Anti-B7H3 antibody-drug conjugates and uses thereof

Abstract

Provided are antibody-drug conjugates that specifically bind to B7H3 and pharmaceutical compositions comprising the same, as well as methods and uses of using the antibody-drug conjugates.

Description

Anti-B7H3 antibody-drug conjugates and uses thereof Technical Field

[0001] The present invention provides an antibody-drug conjugate that specifically binds to B7H3 and a composition comprising the same, as well as methods and uses of the antibody-drug conjugate of the present invention. Background Art

[0002] B7H3, also known as CD276, B7RP-2, and B7-H3, is a member of the B7 family and shares 20%-27% amino acid sequence homology with other B7 family members. Although B7H3 transcripts are widely expressed, expression is limited and maintained at low levels in normal tissues and immune cells. It is also overexpressed in various human malignancies, including melanoma, breast cancer, and prostate cancer. B7-H3 is reportedly expressed on the cell membrane, cytoplasm, or within the nucleus of cancer cells, as well as on 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 its expression on APCs can be induced by GM-CSF and IFNγ.

[0003] B7-H3 is a promising anticancer target. Although the precise function of B7-H3 remains unclear, its diverse immune functions have been demonstrated, including stimulation and inhibition of T cell proliferation and suppression of NK cell function. It is believed that different receptors exist on immune cells that may compete for binding with B7H3 on tumors. In addition to its immune checkpoint function, high B7H3 expression has been reported to correlate with poor cancer prognosis and enhance cell proliferation, migration, invasion, angiogenesis, metastasis, and anticancer drug resistance.

[0004] Like other B7 family molecules, B7H3 is a type 1 transmembrane glycoprotein whose extracellular domain contains an IgV-IgC domain. Unlike the mouse B7H3 gene, which has only one IgV-IgC domain, human B7H3 has two isoforms: one containing a single copy of the IgV-IgC domain (designated 2IgB7H3) and the other containing two IgV-IgC domains (designated 4IgB7H3), a result of gene duplication and differential splicing. 4IgB7H3, rather than 2IgB7H3, is the predominant isoform expressed on immune and malignant cells, suggesting that 4IgB7H3 may play a unique and important role in tumor development and tumor immunity.

[0005] Because B7H3 is overexpressed on many tumor cells, therapeutic molecules targeting B7H3 are currently being developed to treat related indications. However, there is still room for improvement and clinical demand for the development of novel anti-B7H3 antibodies or antibody-drug conjugates.

[0006] Summary of the Invention

[0007] The present application provides an anti-B7H3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof, which may have one or more effects selected from the following groups: (1) having inhibitory activity on the proliferation of tumor cells in vitro; (2) having better affinity with human B7H3; (3) having plasma stability; (4) having an in vivo tumor-suppressing effect; (5) having a bystander killing effect; (6) having anti-transporter transport ability; (7) having in vivo tumor targeting ability; and (8) having good in vivo safety.

[0008] On the one hand, the present application provides an antibody-drug conjugate, comprising: an antibody or an antigen-binding fragment thereof targeting B7H3, a linker unit and a cytotoxic drug; the antibody or antigen-binding fragment thereof targeting B7H3 comprises: a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 2, a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 3, a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 4 or 7, a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 5 and a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 6.

[0009] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the antibody or antigen-binding fragment thereof comprises:

[0010] (I) a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 8 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 9; or a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 10 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 11; or

[0011] (II) a heavy chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, and a light chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9; or a heavy chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10, and a light chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11; or

[0012] (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 its 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 its framework region compared to SEQ ID NO: 9 or 11.

[0013] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the antibody is selected from the group consisting of: a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

[0014] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

[0015] In some embodiments, the antibody in the antibody-drug conjugate of the present invention is a monoclonal antibody.

[0016] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the antibody is a full-length antibody or an antigen-binding fragment of an antibody, and the antigen-binding fragment is, for example, selected from: Fab, Fab′, F(ab′)2, Fv, ScFv, Fab′-SH, sdAb, VHH, diabodies and linear antibodies.

[0017] In some embodiments, the present invention provides an antibody-drug conjugate, wherein 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.

