

[0135] Amino acid sequence of W301088-1.145.16-xlgG1KV320 (the underlined part is the CDR) Heavy chain variable region VH SEQ ID NO: 8 DVQLQESGPGLVKPSQSLSLTCTVT DYSITGDYAWN WIRQFPGNKLEWMG YISYSGSTSYNPSLQS RISITRDTSKNQFFLQLNSVTSEDTATYFCAR SLGRRWYFVV WGAGTTVTVSA HCDR1: DYSITGDYAWN (SEQ ID NO: 1) HCDR2: YISYSGSTSYNPSLQS (SEQ ID NO: 2) HCDR3: SLGRRWYFVV (SEQ ID NO: 3) Light chain variable region VL SEQ ID NO: 9 DIVMTQSPSSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLIY FASTRES GVPDRFIGSGSGTDFTLTISSVQAEDLTDYFC QQHYSAPWT FGGGTKLEIK LCDR1: KSSQSLLNSSNQKNYLA (SEQ ID NO: 7) LCDR2: FASTRES (SEQ ID NO: 5) LCDR3: QQHYSAPWT (SEQ ID NO: 6) Heavy chain SEQ ID NO: 14 DVQLQESGPGLVKPSQSLSLTCTVT DYSITGDYAWN WIRQFPGNKLEWMG YISYSGSTSYNPSLQS RISITRDTSKNQFFLQLNSVTSEDTATYFCAR SLGRRWYFVV WGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Light chain SEQ ID NO: 15 DIVMTQSPSSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLIY FASTRES GVPDRFIGSGSGTDFTLTISSVQAEDLTDYFC QQHYSAPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC

[0136] Amino acid sequence of W301088 - 1.145.16 - z3 - pl - ulgG1KV320 (the underlined part is the CDR) Heavy chain variable region VH SEQ ID NO: 10 QVQLQESGPGLVKPSQTLSLTCTVT DYSITGDYAWN WIRQHPGKGLEWIG YISYSGSTSYNPSLQS RVTISRDTSKNQFSLKLSSVTAADTAVYFCAR SLGRRWYFVV WGQGTTVTVSS HCDR1: DYSITGDYAWN (SEQ ID NO: 1) HCDR2: YISYSGSTSYNPSLQS (SEQ ID NO: 2) HCDR3: SLGRRWYFVV (SEQ ID NO: 3) Light chain variable region VL SEQ ID NO: 11 DIVMTQSPDSLAVSLGERATINC KSSQSLLQSSNQKNYLA WYQQKPGQPPKLLIY FASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHYSAPWT FGGGTKVEIK LCDR1: KSSQSLLQSSNQKNYLA (SEQ ID NO: 4) LCDR2: FASTRES (SEQ ID NO: 5) LCDR3: QQHYSAPWT (SEQ ID NO: 6) Heavy chain SEQ ID NO: 12 QVQLQESGPGLVKPSQTLSLTCTVTDYSITGDYAWNWIRQHPGKGLEWIGYISYSGSTSYNPSLQSRVTISRDTSKNQFSLKLSSVTAADTAVYFCARSLGRRWYFVVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain SEQ ID NO: 13 DIVMTQSPDSLAVSLGERATINCKSSQSLLQSSNQKNYLAWYQQKPGQPPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSAPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0137] The antibodies or antigen-binding sites thereof disclosed herein have certain functional features or characteristics. In some embodiments, the antibodies (including chimeric and humanized antibodies) have one or more of the following characteristics: (a) Specifically binds to human 4IgB7H3-expressing cells with high affinity (e.g., less than 2 nM as measured by FACS), (b) It has a binding affinity for human 2IgB7H3 that is significantly lower than that for human 4IgB7H3, and there is a significant difference between the affinity of an antibody that binds to 2IgB7H3 and the affinity of an antibody that binds to 4IgB7H3. (c) Specifically binds to cynomolgus monkey B7H3 with a high affinity (e.g., less than 5 nM as measured by FACS) that is better than that of the benchmark antibody, and (d) Shows good internalization by B7H3-expressing cancer cells.

[0138] Pharmaceutical compositions and pharmaceutical formulations In yet another aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate described herein, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0139] It should be understood that the antibody-drug conjugates, pharmaceutically acceptable salts thereof, or pharmaceutical compositions presented herein can be incorporated into carriers, excipients, and other reagents suitable for combined administration to form formulations, and thus can bring about improvements in transportation, delivery, tolerance, etc.

[0140] The term "pharmaceutical composition" refers to a formulation that allows the active ingredient contained therein to be present in an effective biologically active form and does not contain additional ingredients that have unacceptable toxicity to the subject receiving the administration of the formulation.

[0141] A pharmaceutical formulation containing the anti-B7H3 antibody described herein, preferably as an aqueous solution or a lyophilized formulation, can be prepared by mixing the anti-B7H3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the present invention having the desired purity with one or more optional pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)).

[0142] The pharmaceutical composition or formulation of the present invention may further comprise one or more additional active ingredients necessary for the specific indication to be treated, preferably active ingredients having complementary activities that do not adversely affect each other. In some embodiments, the additional active ingredient is a chemotherapeutic agent, an immune checkpoint inhibitor, a cell growth inhibitor, an antibiotic, or various known antitumor or anticancer agents that are appropriately present in the combination in an amount effective for the intended purpose. In some embodiments, the pharmaceutical composition of the present invention also comprises a composition of polynucleotides encoding an anti-B7H3 antibody.

[0143] In yet another aspect, the present invention provides a pharmaceutical combination comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0144] In yet another aspect, the present invention provides a kit comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein, and preferably further comprising a drug delivery device.

[0145] Medical Use In yet another aspect, the present invention provides the use of the antibody-drug conjugate, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described herein in the preparation of a medicament for treating and / or preventing a B7H3-mediated disease or condition, preferably wherein the disease or condition is cancer.

[0146] In yet another aspect, the present invention provides a pharmaceutical composition for use in the treatment and / or prevention of a B7H3-mediated disease or condition, preferably wherein the disease or condition is cancer, which comprises the antibody-drug conjugate, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described herein.

[0147] In yet another aspect, the present invention provides a method for treating and / or preventing a B7H3-mediated disease or condition, comprising administering to a subject in need thereof an antibody-drug conjugate described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, preferably, wherein the disease or condition is cancer.

[0148] In yet another aspect, the present invention provides an antibody-drug conjugate described herein, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, for treating and / or preventing a B7H3-mediated disease or condition, preferably, wherein the disease or condition is cancer.

[0149] In some embodiments, the cancer of the present invention is selected from the group consisting of breast cancer, neurological tumors, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, urothelial cancer, lymphoma (e.g., non-Hodgkin lymphoma), chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma.

[0150] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody-drug conjugate described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0151] In yet another aspect, the present invention provides a kit comprising an antibody-drug conjugate described herein or a pharmaceutical composition described herein.

[0152] In some embodiments, the administration routes of the present invention include, but are not limited to, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intra-arterial administration, intra-articular administration (e.g., for joints with arthritis), inhalation, aerosol delivery, intratumoral administration, and the like.

[0153] In some embodiments, the invention provides for the co - administration of an effective amount of one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to a subject. In some embodiments, the therapies include surgical treatment and / or radiation therapy.

[0154] In some embodiments, the methods or uses presented herein further comprise the step of administering an individual one or more therapies (e.g., treatment modalities and / or other therapeutic agents). The antibody - drug conjugate of the invention or a pharmaceutically acceptable salt thereof can be administered alone or in combination with other therapeutic agents as one therapy. For example, the antibody can be co - administered with at least one additional therapeutic agent.

[0155] The antibody-drug conjugate described in this application has inhibitory activity against the in vitro proliferation of tumor cells. The inhibitory activity is such that when the drug conjugate of this application is added to a medium containing tumor cells, the proliferative ability of the tumor cells is reduced by 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more compared to the case where a negative control or a reference drug is added. For example, the inhibitory activity is such that the IC50 value (nM) against tumor cells is 10000 nM or less, 5000 nM or less, 4000 nM or less, 3000 nM or less, 2000 nM or less, 1000 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 150 nM or less, 120 nM or less, 110 nM or less, 100 nM or less, 99 nM or less, 98 nM or less, 97 nM or less, 95 nM or less, 90 nM or less, 80 nM or less, 75 nM or less, 70 nM or less, 65 nM or less, 62 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 25 nM or less, 23 nM or less, 22 nM or less, 20 nM or less, 19 nM or less, 18 nM or less, 18.5 nM or less, 17 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 9 nM or less, 8.5 nM or less, 7 nM or less, 6.7 nM or less, 6 nM or less, 5.9 nM or less, 5.5 nM or less, 5.0 nM or less, 4.8 nM or less, 4.5 nM or less, 4.4 nM or less, 4 nM or less, 3.5 nM or less, 3 nM or less, 2.5 nM or less, 2 nM or less, 1.5 nM or less, 1.0 nM or less, 0.5 nM or less, 0.3 nM or less, 0.29 nM or less, 0.25 nM or less, 0.21 nM or less, 0.20 nM or less, 0.18 nM or less, 0.17 nM or less, 0.15 nM or less, 0.12 nM or less, 0.10 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07 nM or less, 0.06 nM or less, 0.05 nM or less, 0.04 nM or less, 0.03 nM or less, 0.02 nM or less, or 0.01 nM or less. For example, examples of tumor cells include, but are not limited to, solid tumor cells. For example, examples of tumor cells include, but are not limited to, lung cancer cells.For example, as tumor cells, but not limited thereto, Calu-6 lung cancer cells can be mentioned.

[0156] The antibody-drug conjugates described in this specification can have an anti-tumor effect in vivo. The anti-tumor effect can be achieved by administering the antibody-drug conjugate of the present application to an animal, and compared with the case of administering a negative control or a reference drug, after 1 day, 3 days, 5 days, 7 days, 14 days, 20 days, 21 days or 30 days, a reduction of 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 55% or more, 60% or more, 70% or more, 73% or more, 75% or more, 80% or more, 90% or more, or 95% or more in the volume of the tumor in the animal, or a reduction to 1 / 1.1 or less, 1 / 1.3 or less, 1 / 1.5 or less, 1 / 2 or less, 1 / 3 or less, 1 / 5 or less, 1 / 10 or less, 1 / 20 or less, 1 / 22 or less, 1 / 30 or less, 1 / 50 or less, 1 / 100 or less, 1 / 500 or less, 1 / 1000 or less, or 1 / 1500 or less may be brought about. As the animal, but not limited thereto, mammals can be mentioned. For example, as the animal, but not limited thereto, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys or humans can be mentioned. As the administration, but not limited thereto, oral administration, intravenous injection, intravenous drip, intraperitoneal injection or topical administration can be mentioned. For example, as tumor cells, but not limited thereto, solid tumor cells can be mentioned. For example, as tumor cells, but not limited thereto, lung cancer cells, melanoma cells, brain cancer cells, and prostate cancer cells can be mentioned. For example, as tumor cells, but not limited thereto, Calu-6 lung cancer cells, A375 melanoma cells, U87 brain cancer cells, and PC-3 prostate cancer cells can be mentioned.

[0157] The present invention includes any combination of the specific embodiments described. Further embodiments and the full scope of adaptability of the present invention will become apparent from the detailed description presented below. However, the detailed description and specific examples, while indicating preferred embodiments of the present invention, will be understood by those skilled in the art to disclose various changes and modifications within the spirit and scope of the present invention and are presented by way of illustration only. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes, including the citation itself. [Examples]

[0158] The following examples are presented to demonstrate and further illustrate some preferred embodiments and aspects of the present invention and should not be construed as limiting the scope of the present invention.

[0159] Materials and Methods: [Table 2] JPEG2025523382000027.jpg38170

[0160] Sample Test Method 1. ADC DAR Value Analysis Method - HIC - HPLC (Hydrophobic Interaction Chromatography) High - performance liquid chromatograph: Waters e2965 high - speed liquid chromatography system. Chromatography column: MabPac™ HIC - Butyl 5μm 4.6×100mm (manufacturer: Thermo). Mobile phase A: 1.5M (NH4)2SO4 + 50mM K2HPO4 (pH 7.0). Mobile phase B: 50mM K2HPO4 (pH 7.0) / isopropanol (75:25 V / V).

[0161] Elution was carried out according to the following elution procedure. [Table 3]

[0162] Detection conditions: Set the flow rate of the mobile phase to 1 mL per minute, the detection wavelength to 280 nm, and the column temperature to 30 °C.

[0163] 2. SEC purity analysis - SEC-HPLC (size exclusion chromatography) High-performance liquid chromatograph: 1260 Agilent liquid chromatograph. Chromatography column: Waters Xbridge BEH200 SEC (7.8×300 mm, 3.5 μm) Mobile phase: 50 mM NaH2PO4 + 200 mM arginine (pH 6.80) + 10% isopropanol

Table 4

Example

[0164] Generation and characterization of anti-B7H3 antibody 1.1. Preparation of antigen, benchmark antibody and cell line 1.1.1. Generation of antigen The nucleotide sequence encoding the amino acid sequence of the extracellular domain of human 4IgB7H3 (Uniprot ID: Q5ZPR3, amino acids 29 - 461) was first codon-optimized for expression in mammals and then synthesized by Sangon Biotech (Shanghai, China). The DNA segment was subcloned into the pcDNA3.3 expression vector with 6×His at the C-terminus, and then the subcloned expression vector was transfected into Expi293F cells (manufactured by Invitrogen, A14635). After incubating for 5 days, the supernatant was purified using a Ni column (manufactured by Cytiva, 173712). The eluted protein was dialyzed into PBS using a dialysis bag (manufactured by Spectrum, 888 - 10987, MWCO 3.5 kDa). The protein concentration was determined at an absorbance of 280 nm using a NanoDrop device. Electrophoresis of 2 μg of the purified protein was performed on an SDS-PAGE gel (manufactured by Invitrogen) with and without a reducing agent. The purity of the purified protein was quantified by HPLC-SEC using a TSKgel G3000 SWXL size exclusion chromatography column (manufactured by Tosoh, 008541). The purified protein was stored at -80°C. Human 2IgB7H3 was purchased from ACRO (Cat number B73-H52E2), and cynomolgus monkey B7H3 was purchased from ACRO (Cat number B73-C52Ha).