[0018] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0019] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the antibody or antigen-binding fragment thereof comprises:

[0020] (I) a heavy chain with an amino acid sequence as set forth in SEQ ID NO: 12 and a light chain with an amino acid sequence as set forth in SEQ ID NO: 13; or a heavy chain with an amino acid sequence as set forth in SEQ ID NO: 14 and a light chain with an amino acid sequence as set forth in SEQ ID NO: 15; or

[0021] (II) a heavy chain at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12, and a light chain at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; or a heavy chain at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14, and a light chain at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15.

[0022] In some embodiments, the antibody of the present invention is the anti-B7H3 antibody WBP301088.

[0023] In certain preferred embodiments of the present invention, certain groups in the compounds of formula (A-1), (A-2), (A-1a) and (A-1b) or pharmaceutically acceptable salts thereof in the antibody-drug conjugates of the present invention are defined as follows. Unmentioned groups are the same as those described in any embodiment of the present application (referred to as "in some embodiments").

[0024] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the cytotoxic drug comprises or is selected from the following structures: the structure represented by formula (A-1), its tautomers, enantiomers, diastereomers, or mixtures of isomers, or pharmaceutically acceptable salts or solvates thereof,

[0025] in,

[0026] M is -L 2 -L 1 -C(O)-;

[0027] L 2 is -O- or -S-;

[0028] L 1 -(C(R 1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkyl or 3-6 membered saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a replace;

[0029] m is selected from 1, 2, 3 or 4; the heteroatoms in the 3-6 membered saturated heterocyclic group are selected from N, O and S, and the number of heteroatoms is 1-3;

[0030] R 1a are each independently selected from hydrogen, halogen, hydroxy, amino, and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more R;

[0031] R 1b and R 2a are each independently selected from hydrogen, halogen, hydroxy, amino, and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more R;

[0032] Each R is independently hydrogen or halogen.

[0033] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a Selected from: hydrogen, halogen and C1-C6 alkyl; R 1b is selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl. In some embodiments, the present invention provides an antibody-drug conjugate, wherein L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a is hydrogen or -CH3; R 1b Selected from: hydrogen and -CH3; for example R 1a -CH3; R 1b Selected from: hydrogen and -CH3.

[0034] In some embodiments, the present invention provides an antibody-drug conjugate, wherein L 1 -(C(R 1a )(R 1b )) m -CH2-; m is 1 or 2.

[0035] In some embodiments, the present invention provides an antibody-drug conjugate, wherein L 1 Selected from:

[0036] In some embodiments, the present invention provides an antibody-drug conjugate, wherein L 1 is a C3-C6 saturated cycloalkyl or a 3-6 membered saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and the 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a Replacement, R 2a Each is independently selected from the group consisting of: hydrogen, halogen and C1-C6 alkyl.

[0037] In some embodiments, the present invention provides an antibody-drug conjugate, wherein L 1 is optionally replaced by one or more R 2aSubstituted C3-C6 saturated cycloalkyl; R 2a are independently selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl. In some embodiments, the present invention provides an antibody-drug conjugate wherein L 1 It is a C3-C6 saturated cycloalkyl group.

[0038] In some embodiments, the present invention provides an antibody-drug conjugate, wherein L 1 1, 2 or 3 R 2a Replaces: R 2a Each is independently selected from the group consisting of: hydrogen, halogen and C1-C6 alkyl.

[0039] In some embodiments, the present invention provides an antibody-drug conjugate, wherein L 1 Selected from:

[0040] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the -M- is selected from:

[0041] In some embodiments, the present invention provides an anti-B7H3 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0042] In some embodiments, the present invention provides an anti-B7H3 antibody drug conjugate, wherein the cytotoxic drug

[0043] Select from any of the following structures:

[0044] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the linker unit L is -L a -L b -L c -,and

[0045] -L a -for

[0046] -L b -Selected from:

[0047] Preferably

[0048] -L c -for

[0049] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the linker unit L, wherein L a The end is connected to Ab, L c The end is connected to the connector unit M.

[0050] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the linker unit L is

[0051] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the linker unit L is

[0052] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the structure of the antibody-drug conjugate is shown in Formula (A-2):

[0053] in,

[0054] p represents the average number of connections, and p is an integer or decimal selected from 1 to 10;

[0055] Ab, M, and L are the same as those described in any solution of this application.

[0056] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the structure of the antibody-drug conjugate is shown in Formula (A-2):

[0057] in,

[0058] p represents the average number of connections, and p is an integer or decimal selected from 1 to 10;

[0059] Ab is an antibody targeting B7H3 or an antigen-binding fragment of the antibody, comprising: a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 2, a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 3, a LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, a LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and a LCDR3 with an amino acid sequence as shown in SEQ ID NO: 6;

[0060] M is -L 2 -L 1 -C(O)-;

[0061] L 2 is -O- or -S-; preferably -O-;

[0062] L 1 -(C(R 1a )(R1b )) m -CH2-;

[0063] m is selected from 1, 2, 3 or 4; preferably 1 or 2;

[0064] R 1a or R 1b Each independently may be hydrogen, halogen or C1-C6 alkyl optionally substituted by 1, 2 or 3 R;

[0065] R can each independently be hydrogen or halogen;

[0066] L is

[0067] In some embodiments, the present invention provides an antibody-drug conjugate, wherein p is an integer or decimal of 2-8; for example, p is an integer or decimal of 4-8, for example, p is an integer or decimal of 4-6 or 6-8.

[0068] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the structure of the antibody-drug conjugate is as shown in Formula (A-2a) or Formula (A-2b):

[0069] in,

[0070] p represents the average number of connections, and p is selected from an integer or decimal of 1 to 10, preferably an integer or decimal of 2-8, for example, p is an integer or decimal of 4-8, for example, p is an integer or decimal of 4-6 or 6-8;

[0071] Ab is an antibody or an antigen-binding fragment thereof targeting B7H3, comprising: a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 2, a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 3, a LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, a LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and a LCDR3 with an amino acid sequence as shown in SEQ ID NO: 6;

[0072] L 2 is -O- or -S-; preferably -O-;

[0073] X1 is selected from the group consisting of 1, 2 or 3 R 2a Substituted C3-C6 saturated cycloalkyl;

[0074] X2 is selected from -(C(R 1a )(R 1b )) m -CH2-;

[0075] m is selected from 1, 2, 3 or 4; preferably 1 or 2;

[0076] R 1a 、R 1b or R 2a Each independently may be hydrogen, halogen or C1-C6 alkyl optionally substituted by 1, 2 or 3 R;

[0077] Each R independently may be hydrogen or halogen.

[0078] In some embodiments, the present invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate is selected from the following structural formula:

[0079] in,

[0080] p represents the average number of connections, and p is selected from an integer or decimal from 1 to 10, preferably an integer or decimal 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 6 to 8; Ab is the same as described in any scheme of the present application.

[0081] In some embodiments, the present invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is selected from:

[0082] in,

[0083] p represents the average number of connections, and p is an integer or decimal selected from 1 to 10, preferably an integer or decimal of 2-8, for example, p is an integer or decimal of 4-8, for example, p is an integer or decimal of 4-6 or 6-8, for example, p is 7.45, 5.68 or 4.02; WBP301088 is an anti-B7H3 antibody comprising a heavy chain as shown in SEQ ID NO: 12 and a light chain as shown in SEQ ID NO: 13.

[0084] In some embodiments, the present invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is selected from:

[0085] in,

[0086] p represents the average number of connections, and p is an integer or decimal selected from 1 to 10, preferably an integer or decimal of 2-8, for example, p is an integer or decimal of 4-8, for example, p is an integer or decimal of 4-6 or 6-8, for example, p is 7.45, 5.68 or 4.02;

[0087] WBP301088 is an anti-B7H3 antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 12, and the light chain amino acid sequence of which is shown in SEQ ID NO: 13. In some embodiments, the average number of linkages p in the antibody-drug conjugates of the present invention can be an integer or decimal from 2 to 8. For example, the average number of linkages p can be an integer or decimal from 3 to 8. For example, the average number of linkages p can be an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0088] In yet another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate as described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0089] In another aspect, the present invention provides use of an antibody-drug conjugate as described herein, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein in the preparation of a medicament for treating and / or preventing a disease or condition mediated by B7H3, preferably cancer. Preferably, the disease or condition is a disease or condition in which B7H3 is positively expressed.

[0090] In another aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by B7H3, comprising administering to a subject in need thereof an antibody-drug conjugate as described herein, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein, preferably, the disease or condition is cancer. Preferably, the disease or condition is a disease or condition in which B7H3 is positively expressed.