[0165] 1.1.2. Generation of benchmark antibodies The anti-B7H3 antibody enoblituzumab (manufactured by MacroGenics, with the light and heavy chain amino acid sequences being SEQ ID NO: 117 and SEQ ID NO: 119 in the specification of US Patent No. 20120294796, respectively) was used as the benchmark antibody. The nucleic acid sequence encoding the variable domain of the antibody was first codon-optimized for mammalian expression and then synthesized by Sangon Biotech (Shanghai, China). Subsequently, the DNA segment was subcloned into a modified pcDNA3.4 expression vector having the constant region of human IgG1. Plasmids containing the VH gene and the VL gene were co-transfected into Expi293F cells (manufactured by Invitrogen, A14635). After incubating for 5 days, the supernatant was purified using a Protein A column (manufactured by Cytiva, 175438). The eluted protein was dialyzed into PBS using a dialysis bag (manufactured by Spectrum, 888-10987). The protein concentration was determined at an absorbance of 280 nm using a NanoDrop device. Electrophoresis of 2 μg of the purified protein was performed on an SDS-PAGE gel (manufactured by Invitrogen) with and without a reducing agent. The purity of the purified protein was quantified by HPLC-SEC using a TSKgel G3000 SWXL size exclusion chromatography column (manufactured by Tosoh, 008541). The purified antibody was stored at -80°C.

[0166] 1.1.3. Establishment of stable cell lines / cell pools A cell line expressing human 4IgB7H3 was generated. Briefly, CHOK1 cells were transfected with a pcDNA3.3 expression vector containing full-length 4IgB7H3 using a Lipofectamine 2000 transfection kit (Invitrogen, 11668027) according to the manufacturer's protocol. Forty-eight hours after transfection, the cells were subcultured in a selection medium (F12-K containing 10% FBS and 15 μg / mL blasticidin) in a T75 flask. After two or three selection passages, a cell line (W3XX088-CHOK1.hPro1.2A5) stably expressing human 4IgB7H3 was obtained by selection with blasticidin and limiting dilution, and the expression level was determined by FACS using an anti-B7H3 antibody.

[0167] A cell pool expressing cynomolgus B7H3 was generated. Briefly, FlipinCHO cells were transfected with a pcDNA5 / pOG44 expression vector containing full-length cynomolgus B7H3 using a Lipofectamine 2000 transfection kit (Invitrogen, 11668027) according to the manufacturer's protocol. Forty-eight hours after transfection, the cells were subcultured in a selection medium (F12 containing 10% FBS and 600 μg / mL hygromycin B) in a T75 flask. After two or three selection passages, a cell pool (WBP3XX088-FlpinCHO.cPro1.pool) stably and highly expressing cynomolgus B7H3 was obtained by selection with hygromycin B and cell sorting using BD FACSMelody (trademark).

[0168] 1.2. Generation of Antibodies 1.2.1. Immunization of Animals Four 6- to 8-week-old Balb / c mice were immunized with the DNA plasmid encoding human full-length 4IgB7H3 in Example 1.1.3 at 200 - 400 μg per mouse. The adjuvant mixture included Adju-Phos, CpG-ODN, and GM-CSF. The animals were subjected to subcutaneous injection, intramuscular injection, and hydrodynamic tail vein injection once every two weeks. Blood was collected from the mice after the third injection (the first blood collection) and after the fifth injection (the second blood collection). Serum titers were measured by ELISA and FACS.

[0169] The ELISA assay was used to measure the serum antibody titer against the 4IgB7H3 antigen. Plates (manufactured by Nunc) were coated with 100 μL of 0.5 μg / mL His tag overnight at 4°C, and then blocked with blocking buffer (2% BSA in PBS) for 1 hour at ambient temperature. The plates were then washed and incubated with 1 μg / mL antigen for 1 hour at ambient temperature. After washing, mouse sera were serially diluted from a starting dilution of 1:100 in blocking buffer up to 1:3 and added to the culture plates and incubated for 2 hours at ambient temperature. The plates were then washed and subsequently incubated with the secondary antibody, i.e., goat anti-mouse IgG-Fc-HRP (manufactured by Bethyl, A90-231P) for 1 hour. After washing, TMB substrate was added and the interaction was stopped using 2 M HCl. The absorbance at 450 nm was read using a microplate reader (manufactured by Molecular Device). Serum titers were determined at twice the background. [Table 5]

[0170] The FACS assay was used to measure the serum antibody titer against the 4IgB7H3 antigen. Briefly, the modified B7H3-expressing cells of Example 1.1.3 were seeded at 1 × 10 cells per well in a 96-well U-bottom plate. 5Seeds were sown at a density of [quantity not provided], centrifuged at 4°C and 1500 rpm for 4 minutes. Subsequently, the supernatant was removed. Mouse serum was serially diluted from a dilution of 1:100 in 1×PBS / 1% BSA to 1:3, the cells were resuspended, and incubated at 4°C for 1 hour. The cells were washed twice with 180 μL of 1×PBS / 1% BSA. Goat anti-mouse IgG-Fc Alexa 647 (Jackson ImmunoResearch Laboratories, Inc., 109-605-098) as the secondary antibody was added to the resuspended cells, incubated in the dark at 4°C for 30 minutes, and then washed with 180 μL of 1×PBS / 1% BSA. Finally, the cells were resuspended in 100 μL of 1×PBS / 1% BSA, and the fluorescence intensity was measured by FACS (BD Canto II) and analyzed using FlowJo version software. The serum titer was determined at twice the background.

Table 6

[0171] When the serum titer was sufficiently high, 400 μg of the DNA plasmid encoding human full-length 4IgB7H3 of Example 1.1.3 was administered via the hydrodynamic tail vein, and 4×10 [quantity not provided] MCF-7 cells were used for the final boost immunization via the plantar and subcutaneously, and then fused with myeloma cells. Lymph nodes and spleens were collected for cell fusion. 6 After the final boost immunization with [quantity not provided] MCF-7 cells, they were fused with myeloma cells. Lymph nodes and spleens were collected for cell fusion.

[0172] 1.2.2. Hybridoma Generation Lymph nodes and spleens from the immunized mice in Example 1.2.1 were homogenized and filtered to remove blood clots and cell debris. Sp2 / 0 myeloma cells in the logarithmic growth phase were collected and centrifuged. B cells obtained by homogenizing the lymph nodes and spleens from the immunized mice were fused with Sp2 / 0 myeloma cells at a ratio of 1:1 according to the conventional electrofusion procedure in an electrofusion solution. The fused cells were suspended in DMEM medium supplemented with 20% FBS and 1×HAT, and then transferred to a 96-well plate (CORNING). The fused cells were cultured in an incubator set at 37°C and 5% CO2 for 10 - 14 days.

[0173] 1.2.3. Antibody Screening First Screening: A cell-based ELISA assay was used as the first screening to test the binding of hybridoma supernatant to human 4IgB7H3. Briefly, the CHOK1 cell line transfected with human 4IgB7H3 in Example 1.1.3 was seeded at a density of 5×10 3 cells per well in a 96-well plate (manufactured by CORNING), and then maintained in an incubator set at 37°C and 5% CO2 for 2 days. Then, the plate was washed and incubated with the hybridoma supernatant at room temperature for 1 hour. Then, the plate was washed and then incubated with the secondary antibody, goat anti-mouse IgG-Fc-HRP (manufactured by Bethyl, A90-231P), for 1 hour. After washing, the TMB substrate was added and the interaction was stopped using 2M HCl. The absorbance at 450 nm was read using a microplate reader (manufactured by Molecular Device).

[0174] Second Screening: To confirm the binding of the hybridoma supernatant to human and cynomolgus monkey antigens, FACS was performed. The parental CHOK1 cell line and the CHOK1 cell line transfected with 4IgB7H3 in Example 1.1.3 were stained with CellTrace dyes (Violet and FarRed respectively), while the FlipinCHO cell pool transfected with B7H3 in Example 1.1.3 was prepared without dye. Incubated at room temperature for 15 minutes, washed, and then the three types of cells were mixed in equal amounts to make 1×10 6 cells per mL. The mixture of the three types of cells was transferred to a 96-well U-bottom plate (manufactured by BD) at a density of 1×10 5 cells per well. Then, the hybridoma supernatant was transferred to the plate and incubated at 4°C for 1 hour. After washing, the secondary antibody, namely goat anti-mouse IgG Fc PE (manufactured by Jackson, 115-115-164), was added and incubated with the cells in the dark at 4°C for 0.5 hour. Then, the cells were washed, resuspended in 1×PBS / 1% BSA, and analyzed by flow cytometry.

[0175] To test the binding of the hybridoma supernatant to human 2IgB7H3, a conventional ELISA assay was used. The plates were pre-coated with 100 μL / well of human 2IgB7H3 (0.2 μg / mL) at ambient temperature for 1 hour. After blocking for 1 hour with 200 μL / well of 1×PBS / 2% BSA, the plates were washed three times with 1×PBST. The hybridoma supernatant was added to the plates at a volume of 100 μL / well and incubated at ambient temperature for 2 hours. After washing the plates three times with 1×PBST, goat anti-mouse IgG-Fc HRP (Bethyl, A90-231P) was added to the plates at 100 μL / well and incubated at ambient temperature for 1 hour. After washing six times, the TMB substrate was added to the plates at 100 μL / well for color development, left for 5 - 10 minutes, and then the reaction was stopped by adding 100 μL / well of 2M HCl. The absorbance at 450 nm was read using a microplate reader.

[0176] The positive cell lines selected by FACS screening were further confirmed by SPR (surface plasmon resonance). SPR enables real-time label-free detection of biomolecular interactions. SPR occurs when polarized light hits a conductive surface at the interface of two media. This generates an electron charge density wave called a plasmon, and the intensity of the reflected light at a specific angle known as the resonance angle decreases in proportion to the mass on the sensor surface. The binding of the supernatant to human 4IgB7H3 was ranked using a Biacore 8K. An activator was prepared by mixing 400 mM of EDC and 100 mM of NHS (manufactured by GE) and immediately injected into the channel. The CM5 sensor chip was activated with the activator for 420 seconds. Next, goat anti-mouse Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL per minute for 420 seconds. The chip was inactivated with 1 M ethanolamine-HCl. The supernatant was injected into the channel at a flow rate of 10 μL per minute for 30 seconds. As a binding phase, 200 nM of human 4IgB7H3 analyte was injected into the channel at a flow rate of 30 μL per minute for 120 seconds and then dissociated over 300 seconds. After the dissociation phase, glycine (10 mM, pH 1.5) was injected as a regeneration buffer. The sensorgrams for the reference channel and the buffer channel were subtracted from the test sensorgram. The data was then fitted using a 1:1 Langmuir binding model.

[0177] The pHrodo iFL dye can be conjugated to biomolecules such as antibodies and emits high fluorescence only when present in an acidic environment such as those in cell lysosomes and endosomes. This enables the detection of conjugated biomolecules in live cell assays. AffiniPure F(ab’)2 fragment goat anti-human IgG Fcγ and anti-mouse IgG Fcγ were labeled with the pHrodo iFL Red STP ester amine-reactive dye and purified using a Zeba Spin desalting column according to the manufacturer's instructions. MCF-7 cells expressing human full-length B7H3 (2 × 10 cells per well 4Cells (8 μg / mL of poly-D-lysine-coated 96-well clear-bottom black plates were seeded. The cells were grown overnight in an incubator set at 37 °C and 5% CO2. The next day, the supernatant from the WBP301088-1 positive cell line was added to the corresponding wells in a volume of 100 μL. The cells were incubated for 0.5 h in an incubator set at 37 °C and 5% CO2. Human and mouse IgG1 isotype antibodies were used as negative controls. 50 nM of pHrodo iFL Red / F(ab’)2 goat anti-mouse or anti-human IgG Fcγ conjugate (diluted in complete medium) was added in a volume of 100 μL / well. The plate was then incubated in the dark for 5 h in an incubator set at 37 °C and 5% CO2. After incubation, the supernatant was discarded. The plate was washed with complete medium. A mixed dye solution (1 μg / mL of Hoechst 33342 and 0.5 μg / mL of calcein AM diluted in DPBS) was added to the plate in a volume of 100 μL / well and maintained at ambient temperature for 15 min. After incubation, the dye solution was discarded and the plate was washed once with 1× PBS / 1% BSA. The fluorescence intensity of MCF-7 cells was read and analyzed by a Perkin Elmer Operetta CLS high-content analysis system.)

[0178] A total of 8460 wells were tested in the primary screening, 279 hybridomas were selected and expanded in 24-well plates. In the secondary screening, the supernatant from the 24-well plates was used to confirm strong binding to human 4IgB7H3 and cynomolgus B7H3, weak binding to human 2IgB7H3, and strong internalization activity in MCF-7. Based on the results of the secondary screening, the WBP301088-1.145 hybridoma was selected for subcloning.)

[0179] 1.2.4. Antibody Subcloning The positive hybridoma cells selected in Examples 1, 2, and 3 were used for subcloning. Cells in the logarithmic growth phase were counted and added to 1.5 mL of semi-solid HAT medium. The cells were gently mixed for 5 - 10 seconds with a vortex mixer and then seeded into a 6-well plate (manufactured by CORNING). The plate was maintained in an incubator set at 37°C and 5% CO2 for 7 - 8 days. Each visible single colony was picked and placed into a 96-well plate (manufactured by CORNING) with DMEM medium supplemented with 10% FBS. After 2 - 3 days, the cell supernatants were collected and screened by cell-based ELISA (human 4IgB7H3) and conventional ELISA (human 2IgB7H3) using the above antibody screening protocol. After screening for confirmation of antibody subclones by FACS using cells expressing human 4IgB7H3 and cells expressing cynomolgus monkey B7H3, the culture supernatants of the selected single positive clones were collected and the antibodies were purified for further characterization. After subclone screening and confirmation screening, the W301088-1.145.16 hybridoma clone was obtained.

[0180] 1.2.5. Antibody Isotyping Isotyping by ELISA was performed using a 96-well high-binding plate (manufactured by Nunc). Each well was coated with 2 μg / mL of capture antibodies, goat anti-mouse IgG1 (manufactured by Bethyl, A90-205A), goat anti-mouse IgG2a (manufactured by Bethyl, A90-207A), goat anti-mouse IgG2b (manufactured by Bethyl, A90-209A), or goat anti-mouse IgG3 (manufactured by Bethyl, A90-211A) at 4 °C overnight, and blocked with 1× PBS / 2% BSA. The hybridoma supernatant of Example 1.2.4 was incubated in the coated wells, and the specific binding of the antibody to the capture antibody immobilized on the plate was measured using a goat anti-mouse κ / λ light chain antibody conjugated with peroxidase. The HRP signal was detected by adding the TMB substrate, and the reaction was stopped with 2 M HCl after 12 minutes. All incubation steps were carried out at ambient temperature, and the plate was washed with PBS between steps. The absorbance at 450 nm was read using a microplate reader (manufactured by Molecular Device).