[0091] In another aspect, the present invention provides an antibody-drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein for use in treating and / or preventing a disease or condition mediated by B7H3, preferably, the disease or condition is cancer. Preferably, the disease or condition is a disease or condition in which B7H3 is positively expressed.

[0092] In some embodiments, the cancer of the present invention is selected from 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 carcinoma, lymphoma (such as non-Hodgkin's lymphoma), chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma.

[0093] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody-drug conjugate as described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0094] In yet another aspect, the present invention provides a kit comprising the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1: SDS-PAGE results of W301088-1.145.16-z3-p1-uIgG1KV320. TM Unstained protein ladder; Lane 1: W301088-1.145.16-z3-p1-uIgG1KV320, non-reducing; Lane 2: W301088-1.145.16-z3-p1-uIgG1KV320, reducing; Gel information: NuPAGE, Novex 4-12% Bis-Tris gel.

[0096] Figure 2: SEC-HPLC results of W301088-1.145.16-z3-p1-uIgG1KV320.

[0097] Figure 3: DSF curve of W301088-1.145.16-z3-p1-uIgG1KV320.

[0098] Figure 4: HIC-HPLC curve of W301088-1.145.16-z3-p1-uIgG1KV320.

[0099] Figure 5: DLS-k of W301088-1.145.16-z3-p1-uIgG1KV320 D curve.

[0100] Figure 6: FACS binding results of antibodies W301088-1.145.16-xIgG1KV320 and W301088-1.145.16-z3-p1-uIgG1KV320 to MCF-7 cells expressing human B7H3. Enoblituzumab, an anti-B7H3 antibody, was used as a positive control, and hIgG1 was used as a negative control.

[0101] Figure 7: FACS binding results of antibodies W301088-1.145.16-xIgG1KV320 and W301088-1.145.16-z3-p1-uIgG1KV320 to cells transfected with cynomolgus monkey B7H3. Enoblituzumab, an anti-B7H3 antibody, was used as a positive control; hIgG1 isotype was used as a negative control.

[0102] 8A-8C : Binding of antibody W301088-1.145.16-xIgG1KV320 ( FIG. 8A ), antibody W301088-1.145.16-z3-p1-uIgG1KV320 ( FIG. 8B ), and MacroGenics' enoxaparin ( FIG. 8C ) to human 4IgB7H3.

[0103] 9A-9C : Binding of antibody W301088-1.145.16-xIgG1KV320 ( FIG. 9A ), antibody W301088-1.145.16-z3-p1-uIgG1KV320 ( FIG. 9B ), and MacroGenics' enoxaparin ( FIG. 9C ) to human 2IgB7H3.

[0104] Figures 10A-10C: Binding of antibodies W301088-1.145.16-xIgG1KV320 (Figure 10A), W301088-1.145.16-z3-p1-uIgG1KV320 (Figure 10B), and MacroGenics' Enozutuzumab (Figure 10C) to cynomolgus monkey B7H3.

[0105] Figure 11: Internalization of antibodies W301088-1.145.16-xIgG1KV320 and W301088-1.145.16-z3-p1-uIgG1KV320 by MCF-7 cells expressing human B7H3 after antibody binding. The anti-B7H3 antibody Enoblituzumab was used as a positive control; the hIgG1 isotype was used as a negative control.

[0106] Figures 12A-12B: Alignment of the amino acid sequences of the VH and VL regions of antibodies W301088-1.145.16-xIgG1KV320 and W301088-1.145.16-z3-p1-uIgG1KV320. The boxed amino acid segments in the figures represent the CDR regions in the VH and VL regions.

[0107] Figure 13: ELISA test of the binding activity of antibody-drug conjugates and their unconjugated antibodies to human B7H3 antigen. In the figure, "WBP301088" represents the unconjugated antibody W301088-1.145.16-z3-p1-uIgG1KV320.

[0108] Figure 14: ELISA detection of the binding of antibody-drug conjugates and their unconjugated antibodies to various B7 family proteins. In the figure, "WBP301088" represents the unconjugated antibody W301088-1.145.16-z3-p1-uIgG1KV320.