[0181] The results of the isotyping of the W301088-1.145.16 clone are shown in the following table (Table 4).

Table 7

[0182] 1.2.6. Antibody production When a small amount of purified antibody is required, a T75 flask (manufactured by CORNING) is generally used to scale up the hybridoma cell culture. Monoclonal hybridoma cells were seeded into the flasks at 5×10 6 ~1×10 7 cells per T75 flask. The cells were continuously grown for about 7 to 10 days until the cell viability reached about 30% to 40%. The culture supernatant was collected, and cell debris was removed by centrifugation. The supernatant was sterile filtered and stored for antibody purification.

[0183] 1.2.7. Hybridoma sequencing First, total RNA of the W-301088-1.145.16 hybridoma cell sample was extracted according to the instructions of the TaKaRa MiniBEST Universal RNA Extraction Kit. Subsequently, the RNA was converted to cDNA using the SMART RACE cDNA Amplification Kit manufactured by Clonetech. Then, the DNA sequences of the VH domain and VL domain were amplified from the cDNA by 30 cycles of PCR. Each cycle involved denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and then extension at 72°C for 30 seconds. Next, the PCR products were subcloned into a TA-cloning vector and sent to Biosune Biotech (Shanghai, China) for sequencing.

[0184] Once the monoclonal nature of the hybridoma cell sample was confirmed from the sequencing data, the DNA sequences of the VH domain and VL domain were amplified by PCR. The primers were synthesized by Sangon Biotech (Shanghai, China). Then, the DNA segments were subcloned into the pcDNA3.4 expression vector together with the human IgG1 constant region with the LALA mutation and then sequenced at Tsingke Biotechnology (Beijing, China).

[0185] 1.2.8. IgG Conversion Once the monoclonal nature of the hybridoma cell sample was confirmed from the sequencing data, the nucleotide sequences encoding the amino acid sequences of the VH domain and VL domain were codon-optimized for mammalian expression and then the codon-optimized nucleotide sequences were synthesized by Sangon Biotech (Shanghai, China). Then, the DNA segments were subcloned into the pcDNA3.4 expression vector together with the human IgG1 constant region with the L234A / L235A (LALA) mutation.

[0186] Thus, the antibody produced by the W301088-1.145.16 clone was converted into a human IgG1 antibody with the LALA mutant form, thereby obtaining a chimeric antibody designated as antibody W301088-1.145.16-xIgG1KV320. After protein A purification and buffer exchange, this chimeric antibody was characterized by SDS-PAGE and SEC-HPLC. The protein migrated on SDS-PAGE at an apparent molecular weight of 50 kDa and 25 kDa under reducing conditions. These correspond to the IgG heavy chain and light chain, respectively. The purity was higher than 99% (SEC-HPLC).

Table 8

[0187] 1.2.9. Humanization of Antibodies A local copy of the open-source software ANARCI (Dunbar J and Deane CM, "ANARCI: antigen receptor numbering and receptor classification", Bioinformatics, 2015; 32(5): 298-300) was run to assign Kabat numbering to the W301088-1.145.16 antibody. Table 6 shows the identified CDRs according to the definition on Dr. Martin's website (Martin, 2018, supra).

Table 9

Table 10

Table 11

[0188] The VH domain sequence and VL domain sequence of the mouse antibody W301088-1.145.16 were each aligned against the human germline sequence libraries of the VH domain and VL domain of IMGT. The human germline sequences of the VH / VL domains with the minimum number of amino acid differences in the framework compared to the VH / VL domain sequences of the W301088-1.145.16 antibody were selected as the humanized templates for the VH / VL domains. The combinations of IGHV4-31*02 and IGHJ6*01, and IGKV4-1*01 and IGKJ4*01 were the human germline sequences with the highest homology to the VH domain sequence and VL domain sequence of the W301088-1.145.16 antibody, respectively. The CDRs of the W301088-1.145.16 antibody were transplanted into the frameworks of these two human germline templates to construct germline sequences.

[0189] Several "back mutation" positions within the framework were empirically selected to convert the amino acids of the germline sequences to their amino acid counterparts of the original mouse sequences. A series of humanized variants were empirically designed to investigate different combinations of these selected back mutation sites. The germline sequences differed from the original mouse W301088-1.145.16 antibody sequence at positions 16 of the VL domain and 20 of the VH domain. Out of a total of 36 different sites, 14 sites were considered back mutations: VH: Q001D, S025T, H040F, K043N, G044K, I048M, V067I, V071R, A085E, V089T, and Y091F; VL: I021M, P043S, and Y087F. A series of humanized variants were empirically designed to test different combinations of these three back mutations.

[0190] 1.2.10. PTM Removal The VH domain sequences and VL domain sequences of the humanized variants were scanned for several types of extremely important post-translational modification (PTM) sites: asparagine deamidation (N-G and N-S) in the CDRs, aspartic acid isomerization (D-G) in the CDRs, unpaired C throughout the length, and N-linked glycosylation sites (N-X-S / T, where X can be any amino acid other than P) throughout the length. Empirical point mutations were designed to avoid potential risks of PTM modification, removing these extremely important PTM sites in the humanized variants. Finally, PTM-removed antibodies with no effect on expression, binding, and thermal stability compared to the parental antibody were selected.

[0191] Considering that the VL domain sequence of the mouse antibody W301088-1.145.16 contains an N-S-S N-linked glycosylation site, three point mutations in VL: N027dQ, VL:S027eP, and VL:S027fA (numbering according to Kabat) were designed to remove the extremely important PTM sites. These were further characterized by ranking in combination with the humanized variants. off Characterized further by ranking.

[0192] 1.2.11. Ranking of k by SPR off Ranking k of the filtered supernatant offRanking was performed using a Biacore 8K surface plasmon resonance (SPR) instrument. First, the CM5 sensor chip was activated using 400 mM EDC and 100 mM NHS (from GE) at a flow rate of 10 μL per minute for 420 seconds. Next, 30 μg / mL anti-human Fc IgG (from Jackson) in 10 mM NaAc (pH 4.5) was injected into channels 1 - 8 at a flow rate of 10 μL per minute for 420 seconds. Next, the chip was deactivated with 1 M ethanolamine-HCl (from GE) at a flow rate of 10 μL per minute for 420 seconds. The diluted antibody supernatant was injected into Fc2 of the channel at a flow rate of 10 μL per minute. The supernatant of cell culture without plasmid transfection was used as a negative control and injected into Fc1 of the channel at a flow rate of 10 μL per minute. 100 nM human 4IgB7H3 and running buffer were sequentially injected into Fc1 - Fc2 of the channel at a flow rate of 30 μL per minute as the binding phase for 180 seconds and then as the dissociation phase for 3600 seconds. After each flow-through, 10 mM glycine (pH 1.5) was injected to regenerate the chip. Sensorgrams were analyzed using the software sold as a set with the Biacore 8K instrument. The sensorgrams for the reference channel and buffer channel were subtracted from the sensorgram of the sample. Then, the data was fitted using a 1:1 Langmuir binding model.

[0193] 1.2.12. Combination of PTM removal mutations and humanized variants The best k in Example 1.2.11 off The PTM removal point mutations indicating speed were combined with humanized variants having the optimal k off speed by site-directed mutagenesis to construct the final construct for affinity verification.

[0194] k off According to the speed, the PTM removal mutations of VL:N027dQ were combined with four selected humanized mutant amino acids S025T, V071R, Y091F, and V089T to constitute the final antibody. The final antibody was named W301088-1.145.16-z3-p1-uIgG1KV320.

Table 12

[0195] Figures 12A to 12B show the results of amino acid sequence alignment of the VH and VL regions between antibody W301088-1.145.16-xIgG1KV320 and W301088-1.145.16-z3-p1-uIgG1KV320. The amino acid segments enclosed by the frames in these figures are the respective CDR regions within the VH and VL regions.

[0196] 1.2.13. Transient transfection and purification at production scale Plasmid at a final concentration of 1 μg / mL was transiently transfected into 20 mL of Expi293F cells at a cell density of 3.0×10 6 cells per mL and a cell viability of over 95% using the ExpiFectamine™ Transient Transfection Kit. The cell culture was grown in a humidified platform shaker with a rotation speed of 150 rpm. The temperature was maintained at 37°C and the CO2 level was maintained at 8%.

[0197] After incubating the cell culture for 5 days, the supernatant expressing the target antibody was collected, filtered, and purified using a GE MabSelect SuRE Protein A column (Cytiva-175438). The eluted antibody was dialyzed using a D-Tube Dialyzer Maxi (EMD Millipore 71508, MWCO 3.5 kDa) and placed in PBS. The antibody concentration was determined at an absorbance of 280 nm using a NanoDrop device. 2 μg of the purified antibody sample was electrophoresed on an SDS-PAGE gel (Invitrogen NuPAGE™ 4%–12% Bis-Tris protein gel) with and without a reducing agent. The purity of the purified antibody sample was quantified by HPLC-SEC using a TSKgel G3000 SWXL size exclusion chromatography column (Tosoh 008541). The purified antibody was stored at -80°C.

[0198] 1.3. In Vitro Characterization of Antibodies 1.3.1. Purity Detection by SDS-PAGE and SEC-HPLC The purified antibody sample of Example 1.2 was mixed with loading buffer and heated at 75 °C for 10 minutes using a drying oven. After loading the sample, SDS-PAGE was run at a constant voltage (200 V) for 35 minutes. The gel was stained and decolorized using eStain™ L1. The results of SDS-PAGE were obtained using a BIO-RAD imaging system.

[0199] The results are summarized in Table 10 below. The protein migrated on SDS-PAGE at an apparent molecular weight of 50 kDa and 25 kDa under reducing conditions. These correspond to the IgG heavy chain and light chain, respectively. On the other hand, under non-reducing conditions, it migrated to the expected ~150 kDa band (Figure 1).

[0200] 1.3.2. Purity Detection by SEC-HPLC The purity of the antibody of Example 1.2 was detected by an Agilent 1260 Infinity II system (manufactured by Agilent Technologics™) equipped with a TSKgel G3000 SWXL column (manufactured by Tosoh Bioscience - 0008541). An appropriate volume (5 - 100 μL) of the antibody sample was injected into the column and separated for 20 minutes at a flow rate of 1 mL per minute. The running buffer was 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. Peak retention was detected using UV light at a wavelength of 280 nm. The purity of the antibody was analyzed by integrating the areas of all peaks from 4.5 minutes to 10.5 minutes using SEC-HPLC analysis. The operating and analysis software was OpenLab CDS workstation (v2.3.0.443 or v2.2.0.484).

[0201] The results are summarized in Table 10 below. The purity of the W301088-z3-p1 antibody detected by SEC-HPLC was higher than 99% (Figure 2).

[0202] 1.3.3. Thermal stability (T) determined by DSF m Determination The T m (melting temperature) of each antibody was investigated using a QuantStudio (registered trademark) 7 Flex real-time PCR system (manufactured by Applied Biosystems). 19 μL of the antibody solution of Example 1.2 was mixed with 1 μL of SYPRO Orange solution (manufactured by Invitrogen) and transferred to a 96-well plate (manufactured by Applied Biosystems). The plate was sealed with an optical adhesive film (manufactured by Applied Biosystems) and centrifuged at 3,000 rpm for 5 minutes to remove any air bubbles. The plate was heated from 26°C to 95°C at a rate of 0.9°C per minute, and the resulting fluorescence data was collected. The negative derivative of the fluorescence change was calculated for different temperatures, and the maximum value was defined as the melting temperature T m . When the protein has multiple unfolding transitions, the first T m was recorded and designated as T m 1. Data collection and T m calculation were automatically performed by QuantStudio (trademark) real-time PCR software (v1.3).

[0203] The T m value of the W301088-1.145.16-z3-p1-uIgG1KV320 antibody was defined as the maximum value of the negative derivative of the fluorescence change with respect to different temperatures as listed in Table 10 below. The data profile is shown in Figure 3, and the dashed line in the figure indicates the position of the T m 1 value in the fluorescence curve. The T m 1 of the W301088-1.145.16-z3-p1-uIgG1KV320 antibody is 70.1°C, indicating that it has good thermal stability.

[0204] 1.3.4. Detection of hydrophobicity by HIC-HPLC The hydrophobicity of the antibody of Example 1.2 was detected by an HPLC 1260 infinity II system (manufactured by Agilent Technologics (trademark)) using a TSKgel butyl-NPR column (manufactured by Tosoh - 0042168). Each antibody sample was diluted to 0.5 mg / mL using PBS buffer, 20 μL of the diluted sample was injected into the column, and separation was performed for 61 minutes at a flow rate of 0.5 mL per minute. The running buffers were 25 mM sodium phosphate, pH 7.0 (buffer A) and 25 mM sodium phosphate, 1.5 M (NH4)2SO4, pH 7.0 (buffer D). The running gradient from 3 minutes to 53 minutes was 0% to 100% of buffer D. Peak retention was detected using UV light at wavelengths 280 nm and 230 nm. The retention time was analyzed by integrating all peak areas from 20 minutes to 40 minutes using HIC-HPLC analysis. The operation and analysis software was OpenLab CDS workstation (v2.6.0.691).

[0205] The HIC retention time of the W301088-1.145.16-z3-p1-uIgG1KV320 antibody was 23.52 minutes (Table 10 and Figure 4). Therefore, it is shown that the antibody has good hydrophilicity.

[0206] 1.3.5. Determination of diffusion interaction parameter (k D ) by DLS DLS-k DThe measured values were investigated using a DynaPro Plate Reader III (manufactured by Wyatt Technology). During the preparation of the antibody samples, the appearance of the samples was recorded upon thawing, filtration, and concentration. The antibody samples were concentrated to over 20 mg / mL and then diluted with PBS buffer to final concentrations of 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL. Next, 7.5 μL of the sample solution was added to a 1536-well microplate (manufactured by Aurora - ABI1 - 00110A). A ClearSeal film (manufactured by Hampton Research - HR4 - 521) was added to the plate and centrifuged at 3,000 rpm for 5 minutes to drop the sample to the bottom of the well. Each sample was tested in two duplicate wells. The plate was placed in the corresponding position, and data collection was performed by Dynamics Operation Software (v7.8.1.3). Five imaging images were collected for each protein sample, and each imaging time was 5 seconds. For each measurement, the diffusion coefficient was determined and plotted against the protein concentration. k D The value was automatically calculated by the software (v7.8.1.3). The results are shown in Table 10. The data profile is shown in Figure 5. The k D of the W301088 - 1.145.16 - z3 - p1 - uIgG1KV320 antibody in PBS was -5.17 mL / g, indicating that the antibody is an antibody with high solubility. The appearance of the antibody protein during the assay was also recorded, and some particles were observed after thawing and gently shaking. When the buffer was exchanged to 20 mM His, 8% sucrose, 0.02% PS80, pH 6.5, no particles were observed.

[0207] The above results of SDS - PAGE, SEC - HPLC, HIC - HPLC, DSF, and DLS are summarized in Table 10.

Table 13

[0208] 1.3.6. Binding to Human B7H3 (FACS) FACS was used to detect the binding of the antibody to human B7H3. This method enables quantitative analysis and identification of specific molecules expressed on the surface of live cells. Unlabeled cells were used as a control for setting the threshold before the assay, and the percentage change for each group above the fluorescence intensity threshold was analyzed. MCF-7 cells expressing full-length human B7H3 (1×10 5 cells per well) were incubated for 1 hour at 4°C in a refrigerator set at 4°C in a volume of 100 μL / well with various concentrations of the antibody (serially diluted 3.16-fold from 50 nM to 0.0005 nM using 1×DPBS / 1% BSA). The anti-human B7H3 reference antibody enoblituzumab (manufactured by MacroGeneics) was used as a positive control. A human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×DPBS / 1% BSA, Alexa 647 goat anti-human antibody (diluted at a ratio of 1:500 in 1×DPBS / 1% BSA) was added. The cells were incubated for 0.5 hour at 4°C in the dark in a refrigerator set at 4°C. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed by FlowJo. The EC 50 value was calculated by four-parameter non-linear regression analysis using GraphPad Prism 7 software.

[0209] The W301088-1.145.16-z3-p1-uIgG1KV320 antibody showed good binding to B7H3-expressing MCF-7 cells, with an EC 50 value of 1.08 nM and a maximum MFI of 19,900, which was equivalent to the chimeric antibody W301088-1.145.16-xIgG1KV320 and better than the reference antibody enoblituzumab (manufactured by MacroGenics) (Table 11, Figure 6).

Table 14

[0210] 1.3.7. Binding to Cynomolgus B7H3 (FACS) Binding of the antibody to cynomolgus B7H3 was detected using FACS. FlpinCHO cells were transfected with cynomolgus B7H3 (W3XX088-FlpinCHO.cProl.pool) (1×10 5 cells per well), and the transfected cells were incubated for 1 hour at 4°C in a refrigerator at a volume of 100 μL / well with antibodies at various concentrations (serially diluted 3.16-fold from 50 nM to 0.0005 nM using 1×DPBS / 1% BSA). The anti-human B7H3 reference antibody enoblituzumab (manufactured by MacroGeneics) was used as a positive control. A human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×DPBS / 1% BSA, Alexa 647 goat anti-human antibody (diluted at a ratio of 1:500 in 1×DPBS / 1% BSA) was added. The cells were incubated for 0.5 hour in the dark in a refrigerator set at 4°C. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed by FlowJo. The EC 50 value was calculated by four-parameter non-linear regression analysis using GraphPad Prism 7 software.

[0211] The W301088-1.145.16-z3-p1-uIgG1KV320 antibody showed good binding to cynomolgus-transfected cells, and the EC 50 value was 4.68 nM, which was equivalent to the chimeric antibody W301088-1.145.16-xIgG1KV320 and better than enoblituzumab (manufactured by MacroGenics) (Table 12, Figure 7).

Table 15

[0212] 1.3.8. Binding affinity (SPR) for human 4IgB7H3 The affinity of the antibody for human 4IgB7H3 was determined using surface plasmon resonance (SPR). The affinity of the antibody for human 4IgB7H3 was determined by Biacore 8K. An activating agent was prepared by mixing 400 mM of EDC and 100 mM of NHS (manufactured by GE), and immediately injected into the channel. The CM5 sensor chip was activated with the activating agent for 420 seconds. Next, goat anti-human Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL per minute for 420 seconds. The chip was inactivated with 1 M ethanolamine-hydrochloric acid. The antibody diluted in running buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10 μL per minute for 30 seconds. Analytes of seven concentrations (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 2.125 nM, and 1.563 nM) of human 4IgB7H3 were sequentially injected into the channel at a flow rate of 30 μL per minute for 180 seconds, and then dissociated over 3600 seconds. Glycine (10 mM, pH 1.5) was injected as a regeneration buffer after the dissociation phase.

[0213] The sensorgrams for the reference channel and the buffer channel were subtracted from the test sensorgram. The experimental data were fitted using a 1:1 binding model. The molecular weight of human 4IgB7H3 used in the calculation was 47.8 kDa.

[0214] The experimental data were fitted by the 1:1 binding model sold in a set with Biacore 8K evaluation software. As shown in Table 13 and Figures 8A - 8C, the W301088-1.145.16-z3-p1-uIgG1KV320 antibody showed high binding affinity for human 4IgB7H3. The K D value was 7.08×10 -11 M, which was better than the K D value of enobrutuzumab (manufactured by MacroGenics).

Table 16

[0215] 1.3.9. Binding Affinity (SPR) for Human 2IgB7H3 The affinity of the antibody for human 2IgB7H3 was determined using surface plasmon resonance (SPR). The affinity of the antibody for human 2IgB7H3 was determined by Biacore 8K. The activator was prepared by mixing 400 mM of EDC and 100 mM of NHS (manufactured by GE) and immediately injected into the channel. The CM5 sensor chip was activated with the activator for 420 seconds. Then, goat anti-human Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL per minute for 420 seconds. The chip was inactivated with 1 M ethanolamine-hydrochloric acid. The antibody diluted in the running buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10 μL per minute for 30 seconds. Analytes of seven concentrations (500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, and 7.813 nM), human 2IgB7H3, were sequentially injected into the channel at a flow rate of 30 μL per minute for 120 seconds and then dissociated for 300 seconds. Glycine (10 mM, pH 1.5) was injected as the regeneration buffer after the dissociation phase.

[0216] The sensorgrams for the reference channel and the buffer channel were subtracted from the test sensorgram. The experimental data were fitted using a 1:1 binding model. The molecular weight of human 2IgB7H3 used in the calculation was 25.2 kDa.

[0217] The experimental data were fitted by the 1:1 binding model sold in set with the Biacore 8K evaluation software. As shown in Table 14 and Figures 9A - 9C, the W301088-1.145.16-z3-p1-uIgG1KV320 antibody showed a weak binding affinity for human 2IgB7H3 compared to the binding to human 4IgB7H3. The K D value was 4.64×10 -08 M.

Table 17

[0218] 1.3.10. Binding Affinity (SPR) of the Antibody to Cynomolgus Monkey B7H3 The affinity of the antibody for cynomolgus monkey B7H3 was determined using surface plasmon resonance (SPR). The affinity of the antibody for cynomolgus monkey B7H3 was determined by Biacore 8K. An activating agent was prepared by mixing 400 mM of EDC and 100 mM of NHS (manufactured by GE), and immediately injected into the channel. The CM5 sensor chip was activated with the activating agent for 420 seconds. Then, goat anti-human Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL per minute for 420 seconds. The chip was inactivated with 1 M ethanolamine-hydrochloric acid. The antibody diluted in running buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10 μL per minute for 30 seconds. Analytes of nine concentrations (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.563 nM, 0.781 nM, 0.391 nM, and 0.196 nM), cynomolgus monkey B7H3, were sequentially injected into the channel at a flow rate of 30 μL per minute for 180 seconds, and then dissociated over 3600 seconds. After the dissociation phase, glycine (10 mM, pH 1.5) was injected as the regeneration buffer.

[0219] The sensorgrams for the reference channel and the buffer channel were subtracted from the test sensorgram. The experimental data for six analyte concentrations (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.563 nM) were fitted using a 1:1 binding model. The molecular weight of cynomolgus monkey B7H3 used in the calculation was 49 kDa.

[0220] The experimental data were fitted by the 1:1 binding model sold as a set with the Biacore 8K evaluation software. As shown in Table 15 and Figures 10A - 10C, the W301088-1.145.16-z3-p1-uIgG1KV320 antibody showed high binding affinity for cynomolgus monkey B7H3, which was equivalent to the binding of the antibody to human 4IgB7H3. K DThe value was 5.74×10 -11 M, which was better than the K D value of enoblituzumab (manufactured by MacroGenics).

Table 18

[0221] 1.3.11. Internal translocation test (HCS) Antibody internal translocation was determined by operetta CLS (manufactured by PerkinElmer), a high-content imaging and analysis system capable of rapidly and sensitively collecting and analyzing images of samples. MCF-7 cells expressing human full-length B7H3 (1.5×10 cells per well) 4(cells) were seeded into a 96-well clear-bottom black plate pre-coated with 8 μg / mL of poly-D-lysine. After incubating overnight in an incubator set at 37 °C and 5% CO₂, various concentrations of antibodies (serially diluted 3.16-fold from 50 nM to 0.0005 nM using 1×DPBS / 1% BSA) were incubated at 4 °C for 2 hours in a refrigerator at a volume of 100 μL / well. The anti-human B7H3 reference antibody enoblituzumab (manufactured by MacroGeneics) was used as a positive control. A human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×DPBS / 1% BSA, Alexa 647 goat anti-human antibody (diluted at a ratio of 1:500 in 1×DPBS / 1% BSA) was added. The cells were incubated in the dark for 1 hour in a refrigerator set at 4 °C. Subsequently, the cells were washed once and resuspended in 1×DPBS / 1% BSA at 37 °C for 2 hours. After incubation, the supernatant was discarded, and Hoechst 33342 (diluted at a ratio of 1:5000 in 1×DPBS) was added at 100 μL / well and incubated at ambient temperature for 15 minutes. After washing the cells with 1×DPBS, an acid wash buffer (100 mM glycine, 150 mM NaCl, pH 2.5) was added at 100 μL / well and incubated in a refrigerator set at 4 °C for 5 minutes. The cells were washed once with 1×DPBS and fixed using 60 μL of 4% PFA. The mean fluorescence intensity (MFI) of the cells was measured and analyzed by operetta CLS. EC 50 values were calculated by four-parameter non-linear regression analysis using GraphPad Prism 7 software.

[0222] The W301088-1.145.16-z3-p1-uIgG1KV320 antibody was shown to internalize with an EC 50 value of 0.51 nM in B7H3-expressing MCF-7 cells. Therefore, it exhibited good internalization ability, which was equivalent to that of the chimeric antibody W301088-1.145.16-xIgG1KV320 and better than that of the reference antibody enoblituzumab (manufactured by MacroGenics) (Table 16, Figure 11).

Table 19

Example

[0223] Preparation of Antibody-Drug Conjugate (ADC) and Binding Activity against B7H3 Antigen In this example, the anti-B7H3 antibody used in the antibody-drug conjugate was W301088-1.145.16-z3-p1-uIgG1KV320 (abbreviated as WBP301088) or W301088-1.145.16-xIgG1KV320. W301088-1.145.16-z3-p1-uIgG1KV320 (WBP301088) contains the heavy chain amino acid sequence shown in SEQ ID NO: 12 and the light chain amino acid sequence shown in SEQ ID NO: 13.

[0224] 2.1. Preparation of Linker-Cytotoxin (Linker-Payload) 2.1.1. Preparation of Linker-Payload X1

Chemical Structure

[0225] The protocol for preparing Linker-Payload X1 was as follows:

Chemical Structure

[0226] Step 1 Benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a solution of 27a (5.00 g, 43.0 mmol) and NaHCO3 (10.9 g, 129 mmol) in DMF (50 mL) under nitrogen, and the mixture was reacted at 25 °C for 17 hours. After the reaction was completed as indicated by TLC (PE / EA = 2 / 1) (where PE is the abbreviation for petroleum ether and EA is the abbreviation for ethyl acetate), the reaction mixture was added to water (500 mL) and extracted twice with EA (250 mL). The organic phase was separated, washed with a saturated aqueous sodium chloride solution (500 mL), dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (PE:EA = 3:2) to obtain a colorless liquid (5.1 g, yield 57.1%).

[0227] Step 2 A solution of 27b (4.50 g, 21.8 mmol) in THF (10 mL) was added dropwise to a solution of KI2 (4.00 g, 10.9 mmol) and TsOH (800 mg, 4.65 mmol) in THF (30 mL) at 0 °C under nitrogen, and the mixture was reacted at 25 °C for 2 hours. After the reaction was completed as indicated by TLC (PE / EA = 1 / 2), the reaction mixture was added to water (200 mL) and extracted twice with EA (200 mL). The organic phase was separated, dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (PE / EA = 3 / 2) to obtain a white solid (1.56 g, yield 26%).

[0228] Step 3 Pd / C (80 mg) was added to a solution of 27c (800 mg, 1.55 mmol) in a mixture of EtOH (8 mL) and EA (8 mL) at 0 °C under hydrogen, and the resulting mixture was stirred at 0 °C for 2.5 hours. After the reaction was completed as indicated by LCMS, the reaction mixture was filtered through celite, and the filter cake was washed with EA (200 mL). The filtrate was concentrated, dissolved in THF (20 mL), and the resulting solution was concentrated to dryness by rotary evaporation to obtain a white solid (600 mg, yield 91%).

[0229] Step 4 DIEA (152 mg, 1.18 mmol) was added to a solution of 27d (220 mg, 0.515 mmol), KI4 (250 mg, 0.47 mmol) and HATU (214 mg, 0.56 mmol) in DMF (6 mL) at 0 °C under nitrogen, and the mixture was reacted at 0 °C for 2 hours. After the reaction was completed as indicated by LCMS, the reaction mixture was added to an aqueous citric acid solution (pH = 4) (150 mL), and the resulting mixture was filtered. The filter cake was washed with water (175 mL), dewatered, and dried with an oil pump to obtain a brown solid (260 mg, yield 66%).

[0230] Step 5 Diethylamine (8 mL) was added dropwise to a solution of 27e (260 mg, 0.309 mmol) in dichloromethane (DCM) (30 mL) at 0 °C under nitrogen, and the mixture was reacted at 0 °C for 3 hours. After the reaction was completed as indicated by LCMS, the reaction mixture was added to a petroleum ether solution (600 mL) at 0 °C to precipitate a solid. The mixture was allowed to stand until the solid adhered to the bottom of the flask, and the solution was decanted. The residue was dried with an oil pump to obtain a brown solid (90 mg, yield 47.1%).