[0109] Figure 15: Evaluation of the efficacy of antibody-drug conjugates in Calu-6 tumor-bearing mice.

[0110] Figure 16: Evaluation of the efficacy of antibody-drug conjugates in A375 tumor-bearing mice.

[0111] Figure 17: Evaluation of the efficacy of antibody-drug conjugates in U87 tumor-bearing mice.

[0112] Figure 18: Evaluation of the efficacy of antibody-drug conjugates in PC-3 tumor-bearing mice.

[0113] Figure 19A and Figure 19B: Evaluation of the efficacy of antibody-drug conjugates in a human prostate cancer xenograft model.

[0114] Figure 20A and Figure 20B: Evaluation of the efficacy of antibody-drug conjugates in a prostate cancer microtumor xenograft model. DETAILED DESCRIPTION

[0115] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0116] Definition of terms

[0117] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0118] In order to more easily understand the present invention, some scientific and technological terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this article, the scientific and technological terms used herein all have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms of this area, those skilled in the art can specifically refer to Current Protocols in Molecular Biology (Ausubel F. et al., John Wiley & Sons, New York, 2000). The abbreviations of amino acid residues are standard three-letter and / or single-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form, unless otherwise clearly specified in the text.

[0119] The term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, 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% above or below the specified value.

[0120] The term "antibody" or "Ab" is used in its broadest sense herein to include various antibody structures, including polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies). Natural intact antibodies are typically Y-shaped tetrameric proteins comprising two heavy (H) and two light (L) polypeptide chains bound together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be classified into kappa and lambda light chains. Heavy chains can be classified into μ, δ, γ, α, and ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is typically composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions (CDRs)), which are separated by relatively conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form an antigen binding site, respectively. The extent of the framework regions and CDRs can be accurately identified using methods known in the art, such as the Kabat definition, the definition on the Dr. Martin website, the Chothia definition, the AbM definition, the EU definition, and the Contact definition, all of which are well known in the art.See, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Bulletin No. 91-3242; Martin A, “Antibody bioinformatics website of Dr. Andrew Martin′s lab at UCL,” last updated July 31, 2018; Chothia et al. (1989) Nature, Vol. 342: 877; Chothia, C. et al. (1987) J. Mol. Biol., Vol. 196: 901-917; Al-lazikani et al. (1997) J. Molec. Biol., Vol. 273: 927-948; Edelman et al., Proc Natl Acad Sci USA, May 1969, Vol. 63, No. 1: pp. 78-85; and Almagro, J. Mol. Recognit., Vol. 17: pp. 132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. The correspondence or alignment between the numberings according to different definitions can be found, for example, at http: / / www.imgt.org / (see also Giudicelli V et al., "IMGT, the international ImMunoGeneTics database", Nucleic Acids Res., (1997) Vol. 25: pp. 206-211; Lefranc MP et al., "Unique database numbering system for immunogenetic analysis", Immunol Today (1997), Vol. 18: p. 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), Vol. 27: pp. 55-77).The antibodies can be of different antibody isotypes, such as IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtypes), IgAl, IgA2, IgD, IgE, or IgM antibodies.

[0121] The term "B7H3," also known as the CD276 antigen, refers to a type I transmembrane protein that belongs to the B7 family and has an extracellular domain consisting of a single IgV-IgC domain. B7 family proteins 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 a variety of cancers. The amino acid sequence of a B7H3 protein includes a full-length B7H3 protein (such as a human 4IgB7H3 protein or a human 2IgB7H3 protein), or the extracellular domain of B7H3 (B7H3 ECD) or a fragment containing the B7H3 ECD; a B7H3-ECD fusion protein. Exemplary sequences of B7H3 proteins are shown in Uniprot ID: Q5ZPR3 (human 4IgB7H3), Genebank accession numbers NP_001019907 (human), NP_001316557 (human), NP_001316558 (human), NP_079516 (human), and NP_598744 (mouse). The amino acid sequence homology of cynomolgus monkey B7H3 to human and mouse B7H3 is approximately 97% and 88%, respectively.

[0122] The term "antibody that binds to B7H3" or "anti-B7H3 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize a B7H3 protein, as well as antibodies and antigen-binding fragments thereof that specifically bind to a B7H3 protein. As used herein, "anti-B7H3 antibody" includes monovalent antibodies having a single specificity, as well as bispecific antibodies comprising a first antigen-binding site that binds to B7H3 and a second antigen-binding site that binds to a second antigen.