[0231] Step 6 HATU (74 mg, 0.19 mmol) was added to a solution of 27f (90 mg, 0.13 mmol), KI-1 (92 mg, 0.19 mmol) and DIEA (50 mg, 0.39 mmol) in DMF (2.5 mL) at 0 °C under nitrogen, and the mixture was reacted at 0 °C for 2 hours. After the reaction was completed as detected by LCMS, the reaction mixture was added to an aqueous citric acid solution (pH 4) (30 mL) at 0 °C, whereby a flocculent solid precipitated. The solid was collected by filtration and purified by preparative thin-layer chromatography (DCM / MecOH = 10 / 1) to obtain X1 as a pale yellow solid (9.2 mg, yield 6%).

[0232] MS m / z (ESI): 1074 [M+1] H-NMR (400 MHz, MeOD): 7.65 (d, 1H), 7.62 (s, 1H), 7.30 - 7.21 (m, 5H), 6.79 (s, 2H), 5.69 - 5.65 (m, 1H), 5.57 (d, 1H), 5.43 - 5.10 (m, 3H), 4.70 (d, 2H), 4.48 - 4.39 (m, 2H), 4.10 - 4.05 (m, 1H), 4.01 - 3.75 (m, 5H), 3.46 (t, 2H), 3.22 - 3.15 (m, 2H), 3.07 - 3.00 (m, 1H), 2.75 (m, 1H), 2.62 (m, 1H), 2.45 (s, 3H), 2.37 - 2.20 (m, 6H), 2.10 - 2.02 (m, 2H), 2.00 - 1.92 (m, 2H), 1.68 - 1.57 (m, 6H), 1.01 (t, 3H)

[0233] 2.1.2. Preparation of Linker-Payload X2

Chemical formula

[0234] The protocol for preparing Linker-Payload X2 was as follows:

Chemical formula

[0235] Step 1 34a (5 g, 48.0 mmol) and K2CO3 (19.9 g, 144.0 mmol) were dissolved in DMF (20 mL), and the resulting solution was added dropwise to benzyl bromide (12.3 g, 72.0 mmol). The mixture was reacted at 25 °C for 17 hours. After the starting material was completely consumed as detected by TLC (PE / EA = 3 / 1), the reaction mixture was added to water (200 mL) and extracted with EA (250 mL). The organic phase was separated, washed with saturated NaCl, dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (PE:EA = 2:1) to obtain 34b as a colorless liquid (8.7 g, yield 93%). MS-ESI: m / z 195.1 [M + H]+.

[0236] Step 2 43c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) were dissolved in THF (20 mL), and the resulting solution was cooled to 0 °C under nitrogen. A solution of 34b (7.7 g, 39.6 mmol) in THF (10 mL) was added dropwise. After the addition, the mixture was reacted at 0 °C for 2 hours. After most of the starting materials were consumed as indicated by TLC (PE / EA = 2 / 1), the reaction mixture was poured into water (100 mL) and extracted with DCM (100 mL). The organic phase was separated, washed with saturated NaCl, dried over anhydrous Na2SO4, and purified by column chromatography (PE / EA = 1 / 1) to obtain 34d as a colorless sticky substance (3.9 g, yield 39%). MS-ESI: m / z 503.3 [M+H]+.

[0237] Step 3 Pd / C (1 g, 10 wt.%) was added to a solution of 34d (1.9 g, 3.78 mmol) in a mixture of EtOH (100 mL) and EA (100 mL) at 0 °C under hydrogen, and the resulting mixture was reacted at 0 °C for 3 hours. After the reaction was completed as indicated by TLC (PE / EA = 2 / 1), the reaction mixture was filtered through celite, and the filter cake was washed with EA / EtOH (1:1, 100 mL × 3). The filtrate was concentrated. The residue was dissolved in THF (50 mL × 3), and the resulting solution was concentrated to dryness by rotary evaporation. This procedure was repeated three times to obtain 34e as a gray solid (1 g, yield 64%). MS-ESI: M / z 435.2 [M+Na]+.

[0238] Step 4 DIEA (303 mg, 2.35 mmol) was added dropwise to a solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol) and HATU (429 mg, 1.13 mmol) in DMF (20 mL) at 0 °C under nitrogen. After the addition, the mixture was reacted at 0 °C for 2 hours. After completion of the reaction as indicated by LCMS, the reaction mixture was added dropwise to water (300 mL). The resulting mixture was stirred, then left standing for 5 minutes, filtered, and the filter cake was dissolved in a DCM / MeOH (10:1, 100 mL) solution. The resulting solution was dried and concentrated to dryness by rotary evaporation. The residue was mixed with silica gel and purified by column chromatography (EA:MeOH = 30:1) to obtain 34f as a yellow solid (600 mg, yield 77%). MS-ESI: M / z 830.3 [M+H]+.

[0239] Step 5 Diethylamine (5 mL) was added dropwise to a solution of 34f (150 mg, 0.18 mmol) in DCM (5 mL) at 0 °C under nitrogen. The mixture was reacted at 0 °C for 2 hours. After completion of the reaction as indicated by LCMS, a petroleum ether solution (100 mL × 6) was added to the reaction mixture to precipitate the solid. The resulting mixture was left standing until the solid settled, and the solution was decanted. The residue was dried with an oil pump to obtain 34g as a white powder (120 mg, yield 76%). The product content was determined by LCMS to be 70%. MS-ESI: M / z 608.3 [M+H]+.

[0240] Step 6 A solution of HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to a solution of 34g (60 mg, 0.099 mmol), 43h (51 mg, 0.108 mmol) and DIEA (32 mg, 0.25 mmol) in DMF (1 mL) at 0 °C under nitrogen. The mixture was reacted at 0 °C for 2 hours. After the starting materials were completely consumed as indicated by LCMS, the reaction mixture was directly purified by reverse-phase column chromatography (eluent: (MeCN / MeOH = 1 / 1):H2O = 60%:40%) to obtain X2 as a yellow solid (14.8 mg, yield 14%).

[0241] MS-ESI: m / z 1062.4 [M+H]+。 1H NMR (400 MHz, methanol-d4) δ 7.69~7.61 (m, 2H), 7.22~7.16 (m, 2H), 7.16~7.09 (m, 3H), 6.76 (s, 2H), 5.70~5.64 (m, 1H), 5.60 (d, J = 16.4 Hz, 1H), 5.40~5.31 (m, 2H), 5.26 (d, J = 19.0 Hz, 1H), 4.65~4.50 (m, 7H), 4.25~4.16 (m, 1H), 3.87 (d, J = 16.7 Hz, 1H), 3.83~3.76 (m, 3H), 3.72 (d, J = 17.0 Hz, 2H), 3.44 (t, J = 7.1 Hz, 2H), 3.25~3.17 (m, 2H), 3.10~3.02 (m, 1H), 2.92~2.83 (m, 1H), 2.45~2.39 (m, 5H), 2.32~2.20 (m, 5H), 1.97~1.89 (m, 2H), 1.63~1.50 (m, 4H), 1.34~1.20 (m, 6H), 0.99 (t, J = 7.3 Hz, 3H).

[0242] 2.1.3. Preparation of Linker-Payload X3

Chem.

[0243] The protocol for preparing Linker-Payload X3 was as follows:

Chem.

[0244] Step 1 Bromopropene (960 mg, 7.92 mmol) was added to 32a (2.00 g, 6.6 mmol) and K2CO3 (1.82 g, 13.2 mmol) in MeCN (20 mL), and the mixture was stirred at 20 °C for 5 h. After the reaction was completed as indicated by TLC (PE / EA = 1 / 2), the reaction mixture was poured into water (100 mL), adjusted to pH 5, and extracted three times with EA (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness by rotary evaporation, and the residue was purified by column chromatography (PE / EA = 2 / 1) to give 32b as a white solid (1.83 g, 81% yield).

[0245] Step 2 TFA (10 mL) was added to 32b (1.38 g, 4.02 mmol) in DCM (10 mL), and the mixture was stirred at 25 °C for 17 h. After the reaction was completed as indicated by TLC (PE / EA = 1 / 3), the reaction mixture was concentrated to dryness by rotary evaporation to give 32c as a yellow sticky substance (0.91 g, yield not calculated).

[0246] Step 3 41d (1.92 g, 4.87 mmol) was added to 32c (910 mg, 4.87 mmol) and NaHCO3 (613 mg, 7.3 mmol) in DME / H2O (20 mL / 10 mL), and the mixture was stirred at 25 °C for 3 h. After the reaction was completed as indicated by TLC (DCM / MeOH = 1 / 1), the reaction mixture was poured into water (100 mL), adjusted to pH 5 with aqueous HCl (1 N), and extracted twice with EA (150 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness by rotary evaporation, and the residue was purified by column chromatography (DCM / MeOH = 20 / 1) to give 32e as a white solid (1.53 g, 67% yield). MS-ESI: M / z 467.4 [M+H]+.

[0247] Step 4 Pd / C (600 mg) was added to 32f (3 g, 5.83 mmol) in MeOH (50 mL), and the mixture was stirred at 25 °C for 5 h under hydrogen. After completion of the reaction as indicated by TLC (EA), the reaction mixture was filtered and the filtrate was concentrated to dryness by rotary evaporation to give 32g as a white solid (1.9 g, yield 77%).

[0248] Step 5 HATU (707 mg, 1.86 mmol) was added to 32g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol) and triethylamine (342 mg, 3.38 mmol) in DMF (10 mL), and the mixture was stirred at 0 °C for 3.5 h. After completion of the reaction as indicated by TLC (EA), the reaction mixture was poured into H2O (80 mL) and extracted twice with EA (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness by rotary evaporation, and the residue was purified by column chromatography (EA) to give 32h as a white solid (1.186 g, yield 83%). MS-ESI: M / z 842.3 [M+H]+.

[0249] Step 6 32h (1.186 g, 1.41 mmol) in DCM / diethylamine (20 mL, 20 / 1) was stirred at 25 °C for 17 h. After completion of the reaction as indicated by TLC (DCM / MeOH = 10 / 1), the reaction mixture was poured into petroleum ether (200 mL), and the resulting mixture was filtered to give 32i as a white solid (768 mg, yield 88%). MS-ESI: M / z 620.3 [M+H]+.

[0250] Step 7 HATU (414 mg, 1.09 mmol) was added to 32i (676 mg, 1.09 mmol), 32e (508 mg, 1.09 mmol) and DIEA (423 mg, 3.27 mmol) in DMF (10 mL), and the mixture was stirred at 20 °C for 17 h. After completion of the reaction as indicated by TLC (PE / EA = 1 / 5), the reaction mixture was poured into water (30 mL). The resulting mixture was filtered, and the filter cake was purified by column chromatography (DCM / MeOH = 50 / 1) to give 32j as a white solid (511 mg, 44% yield). MS-ESI: M / z 1068.3 [M+H]+.

[0251] Step 8 A solution of 32j (482 mg, 0.451 mmol) in diethylamine / DCM (10 mL, 1 / 5) was stirred at 10 °C for 17 h. After completion of the reaction as indicated by TLC (EA), the reaction mixture was poured into PE (300 mL), and the resulting mixture was filtered to give 32k as a white solid (301 mg, yield not calculated).

[0252] Step 9 Morpholine (93 mg, 1.07 mmol) was added to 32k (301 mg, 0.356 mmol) and Pd(PPh3)4 (82 mg, 0.071 mmol) in THF (5 mL), and the mixture was stirred at 25 °C for 5 h. After completion of the reaction as indicated by LCMS, the reaction mixture was purified by preparative chromatography to give 32l as a white solid (108 mg, 38% yield). MS-ESI: M / z 806.3 [M+H]+.

[0253] Step 10 Bromoacetyl bromide (27 mg, 0.134 mmol) was added to 32l (108 mg, 0.134 mmol) and triethylamine (41 mg, 0.402 mmol) in THF (2 mL) and DMF (2 mL), and the mixture was stirred at 0 °C for 1 h. After completion of the reaction as indicated by TLC (DCM / MeOH = 10 / 1), the reaction mixture was directly purified by preparative chromatography to give X3 as a white solid (15 mg, 12% yield).

[0254] MS-ESI: m / z 926.3 [M+H]+。 1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.54 - 8.42 (m, 3H), 8.27 - 8.16 (m, 2H), 7.78 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.61 - 5.51 (m, 1H), 5.42 (s, 2H), 5.20 - 5.05 (m, 2H), 4.56 - 4.42 (m, 2H), 4.32 - 4.22 (m, 1H), 3.96 - 3.87 (m, 3H), 3.79 (d, J = 5.6 Hz, 2H), 3.70 (d, J = 5.9 Hz, 2H), 3.25 - 3.08 (m, 2H), 2.61 - 2.53 (m, 2H), 2.45 - 2.36 (m, 4H), 2.36 - 2.22 (m, 3H), 2.20 - 2.03 (m, 4H), 1.99 - 1.68 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0255] 2.1.4. Preparation of Linker-Payload X4

Chemical Structure

[0256] The protocol for preparing Linker-Payload X4 was as follows:

Chemical Structure

[0257] Step 1 Pd / C (400 mg, 10 wt.%) was added to 33a (2.00 g, 2.58 mmol) in MeOH (20 mL), and the mixture was stirred at 20 °C for 5 h. After the reaction was completed as indicated by TLC (EA), the reaction mixture was filtered, and the filtrate was concentrated to dryness by rotary evaporation to obtain 33b as a white solid (1.3 g, yield 74%).

[0258] Step 2 HATU (305 mg, 0.802 mmol) was added to 33b (0.55 g, 0.802 mmol), KI4 (427 mg, 0.802 mmol) and DIPEA (310 mg, 2.40 mmol) in DMF (5 mL), and the mixture was stirred at 0 °C for 2 h. After completion of the reaction as indicated by TLC (DCM / MeOH = 1 / 10), the reaction mixture was poured into water (40 mL). The resulting mixture was filtered, and the filter cake was purified by column chromatography (DCM / MeOH = 20 / 1) to give 33c as a yellow solid (360 mg, yield 41%).

[0259] Step 3 Diethylamine (2 mL) was added to 33c (360 mg, 0.326 mmol) in DCM (10 mL), and the mixture was stirred at 25 °C for 17 h. After completion of the reaction as indicated by TLC (DCM / MeOH = 5 / 1), the reaction mixture was poured into PE (100 mL), and the resulting mixture was filtered to give 33d as a white solid (205 mg, yield 71%). MS-ESI: M / z 881.3 [M+H]+.

[0260] Step 4 A solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL) was added to 33d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) and water (1 mL), and the mixture was stirred at 0 °C for 1 h. The reaction mixture was purified directly by preparative chromatography to give X4 as a white solid (15 mg, yield 6%).

[0261] MS-ESI: m / z 1001.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.57~8.50 (m, 1H), 8.50~8.43 (m, 2H), 8.35~8.29 (m, 1H), 8.19~8.12 (m, 2H), 7.80 (d, J = 10.8 Hz, 1H), 7.27~7.14 (m, 7H), 6.53 (s, 1H), 5.59~5.51 (m, 1H), 5.44~5.39 (m, 2H), 5.20~5.07 (m, 2H), 4.56~4.44 (m, 3H), 3.92 (s, 3H), 3.80~3.68 (m, 5H), 3.41 (s, 1H), 3.21~3.12 (m, 2H), 2.83~2.74 (m, 1H), 2.58~2.55 (m, 3H), 2.39 (s, 4H), 2.18~2.03 (m, 4H), 1.93~1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0262] 2.2. Preparation of Anti-B7H3 Antibody-Drug Conjugate 2.2.1. Preparation of Reference ADC-1 (manufactured by Daiichi Sankyo, DS7300) DS-7300 is composed of a humanized anti-B7-H3 IgG1 monoclonal antibody that forms a conjugate via a cleavable linker based on a tetrapeptide and the topoisomerase I inhibitor payload exatecan derivative deruxtecan.

[0263] A reducing agent and a protecting agent were formulated using ultrapure water: a 2 mg / mL aqueous solution of TCEP (tris-2-carboxyethyl-phosphine, manufacturer: Thermo) and a 100 mmol / L aqueous solution of EDTA (disodium ethylenediaminetetraacetate, manufacturer: Sigma).

[0264] The linker-payload (deruxtecan) was dissolved in anhydrous DMA (N,N-dimethylacetamide, manufacturer: Sinopharm) to prepare a 10 mg / mL solution of the linker-payload in DMA.

[0265] 16 mg of the reference monoclonal antibody at 7.4 mg / mL (the antibody sequence included the heavy chain shown in SEQ ID NO: 9 and the light chain shown in SEQ ID NO: 16 in the patent document "Specification of Chinese Patent No. 104755494"; the antibody was prepared by transfection of CHO cells followed by conventional antibody expression and purification, and the purity exceeded 95%) was weighed and placed in a 50 mL centrifuge tube, and diluted to 5 mg / mL by adding 30 mM His-HAc, pH 5.5 buffer. An aqueous solution of 100 mM EDTA was added in an amount of 5% of the total volume of the reaction mixture, and the resulting mixture was shaken well to mix thoroughly. Then, the antibody was reduced by adding an aqueous solution of 2 mg / mL TCEP at a molar ratio of TCEP to antibody of 2.7:1, the resulting mixture was shaken well to mix thoroughly, and reacted at 37 °C for 2 hours on a cooling thermomixer. The above linker-payload solution in DMA was added at a final molar ratio of drug to antibody of 9:1, and additional DMA was added in an amount of 10% of the total volume of the reaction mixture. The resulting mixture was shaken well to mix thoroughly, and reacted at 4 °C for 1 hour on a cooling thermomixer. Exchange of the sample storage buffer was carried out using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). First, ultrafiltration was carried out 3 times using 30 mM His-HAc, pH 5.5 buffer containing 10% DMSO, then ultrafiltration was carried out 6 times using His-HAc, pH 5.5 buffer without 30 mM DMSO, and finally filtration was carried out through a 0.2 μm PES membrane to remove bacteria, obtaining the antibody-drug conjugate reference ADC-1 (11 mg, concentration 5.825 mg / mL, yield 68.75%).

[0266] Tests showed that the drug loading (DAR) of the antibody-drug conjugate reference ADC-1 was 4.59 and the SEC purity was 99.64%.

[0267] 2.2.2. Preparation of the antibody-drug conjugate WBP301088-X2

Chemical formula

[0268] The reducing agent and the protecting agent were formulated using ultrapure water: 2 mg / mL aqueous TCEP solution (tris-2-carboxyethyl-phosphine, manufacturer: Thermo) and 100 mmol / L aqueous EDTA solution (disodium ethylenediaminetetraacetate, manufacturer: Sigma).

[0269] The linker-payload X2 prepared in Example 2.1.2 was dissolved in anhydrous DMA (N,N-dimethylacetamide, manufacturer: Sinopharm) to prepare a 10 mg / mL linker-payload solution in DMA.

[0270] 1) The antibody-drug conjugate WBP301088-X2 (DAR4) was prepared as follows: Weighed 20 mg of the WBP301088 monoclonal antibody at 11.3 mg / mL and placed it in a 50 mL centrifuge tube, and diluted it to 5 mg / mL by adding 30 mM His-HAc, pH 5.5 buffer. Added 100 mM aqueous EDTA in an amount of 5% of the total volume of the reaction mixture, and mixed the resulting mixture thoroughly by shaking. Then, the antibody was reduced by adding 2 mg / mL aqueous TCEP at a molar ratio of TCEP to antibody of 4.5:1, and the resulting mixture was mixed thoroughly by shaking and reacted at 37 °C for 2 hours on a cooled thermomixer. Added the above linker-payload solution in DMSO at a final molar ratio of drug to antibody of 12:1, and added additional DMA in an amount of 10% of the total volume of the reaction mixture. The resulting mixture was mixed thoroughly by shaking and reacted at 4 °C for 1 hour on a cooled thermomixer. The exchange of the sample storage buffer was carried out using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). First, ultrafiltration was carried out 3 times using 30 mM His-HAc, pH 5.5 buffer containing 10% DMSO, then ultrafiltration was carried out 6 times using His-HAc, pH 5.5 buffer without DMSO, and finally filtration was carried out through a 0.2 μm PES membrane to remove bacteria, obtaining the antibody-drug conjugate WBP301088-X2 (DAR4) (15 mg, concentration 7.162 mg / mL, yield 75%).

[0271] Tests showed that the drug loading (DAR) of the antibody-drug conjugate WBP301088-X2 (DAR4) was 4.02 and the SEC purity was 99.20%.

[0272] 2) The antibody-drug conjugate WBP301088-X2 (DAR6) was prepared as follows: Weighed 20 mg of the WBP301088 monoclonal antibody at 11.3 mg / mL and placed it in a 50 mL centrifuge tube, then diluted it to 5 mg / mL by adding 30 mM His-HAc, pH 5.5 buffer. Added 100 mM aqueous EDTA in an amount of 5% of the total volume of the reaction mixture, and mixed the resulting mixture thoroughly by shaking. Then, reduced the antibody by adding 2 mg / mL aqueous TCEP at a molar ratio of TCEP to antibody of 8:1, mixed the resulting mixture thoroughly by shaking, and reacted it at 37 °C for 2 hours on a cooling thermomixer. Added the above linker-payload solution in DMSO at a final molar ratio of drug to antibody of 12:1, and added additional DMA in an amount of 10% of the total volume of the reaction mixture. Mixed the resulting mixture thoroughly by shaking and reacted it at 4 °C for 1 hour on a cooling thermomixer. The exchange of the sample storage buffer was carried out using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). First, carried out ultrafiltration 3 times using 30 mM His-HAc, pH 5.5 buffer containing 10% DMSO, then carried out ultrafiltration 6 times using 30 mM His-HAc, pH 5.5 buffer without DMSO, and finally carried out filtration through a 0.2 μm PES membrane to remove bacteria, obtaining the antibody-drug conjugate WBP301088-X2 (DAR6) (10.8 mg, concentration 4.933 mg / mL, yield 54%).

[0273] Tests showed that the drug loading (DAR) of the antibody-drug conjugate WBP301088-X2 (DAR6) was 5.68 and the SEC purity was 99.57%.

[0274] 3) Prepared the antibody-drug conjugate WBP301088-X2 (DAR8) as follows: Weighed 20 mg of the WBP301088 monoclonal antibody at 11.3 mg / mL and placed it in a 50 mL centrifuge tube. Diluted it to 5 mg / mL by adding 30 mM His-HAc, pH 5.5 buffer. Added 100 mM aqueous EDTA in an amount of 5% of the total volume of the reaction mixture and mixed the resulting mixture thoroughly by shaking. Then, reduced the antibody by adding 2 mg / mL aqueous TCEP at a molar ratio of TCEP to antibody of 15:1, mixed the resulting mixture thoroughly by shaking, and reacted it at 37 °C for 2 hours on a cooled thermomixer. Added the above linker-payload solution in DMA at a final molar ratio of drug to antibody of 18:1 and added additional DMA in an amount of 10% of the total volume of the reaction mixture. Mixed the resulting mixture thoroughly by shaking and reacted it at 4 °C for 1 hour on a cooled thermomixer. Performed the exchange of the sample storage buffer using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). First, performed ultrafiltration 3 times using 30 mM His-HAc, pH 5.5 buffer containing 10% DMSO, then performed ultrafiltration 6 times using His-HAc, pH 5.5 buffer without DMSO, and finally performed filtration through a 0.2 μm PES membrane to remove bacteria, obtaining the antibody-drug conjugate WBP301088-X2 (DAR8) (10.2 mg, concentration 4.278 mg / mL, yield 51.0%).

[0275] Tests showed that the drug loading (DAR) of the antibody-drug conjugate WBP301088-X2 (DAR8) was 7.45 and the SEC purity was 98.73%.

[0276] 2.3. Preparation of Cytotoxic (Payload)

Chemical formula

[0277] The protocol for preparing payload P-III-30 was as follows:

Chemical formula

[0278] Step 1 DIEA (60.6 mg, 0.47 mmol) was added dropwise under nitrogen to a solution of KI4 (100 mg, 0.19 mmol), HATU (85.7 mg, 0.23 mmol) and 23a (21.5 mg, 0.21 mmol) in DMF (2 mL). After the addition, the mixture was reacted at 0 °C for 2 hours. After the starting materials were completely consumed as indicated by LCMS, the reaction mixture was added dropwise to water (20 mL), stirred, and the solid was precipitated. The resulting mixture was filtered to obtain P-III-30 as a gray solid (60.2 mg, yield 61%). MS-ESI: M / z 522.2 [M+H]+.

[0279] 1H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.62 - 5.53 (m, 1H), 5.42 (s, 2H), 5.30 - 5.16 (m, 2H), 4.63 (d, J = 4.6 Hz, 1H), 4.09 - 3.99 (m, 1H), 3.22 - 3.11 (m, 2H), 2.40 (s, 3H), 2.28 (dd, J = 13.7, 7.2 Hz, 1H), 2.22 - 2.08 (m, 3H), 1.94 - 1.78 (m, 2H), 1.08 (d, J = 6.1 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0280] 2.4. Detection of the binding activity of the antibody-drug conjugate and the unconjugated antibody to the human B7H3 antigen by ELISA In this example, it was determined using ELISA whether there was a change in the specific binding of the antibody to the B7H3 antigen protein before and after the formation of the conjugate with the drug. Information on the antigens and test antibodies used is as follows. [Table 20]

[0281] 2.4.1. Detection of binding activity based on ELISA A 96-well ELISA plate was coated with 2 μg / mL of recombinant human B7H3 antigen (see the above table) at 30 μL / well and maintained at 4°C overnight. The next day, the well plate was washed three times with PBST and then blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, serially diluted antibody WBP301088, antibody-drug conjugate WBP301088-X2 (DAR6), or isotype control antibody hIgG1 isotype was added and incubated for 1 hour. The plate was then washed three times with PBST, and then HRP-labeled anti-human secondary antibody diluted at a ratio of 1:5000 was added at 100 μL / well and incubated for 1 hour. After completion of the incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. Based on the results of the color development, the reaction was stopped by adding 2 M HCl, and the plate was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190). The results are shown in Figure 13, indicating that there was no significant change in the binding activity of the antibody to the human B7H3 antigen protein before and after complex formation with the drug.

[0282] 2.4.2. ELISA-based detection of binding activity to B7 family proteins Human PD-L1 protein (see the above table), human B7-H2 protein, human B7-H3 protein, human B7-H4 protein, human B7-H5 protein, human B7-H6 protein, human B7-1 protein, human B7-2 protein, human PD-L2 protein, human B7-H7 protein, and human BTNL2 protein were separately immobilized on 96-well plates by incubating overnight at 4°C. Subsequently, the 96-well plates were blocked by incubating at 37°C for 1 hour with 1% BSA in PBS. After blocking, the 96-well plates were washed three times with PBST (PBS containing 0.05% Tween 20). Serial diluted antibody WBP301088, antibody-drug conjugate WBP301088-X2 (DAR6), or isotype control antibody hIgG1 isotype was prepared in binding buffer (PBS containing 0.05% Tween 20 and 0.5% BSA) and incubated with the B7 family proteins immobilized on the 96-well plates at 37°C for 1 hour. After incubation, the 96-well plates were washed three times with PBST, incubated with a secondary antibody (see the above table) in binding buffer at 37°C for 1 hour, and then washed again. TMB was added for color development and the reaction was stopped with 1 M H2SO4 . The results are shown in Figure 14.

[0283] As can be seen from Figure 14, there was no significant change in the binding activity of the antibody to the human B7H3 antigen protein before and after the formation of the complex with the drug. Also, the antibody and the antibody-drug conjugate specifically bound only to human B7-H3 and did not bind to any of the other B7 family proteins.

Example

[0284] Test on the inhibitory effect of the antibody-drug conjugate on the proliferation of tumor cells in vitro The anti-B7H3 ADC WBP301088-X2 (DAR6) and the reference molecule ADC-1 were incubated with human tumor cells that were positive for B7H3 expression for 7 days, and their inhibitory effects on cell proliferation were evaluated by the CellTiter-Glo® luminescent cell viability assay (i.e., the CTG method).

[0285] Growing cells in the logarithmic phase were collected, seeded at a density of 1000 - 3000 cells per well, and the cell plates were incubated overnight in an incubator at 37°C and 5% CO2. On the second day of the experiment, WBP301088-X2 (DAR6) was serially diluted 3-fold in complete medium to obtain drugs with 9 concentration gradients (starting from the highest concentration of 1000 nM), and then the drugs were added to the cell culture plates at 50 μL / well. Complete medium was used as a blank control, and three replicate experimental wells were prepared. The plates were incubated in an incubator at 37°C and 5% CO2 for an additional 5 days. After incubation, the cell culture plates were taken out and equilibrated to room temperature. To each well, 50 μL of CTG assay reagent was added, and the mixture was thoroughly mixed by shaking, and then the plates were left in the dark for 10 minutes. Subsequently, the signals were detected and their values were read using a microplate reader. An S-shaped dose-response curve was plotted using a non-linear regression model with GraphPad Prism software, and the IC 50 values were calculated. The cell viability was calculated as (Lum 試験薬物 -Lum ブランク対照 ) / (Lum 溶媒ブランク対照 -Lum ブランク対照 )×100%.