[0123] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0124] The term "full-length antibody" refers to a naturally occurring immunoglobulin molecule comprising four peptide chains: two heavy (H) chains (about 50-70 kDa at full length) and two light (L) chains (about 25 kDa at full length) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementary determining regions (CDRs) and CDRs separated by more conserved regions called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0125] The term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains, which are designated HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 for each heavy and light chain variable region. The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (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)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and affinity clustering based on a large number of crystal structures. The boundaries of the CDRs of the antibodies of the present invention can be determined by a person skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0126] The term "antigen-binding fragment" or "antibody fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, generally including at least a fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as scFv; nanobodies formed from antibody fragments; and multispecific antibodies. When the binding activity to the antigen is expressed on a molar concentration basis, the antigen-binding fragment or derivative generally retains at least 10% of the antigen-binding activity of the parent antibody. 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-binding fragments of an antibody may include conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants") or non-conservative amino acid substitutions that do not significantly alter their biological activity.

[0127] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from an antibody such as a rodent ("parent antibody"), while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0128] The term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0129] In the present application, the term "halogen" generally refers to fluorine, chlorine, bromine, and iodine, for example, it can be fluorine or chlorine.

[0130] In the present application, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. An alkyl group can be substituted or non-substituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight or branched aliphatic hydrocarbon group having a residue derived from the same carbon atom or two different carbon atoms of the parent alkane by removing a hydrogen atom, and can be a straight or branched group containing 1 to 20 carbon atoms, for example, a chain alkyl group containing 1 to 12 carbon atoms, for example, 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. The alkyl group may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituent may be substituted at any available point of attachment, and the substituent may be independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, 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 Aliphatic groups, for example, when substituted with cycloalkyl groups, are cycloalkylalkyl groups.

[0131] In this application, the term "alkylene" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane group, and can be a straight-chain or branched group containing 1 to 20 carbon atoms. For example, the term "methylene" can refer to a residue derived from a group containing 1 carbon atom by removing two hydrogen atoms. The methylene group can be substituted or unsubstituted, substituted or unsubstituted; for example, an alkylene group containing 1 to 12 carbon atoms, for example, an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-pentylene (-CH2CH2CH2CH2CH2-), and the like. Alkylene may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituent may be substituted at any available point of attachment, and the substituent may be independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, 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 Aliphatic groups. Methylene or alkylene groups may be substituted or unsubstituted.

[0132] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl are as defined herein. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0133] In this application, the term "alkenyl" generally refers to a straight or branched hydrocarbon group containing one or more double bonds. Illustrative examples of alkenyl include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, and hexenyl. Illustrative examples of C2-6 alkenyl groups with one or more double bonds include butadienyl, pentadienyl, hexadienyl, and hexatrienyl, as well as branched forms thereof. The position of the unsaturated bond (double bond) can be at any position of the carbon chain. Alkenyl groups can be substituted or unsubstituted. For example, when substituted, the substituent may be substituted at any available point of attachment and is preferably independently optionally selected from the group consisting of one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, heterocycloalkylthio and oxo, for example, it can be 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 Aliphatic group.

[0134] In this application, the term "alkenylene" generally refers to a residue derived from an alkene carbon atom by removing two hydrogen atoms. For example, it can be allylene, vinylene, butenylene, pentenylene and hexenylene. Alkenylene can be substituted or unsubstituted. For example, when substituted, the substituent may be substituted at any available point of attachment and is preferably independently optionally selected from the group consisting of one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, heterocycloalkylthio and oxo, for example, it can be 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 Aliphatic group.

[0135] In this application, the term "alkynyl" generally refers to a straight or branched hydrocarbon group containing one or more triple bonds. Alkynyl can be an unsaturated straight or branched alkynyl group, such as ethynyl, 1-propynyl, propargyl, butynyl, etc. Alkynyl can be substituted or unsubstituted. For example, when substituted, the substituent may be substituted at any available point of attachment and is preferably independently optionally selected from the group consisting of one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, heterocycloalkylthio and oxo, for example, it can be 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 Aliphatic group.