[0286] The experimental results are shown in the following table. CAL-120 was obtained from DSMZ, and the other cell lines were obtained from ATCC.

Table 21

[0287] As can be seen from the results in Table 17, the antibody-drug conjugate WBP301088-X2 (DAR6) of the present application showed better inhibitory activity against the growth of various tumor cells with positive B7H3 expression than the reference ADC-1.

Example

[0288] Evaluation of the efficacy of an antibody-drug conjugate in mice bearing Calu-6 human lung cancer cell tumors To investigate the inhibitory effect of WBP301088-X2 (DAR6) on in vivo tumor formation, xenograft tumors in mice were formed using Calu-6 human lung cancer cells with positive B7H3 expression, and then the in vivo antitumor effect of WBP301088-X2 (DAR6) was evaluated.

[0289] Female BALB / c nude mice aged 6 - 8 weeks (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were used as test animals. 10 × 10 6 Calu-6 lung cancer cells were subcutaneously inoculated on the right side of the back of female BALB / c nude mice aged 6 - 8 weeks. The growth of tumors in the mice was observed. When the tumor volume reached approximately 139 mm 3 , the tumor-bearing mice were randomly divided into groups of 6 mice each for the blank control group and the treatment group. On the day of grouping (day 0), WBP301088-X2 or the reference ADC-1 was administered by intravenous (i.v.) injection, with a total of 1 injection. The administration doses were 1 mg / kg and 3 mg / kg. The experiment was terminated on day 28. The grouping and dosing of the experiment were as follows. The tumor volume and body weight of the mice were measured twice a week, and the data were recorded.

[0290] Blank control (negative control group): Normal saline WBP301088-X2 (treatment group): 1 mg / kg WBP301088-X2 (treatment group): 3 mg / kg Reference ADC-1 (treatment group, positive control group): 1 mg / kg Reference ADC-1 (treatment group, positive control group): 3 mg / kg

[0291] All samples were prepared by diluting with normal saline.

[0292] At the end of the experiment, the mice were euthanized and the tumor growth inhibition rate TGI was calculated as follows: TGI (%) = [1 - (T i - T0) / (V i - V0)]×100 T i : The average tumor volume of the treatment group and the positive control group on the i-th day after administration; T0: The average tumor volume of the treatment group and the positive control group on the 0th day of administration; V i : The average tumor volume of the negative control group on the i-th day after administration; V0: The average tumor volume of the negative control group on the 0th day of administration.

[0293] The experimental results are shown in Figure 15, Table 18 and Table 19.

Table 22

Table 23

[0294] From the results of Figure 15, Table 18 and Table 19, it is shown that the antibody-drug conjugate WBP301088-X2 of the present application showed significant dose-dependent antitumor activity after a single-dose administration, and its antitumor effect was significantly better than that of reference ADC-1 for both the 1 mg / kg dose and the 3 mg / kg dose.

Example

[0295] Evaluation of the Efficacy of an Antibody-Drug Conjugate in Mice Bearing A375 Human Melanoma Tumors To investigate the inhibitory effect of WBP301088-X2 (DAR6) on in vivo tumor formation, xenograft tumors in mice were formed using A375 human melanoma cells that were positive for B7H3 expression, and then the in vivo antitumor effect of WBP301088-X2 (DAR6) was evaluated.

[0296] Female NOD / SCID mice (purchased from Jiangsu GemPharmatech Co., Ltd.) at 6 - 8 weeks of age were used as test animals. 5×10 6 individual A375 human melanoma cells were subcutaneously inoculated on the right side of the back of 6 - 8 week-old female NOD / SCID mice. Tumor growth was observed. When the tumor volume reached approximately 89 mm 3 ³, the tumor-bearing mice were randomly divided into groups of 6 mice each for the blank control group and the treatment group. On the day of grouping (day 0), WBP301088-X2 or reference ADC-1 was administered by intravenous (i.v.) injection, with a total of 1 injection. The administration doses were 1 mg / kg, 3 mg / kg, and 10 mg / kg. The experiment was terminated on day 20. The tumor volume and body weight of the mice were measured twice a week, and the data were recorded.

[0297] Blank control (negative control group): Normal saline WBP301088-X2 (treatment group): 1 mg / kg WBP301088-X2 (treatment group): 3 mg / kg WBP301088-X2 (treatment group): 10 mg / kg Reference ADC-1 (treatment group): 3 mg / kg Reference ADC-1 (treatment group): 10 mg / kg

[0298] All samples were prepared by diluting with normal saline.

[0299] At the end of the experiment, the mice were euthanized, and TGI TV(Relative tumor growth inhibition rate) was calculated. TGI TV (Relative tumor growth inhibition rate) was calculated by the following formula:

Equation

Equation

[0300] The experimental results are shown in Figure 16, Table 20 and Table 21.

Table 24

Table 25

[0301] From the results of Figure 16, Table 20 and Table 21, it is shown that the antibody-drug conjugate WBP301088-X2 of the present application showed significant dose-dependent antitumor activity after a single-dose administration.

Example

[0302] Evaluation of the efficacy of an antibody-drug conjugate in mice bearing U87 human brain cancer cell tumors To investigate the inhibitory effect of WBP301088-X2 (DAR6) on in vivo tumor formation, U87 human brain cancer cells with positive B7H3 expression were used to form xenograft tumors in mice, and then the in vivo antitumor effect of WBP301088-X2 (DAR6) was evaluated.

[0303] Six- to eight-week-old female NCG mice (purchased from Jiangsu GemPharmatech Co., Ltd.) were used as test animals. 5×10 6 cells of U87 human brain cancer were subcutaneously inoculated on the right side of the neck or back of six- to eight-week-old female NCG mice. The growth of tumors in the mice was observed. When the tumor volume reached approximately 187 mm 3 , the tumor-bearing mice were randomly divided into groups of six mice each for the blank control group and the treatment group. On the day of grouping (day 0), WBP301088-X2 or reference ADC-1 was administered by intravenous (i.v.) injection, and the administration was carried out in a total of one injection. The administration doses were 1 mg / kg and 3 mg / kg. The experiment was terminated on day 20. The grouping and dosing of the experiment were as follows. The tumor volume and body weight of the mice were measured twice a week, and the data were recorded.

[0304] Blank control (negative control group): Normal saline WBP301088-X2 (treatment group): 1 mg / kg WBP301088-X2 (treatment group): 3 mg / kg Reference ADC-1 (treatment group, positive control group): 1 mg / kg Reference ADC-1 (treatment group, positive control group): 3 mg / kg

[0305] All samples were prepared by diluting with normal saline.

[0306] At the end of the experiment, the mice were euthanized, and the tumor growth inhibition rate TGI was calculated as follows: TGI (%) = [1 - (T i - T0) / (V i - V0)]×100 T i: Mean tumor volume of the treatment group and the positive control group on the i-th day after administration; T0: Mean tumor volume of the treatment group and the positive control group on the 0-th day of administration; V i : Mean tumor volume of the negative control group on the i-th day after administration; V0: Mean tumor volume of the negative control group on the 0-th day of administration).

[0307] The experimental results are shown in Figure 17, Table 22 and Table 23.

Table 26

Table 27

[0308] From the results of Figure 17, Table 22 and Table 23, it is shown that the antibody-drug conjugate WBP301088-X2 of the present application exhibited significant dose-dependent antitumor activity after a single-dose administration, and for the dose of 3 mg / kg, the antitumor effect of the antibody-drug conjugate WBP301088-X2 was significantly better than that of the reference ADC-1.

Example

[0309] Evaluation of the efficacy of an antibody-drug conjugate in mice bearing PC-3 human prostate cancer cell tumors To investigate the inhibitory effect of WBP301088-X2 (DAR6) on in vivo tumor formation, PC-3 human prostate cancer cells with positive B7H3 expression were used to form xenograft tumors in mice, and then the in vivo antitumor effect of WBP301088-X2 (DAR6) was evaluated.

[0310] Female BALB / c nude mice aged 6 - 8 weeks (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were used as test animals. 5×10 6 PC-3 human prostate cancer cells were subcutaneously inoculated into the necks or the right sides of the backs of 6 - 8 week-old female BALB / c nude mice. The growth of the tumors was observed. When the tumor volume reached approximately 155 mm 3 the tumor-bearing mice were randomly divided into groups of 6 mice each for the blank control group and the treatment group. On the day of grouping (day 0), WBP301088-X2 or reference ADC-1 was administered by intravenous (i.v.) injection, with a total of 1 injection. The administration doses were 2 mg / kg and 6 mg / kg. The experiment was terminated on day 21. The grouping and dosing of the experiment were as follows. The tumor volume and body weight of the mice were measured twice a week, and the data were recorded.

[0311] Blank control (negative control group): Normal saline WBP301088-X2 (treatment group): 2 mg / kg WBP301088-X2 (treatment group): 6 mg / kg Reference ADC-1 (treatment group, positive control group): 2 mg / kg Reference ADC-1 (treatment group, positive control group): 6 mg / kg

[0312] All samples were prepared by diluting with normal saline.

[0313] At the end of the experiment, the mice were euthanized, and the tumor growth inhibition rate TGI was calculated as follows: TGI (%) = [1 - (T i - T0) / (V i- V0)]×100) T i : Mean tumor volume of the treatment group and the positive control group on the i-th day after administration; T0: Mean tumor volume of the treatment group and the positive control group on day 0 of administration; V i : Mean tumor volume of the negative control group on the i-th day after administration; V0: Mean tumor volume of the negative control group on day 0 of administration.

[0314] The experimental results are shown in Figure 18, Table 24 and Table 25.

Table 28

Table 29

[0315] From the results of Figure 18, Table 24 and Table 25, it is shown that the antibody-drug conjugate WBP301088-X2 of the present application showed significant dose-dependent antitumor activity after a single-dose administration, and its antitumor effect was significantly better than that of reference ADC-1 for both the 2 mg / kg dose and the 6 mg / kg dose.

Example

[0316] Evaluation of the efficacy of an antibody-drug conjugate in a patient-derived xenograft (PDX) model of prostate cancer To investigate the efficacy of WBP301088-X2 (DAR6) in a xenograft model derived from prostate cancer patients, patient-derived prostate cancer PR9586 and PR9587 tumor tissues (obtained from Crown Biotechnology (Zhongshan) Co., Ltd.) were xenografted subcutaneously into NPG male mice and NOG male mice to evaluate the antitumor effect of WBP301088-X2 (DAR6).

[0317] 1. Test compounds and materials Blank control (control group): Normal saline WBP301088-X2 (treatment group): 10 mg / kg

[0318] 2. Preparation method of test compounds: All samples were prepared by diluting with normal saline.

[0319] 3. Experimental animals: NPG mice were purchased from Beijing Vitalstar Biotechnology Co., Ltd., and NOG mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0320] 4. Procedure: Patient-derived prostate cancer PR9586 and PR9587 tumor tissues were separately subcutaneously inoculated into NPG mice or NOG mice. When the tumors grew to about 500 - 800 mm 3 in size, the tumor tissues were collected and the mice were euthanized. The collected tumor tissues were cut into tumor pieces with a diameter of 2 - 3 mm and then subcutaneously inoculated on the right front side of the scapula of NPG mice or NOG mice. When the average tumor volume of the NPG mice or NOG mice inoculated with the tumor pieces reached about 100 - 200 mm 3 in size, the mice were randomly divided into groups of 6 mice each in the blank control group and the treatment group.

[0321] On the day of grouping (day 0), WBP301088-X2 was administered by intravenous (i.v.) injection, and the administration was carried out at a single dose of 10 mg / kg once every two weeks for a total of two injections. The experiment was terminated on day 25 (mice inoculated with PR9586) or day 48 (mice inoculated with PR9587). After the start of administration, the body weight and tumor size of the mice were measured twice a week. Tumor volume calculation formula: Tumor volume (mm 3 ) = 1 / 2×(a×b 2 )(where a represents the major axis and b represents the minor axis).

[0322] At the end of the experiment, the mice were euthanized and the tumor growth inhibition rate TGI was calculated as follows: TGI (%) = [1 - (T i - T0) / (V i )]×100 (where T i : the average tumor volume of the treatment group on the i-th day after administration; T0: the average tumor volume of the treatment group on day 0 of administration; V i : the average tumor volume of the negative control group on the i-th day after administration; V0: the average tumor volume of the negative control group on day 0 of administration).

[0323] 5. Experimental results: The experimental results are shown in Figure 19A, Figure 19B, Table 26 and Table 27.

Table 30

Table 31

[0324] The results indicate that the antibody-drug conjugate WBP301088-X2 of the present application showed significant antitumor activity after administration.

Example

[0325] Evaluation of the Efficacy of Antibody-Drug Conjugates in a Xenograft Model Derived from Prostate Cancer Micrometastasis Patients To investigate the efficacy of WBP301088-X2 (DAR6) in a xenograft model derived from prostate cancer patients, the antitumor effect of WBP301088-X2 (DAR6) in a xenograft mouse model derived from prostate cancer micrometastasis patients was evaluated.

[0326] 1. Test Compounds and Materials Blank control (control group): Normal saline WBP301088-X2 (treatment group): 10 mg / kg Collagenase digestant:

Table 32

[0327] The components in the table were mixed according to the manufacturer's instructions to prepare the collagenase digestant.

[0328] 2. Preparation Method of Test Compounds: All samples were prepared by diluting with normal saline.

[0329] 3. Experimental Animals: CB17 SCID mice, male, 6 - 8 weeks old, weighing approximately 18 - 22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0330] 4. Procedure: Tumor tissues of the patient-derived prostate cancer xenograft models LD1-2027-410644 and LD1-2034-362055 (obtained from Shanghai LIDE Biotech., Co. Ltd.) were subcutaneously inoculated into mice. When the tumors reached 500 - 800 mm 3Once it had grown to this stage, the tumor tissue was surgically and aseptically removed from the mouse within the biosafety cabinet. Non-tumor tissue and necrotic tissue were removed to ensure the purity of the tumor tissue to be inoculated. The mouse was euthanized. The tumor tissue to be treated was cut into small tumor pieces of 1 - 3 mm 3 and the tumor pieces were digested at 37°C for 1 - 2 hours using a collagenase digestive agent. The supernatant was removed by centrifugation at 1200 rpm for 3 minutes, and the cells were resuspended in 10 mL of PBS containing 1% FBS and counted using a hemocytometer. Mouse cells were taken out, and the supernatant was removed by centrifugation at 1200 rpm for 3 minutes. The cells were resuspended in RPMI1640 cell culture medium and counted using a hemocytometer. The cell density was adjusted.

[0331] The cell suspension was placed into capsules. CB17 SCID mice were randomly divided based on body weight into groups of 3 mice each for the blank control group and the treatment group. One capsule was subcutaneously inoculated on the left and right sides of each mouse, for a total of 6 capsules per group. Each capsule contained 5000 tumor cells. The inoculation day was recorded as day 0, and the animals were subjected to a single administration by tail vein injection. The experiment was conducted for 10 days.

[0332] At the end of the experiment, the mice were euthanized. The capsules were taken out, and CellTiter - Glo was used, that is, the capsules were cut into small pieces and a cell viability assay was performed. The same volume of PBS and CellTiter - Glo™ reagent were added, and the luminescence value was measured. In the optical signal and system, the luminescence value is directly proportional to the amount of ATP, and the amount of ATP is positively correlated with the number of living cells, so the cell viability can be known by detecting the ATP content.

[0333] 5. Experimental Results: The experimental results are shown in Figure 20A, Figure 20B, and Table 28.

Table 33

[0334] From the results of FIGS. 20A, 20B, and Table 28, it is shown that WBP301088-X2 exhibited a significant antitumor effect.

Example

[0335] Pharmacokinetics study of single administration of antibody-drug conjugate Purpose After a single intravenous injection of the antibody-drug conjugate WBP301088-X2 (DAR6) in cynomolgus monkeys, the concentrations of WBP301088-X2 (DAR6), total WBP301088-antibody, and payload P-III-30 in serum were measured at different time points, and the pharmacokinetic parameters of WBP301088-X2 (DAR6), total WBP301088-antibody, and payload P-III-30 were calculated, thereby obtaining the basis for preclinical and clinical trials.

[0336] Procedure The body weights of 18 cynomolgus monkeys (half female and half male) were measured on the day before dosing, stratified based on body weight, and then randomly divided into three groups (3 females and 3 males / group): low-dose group (1 mg / kg), middle-dose group (3 mg / kg), and high-dose group (10 mg / kg). Each animal was administered a WBP301088-X2 solution by intravenous injection.

[0337] Blood was collected from the forelimb veins of cynomolgus monkeys in each group, and the treated blood samples collected at 0 hours before dosing and at 0.5 hours, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours after dosing were used as PK samples. After completing blood collection at each time point, serum was obtained by centrifugation for sample analysis.

[0338] The concentrations of total WBP301088-antibody and WBP301088-X2 in the serum samples of cynomolgus monkeys were quantitatively detected by ELISA method, and the concentration of P-III-30 in the serum samples of cynomolgus monkeys was quantitatively detected by LC-MS / MS method. The experimental results are shown in Table 29.

Table 34

[0339] Conclusion Under the conditions of this experiment, after single intravenous administration of WBP301088-X2 to cynomolgus monkeys at 1 mg / kg, 3 mg / kg and 10 mg / kg, there was no significant difference in pharmacokinetics between the antibody-drug conjugate and the whole antibody (antibody WBP301088), and all showed dose-dependent increases. Therefore, it was shown that WBP301088-X2 had good stability in plasma in vivo. In addition, P-III-30 was detected at a low concentration in plasma. Thereby, it was shown that the ADC was slowly released in monkeys, the complex formation method was stable, the systemic exposure of P-III-30 was low, and WBP301088-X2 had high safety.

Example

[0340] Pharmacokinetics and Toxicity Tests of Multiple Administrations of Antibody-Drug Conjugates Objective Cynomolgus monkeys were intravenously administered WBP301088-X2 (DAR6) once every 3 weeks for a total of 2 times. The nature, degree of potential toxic reactions caused by the antibody-drug conjugate, as well as the relationship between dose and effect and time and effect were observed, the target organ or tissue of toxicity was determined, and a reference for subsequent tests was obtained.

[0341] Procedure Six cynomolgus monkeys weighing 2.3 - 3.4 kg (half female and half male) were divided into groups: a low-dose group (30 mg / kg) and a high-dose group (80 mg / kg). Each animal was administered at a dosing volume of 5 mL / kg once every 3 weeks for a total of 2 times. The tolerance of the animals and the manifestation of drug-related toxicity were investigated from multiple aspects such as clinical observation, body weight and food intake, hematological examination, blood biochemical examination, urine and gross anatomical structure.

[0342] Conclusion Cynomolgus monkeys were intravenously administered WBP301088-X2 (DAR6) at 30 mg / kg and 80 mg / kg once every three weeks for a total of two times, and dissected four days after the second administration (i.e., on the 25th day after the start of administration).

[0343] The results showed that during the test period, all animals had good tolerance to the antibody-drug conjugate, and no abnormalities were observed in body weight, food intake, electrocardiogram parameters, coagulation parameters, and gross anatomical structures. Therefore, it was shown that the antibody-drug conjugate had good safety.

[0344] To illustrate the present invention, certain specific representative embodiments and details have been shown. However, it will be apparent to those skilled in the art that various modifications and alterations can be made to these embodiments and details without departing from the scope of the subject matter of the present invention. In this regard, the scope of the present invention is limited only by the claims.

Claims

1. An antibody-drug conjugate comprising a B7H3-targeted antibody or an antigen-binding fragment thereof, a linker unit, and a cytotoxic drug, wherein the B7H3-targeted antibody or its antigen-binding fragment comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4 or 7, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:

6.

2. A B7H3 target antibody or its antigen-binding fragment, (I) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 8 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 9; or a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 11, or (II) Heavy chain variable regions having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8 and light chain variable regions having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9; or heavy chain variable regions having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10 and light chain variable regions having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11, or (III) A heavy chain variable region having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions within the framework region compared to SEQ ID NO: 8 or 10, and a light chain variable region having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions within the framework region compared to SEQ ID NO: 9 or 11. The antibody-drug conjugate according to claim 1, comprising:

3. The antibody-drug conjugate according to claim 1, wherein the antibody is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies.

4. The antigen-binding fragments are Fab, Fab', and F(ab'). ２ The antibody-drug conjugate according to claim 1, selected from the group consisting of Fv, ScFv, Fab'-SH, sdAb, VHH, bispecific antibodies, and linear antibodies.

5. The antibody-drug conjugate according to claim 1, wherein the antibody comprises an immunoglobulin constant region, and the immunoglobulin constant region is a human IgG constant region, for example, a human IgG1 constant region.

6. A B7H3 target antibody or its antigen-binding fragment, (I) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 12 and a light chain containing the amino acid sequence shown in SEQ ID NO: 13; or a heavy chain containing the amino acid sequence shown in SEQ ID NO: 14 and a light chain containing the amino acid sequence shown in SEQ ID NO: 15, or (II) A heavy chain having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12 and a light chain having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13; or a heavy chain having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14 and a light chain having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15 The antibody-drug conjugate according to claim 1, comprising:

7. Cytotoxic drugs, formula (A-1) 【Chemistry 1】 (In the ceremony M is -L ２ -L １ -C(O)-, L ２ is -O- or -S-, L １ is -(C(R １ａ )(R １ｂ )) ｍ -CH ２ -, C ３ -C ６ is a saturated cycloalkyl or a 3- to 6-membered saturated heterocyclyl, and the C ３ -C ６ saturated cycloalkyl and 3- to 6-membered saturated heterocyclyl may each independently be substituted with one or more R ２ａ and m is selected from the group consisting of 1, 2, 3, and 4, and the 3-6 member saturated heterocycline contains 1 to 3 heteroatoms selected from the group consisting of N, O, and S. R １ａ , R １ｂ and R ２ａ These are, independently, hydrogen, halogen, hydroxyl, amino, and C. １ -C ６ Selected from the group consisting of alkyl groups, the C １ -C ６ The alkyl group may be substituted with one or more R groups. Each R is independently either hydrogen or a halogen. The antibody-drug conjugate according to claim 1, comprising the structure shown in, or a tautomer, enantiomer, diastereoisomer, or mixture thereof, or a pharmaceutically acceptable salt or solvated compound thereof.

8. L １ However, -(C(R １ａ ) (Caution １ｂ )) ｍ -CH ２ - and R １ａ However, hydrogen, halogens, and C １ -C ６ Selected from the group consisting of alkyl groups, R １ｂ However, hydrogen, halogens, and C １ -C ６ The antibody-drug conjugate according to claim 7, selected from the group consisting of alkyl groups.

9. L １ However, -(C(R １ａ ) (Caution １ｂ )) ｍ -CH ２ The antibody-drug conjugate according to claim 7, wherein m is 1 or 2.

10. L １ but, 【Chemistry 2】 An antibody-drug conjugate according to claim 9, selected from the group consisting of the following.

11. L １ However, C ３ -C ６ A saturated cycloalkyl or a 3-6 member saturated heterocycline, and the C ３ -C ６ A saturated cycloalkyl group and a saturated heterocycline group with 3 to 6 members each independently contain one or more R groups. ２ａ It may also be replaced with each R ２ａ However, independently, hydrogen, halogen, and C １ -C ６ The antibody-drug conjugate according to claim 7, selected from the group consisting of alkyl groups.

12. L １ However, one or more R ２ａ C may be replaced with ３ -C ６ It is a saturated cycloalkyl, and each R ２ａ However, independently, hydrogen, halogen, and C １ -C ６ The antibody-drug conjugate according to claim 11, selected from the group consisting of alkyl groups.

13. L １ However, one, two, or three R ２ａ It may be replaced with 【Transformation 3】 And each R ２ａ However, independently, hydrogen, halogen, and C １ -C ６ The antibody-drug conjugate according to claim 12, selected from the group consisting of alkyl groups.

14. L １ but, 【Chemistry 4】 An antibody-drug conjugate according to claim 13, selected from the group consisting of the following.

15. The linker unit is -L ａ -L ｂ -L ｃ - and -L ａ -but, 【Transformation 5】 And, -L ｂ -but, 【Transformation 6】 Preferably, 【Transformation 7】 Selected from the group consisting of, -L ｃ -but, 【Transformation 8】 The antibody-drug conjugate according to claim 1.

16. The linker unit, 【Chemistry 9】 Preferably, 【Chemistry 10】 The antibody-drug conjugate according to claim 15.

17. Formula (A-2): 【Chemistry 11】 (In the ceremony p represents the average number of connections, and p is selected from a group consisting of integers or decimals from 1 to 10. Ab is as described in claim 1, M is as described in claim 7, L (linker unit) is as described in claim 15. The antibody-drug conjugate according to claim 1, having the structure shown in [image / figure].

18. The antibody-drug conjugate according to claim 17, wherein p is an integer or decimal between 2 and 8, for example, p is an integer or decimal between 4 and 8, for example, p is an integer or decimal between 4 and 6 or 6 and 8.

19. Formula (A-2a) or Formula (A-2b): 【Chemistry 12】 (In the ceremony p represents the average number of connections, and p is selected from the group consisting of integers or decimals from 1 to 10, preferably from 2 to 8. For example, p may be an integer or decimal from 4 to 8, or for example, p may be an integer or decimal from 4 to 6 or 6 to 8. Ab is a B7H3 target antibody or its antigen-binding fragment, comprising HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 7, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:

6. L ２ is -O- or -S-, preferably -O-, X １ is one, two, or three R ２ａ C may be replaced with ３ -C ６ Selected from the group consisting of saturated cycloalkyls, X ２ is -(C(R １ａ ) (Caution １ｂ )) ｍ -CH ２ - Selected from the group consisting of, m is selected from the group consisting of 1 and 2. R １ａ , R １ｂ and R ２ａ These are each independently of hydrogen, halogen, and C which may be substituted with one, two, or three R atoms. １ -C ６ Selected from the group consisting of alkyl groups, Each R is independently either hydrogen or a halogen. The antibody-drug conjugate according to claim 18, having the structure shown in [image / figure].

20. The following structural formula: 【Chemistry 13】 【change】 【change】 (In the ceremony p represents the average number of connections, and p is selected from the group consisting of integers or decimals from 1 to 10, preferably from 2 to 8. For example, p may be an integer or decimal from 4 to 8, or for example, p may be an integer or decimal from 4 to 6 or 6 to 8. Ab is as described in claim 1. An antibody-drug conjugate according to claim 1, selected from the group consisting of the following.

21. Antibody-drug conjugates 【Chemistry 14】 【change】 (In the ceremony p represents the average number of linked molecules, and p is selected from the group consisting of integers or decimals from 1 to 10, preferably from 2 to 8. For example, p is an integer or decimal from 4 to 8, for example, p is an integer or decimal from 4 to 6 or from 6 to 8. Ab is an anti-B7H3 antibody, and the antibody comprises HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; for example, the antibody comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 11; for example, the antibody comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 12 and a light chain described in SEQ ID NO:

13. The antibody-drug conjugate according to claim 20, selected from the group consisting of and pharmaceutically acceptable salts thereof. 【Request Item 22】 【Chemistry 15】 (In the ceremony p represents the average number of connections, and p is selected from the group consisting of integers or decimals from 1 to 10, preferably from 2 to 8. For example, p is an integer or decimal from 4 to 8, for example, p is an integer or decimal from 4 to 6 or from 6 to 8, for example, p is 7.45, 5.68 or 4.

02. WBP301088 is an anti-B7H3 antibody containing the heavy chain amino acid sequence shown in SEQ ID NO: 12 and the light chain amino acid sequence shown in SEQ ID NO:

13. An antibody-drug conjugate according to claim 21, selected from the group consisting of the following.

23. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 22 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

24. Use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 22 in the preparation of a medicament for the treatment and / or prevention of a B7H3-mediated disease or condition, wherein the disease or condition is cancer, for example, the disease or condition is a disease or condition in which B7H3 expression is positive.

25. The use according to claim 24, wherein the cancer is selected from the group consisting of breast cancer, neurological tumors, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, urothelial carcinoma, lymphoma (e.g., non-Hodgkin lymphoma), chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma.

26. A therapeutic and / or prophylactic agent for a B7H3-mediated disease or condition, comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 22, wherein the disease or condition is cancer, for example, the disease or condition is a disease or condition in which B7H3 expression is positive.

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