[0136] In this application, the term "alkynylene" generally refers to a residue derived from a carbon atom of an alkyne by removing two hydrogen atoms. For example, it may be ethynylene, propynylene, propargylene, butynylene, etc. Alkynylenes may be substituted or unsubstituted. For example, when substituted, the substituent may be substituted at any available point of attachment and is preferably independently optionally selected from the group consisting of one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, heterocycloalkylthio and oxo, for example, it can be 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 Aliphatic group.

[0137] In this application, the term "aryl" generally refers to a residue derived from an aromatic ring by removing a hydrogen atom. The term "aromatic ring" can refer to a 6- to 14-membered all-carbon monocyclic ring or a fused polycyclic ring (i.e., a ring sharing adjacent pairs of carbon atoms) with a conjugated π electron system, which can be 6- to 10-membered, such as benzene and naphthalene. The aromatic ring can be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring connected to the parent structure is the aryl ring. The aryl group can be substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups independently selected from the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. The aryl group can be substituted or unsubstituted.

[0138] As used herein, the term "heteroaryl" generally refers to a residue derived from a carbon atom in a heteroaromatic ring by removing a hydrogen atom. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms may be selected from the group consisting of oxygen, sulfur, and nitrogen. A heteroaryl group may be 5 to 10-membered, and may be 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. Heteroaryl can be optionally substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. Heteroaryl can be substituted or unsubstituted.

[0139] As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein 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, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutane, cyclopentane, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cycloheptatrienyl, cyclooctanyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls. Cycloalkyls may be substituted or unsubstituted. When substituted, the substituents may be present at any available point of attachment, preferably independently selected from one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0140] As used herein, the term "partially unsaturated" generally refers to a cyclic structure containing at least one double or triple bond between the ring molecules. The term "partially unsaturated" encompasses cyclic structures with multiple sites of unsaturation but is not intended to include aromatic or heteroaromatic rings as defined herein. The term "unsaturated" refers to a moiety having one or more degrees of unsaturation.

[0141] As used herein, 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 is a heteroatom selected from nitrogen, oxygen, or sulfur, and the remaining ring atoms being carbon. Preferably, the substituent contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; more preferably, it contains 3 to 6 ring atoms, of which 1 to 3 are heteroatoms; and most preferably, it contains 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl. Polycyclic heterocyclyls include spirocyclic, fused, and bridged heterocyclyls. The heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring; the ring attached to the parent structure is the heterocyclyl. The heterocyclic group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available attachment point. The substituent is preferably independently selected from one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0142] In this application, the term "ring-forming atoms" generally refers to atoms contained in a ring structure. For example, ring-forming atoms can be carbon atoms in a benzene ring or nitrogen atoms in a pyridine ring. When a ring-forming atom is attached to a hydrogen atom, the ring-forming atom can be substituted or unsubstituted.

[0143] As used herein, the term "each independently" generally refers to a variable being applicable in any instance, regardless of the presence or absence of variables with the same or different definitions in the same compound. For example, the variable may refer to the type or number of substituents in a compound, or the type of atoms in the compound. For example, if R occurs twice in a compound and R is defined as "independently carbon or nitrogen," both R may be carbon, both R may be nitrogen, or one R may be carbon and the other R may be nitrogen.

[0144] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0145] In this application, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, which are independently replaced by a corresponding number of substituents. The substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) whether substitution is possible or not without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0146] In this application, the term 0 or more (e.g., 0 or more, 0 or 1, 0) methylene units are "replaced" generally means that when the structure contains 1 or more methylene units, the one or more methylene units may be unsubstituted or replaced by one or more groups that are not 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-, -SONH-, -C(=S)-, -C(=NH)-, -N=N-, -C=N-, -N=C- or -C(=N2)-).

[0147] In this application, the "connection" of group X to group Y can generally be in any orientation, which generally means that when group X is used to connect group Y and group Z, the two or more connection sites of group X can be connected to group Y or group Z at will.

[0148] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application can be an organic compound, for example, the compound of the present application can be a compound with a molecular weight of 500 Daltons or less, a compound with a molecular weight of 1000 Daltons or less, a compound with a molecular weight of 1000 Daltons or more, or a compound with a molecular weight of 1000 Daltons or more, or a compound with a molecular weight of 10000 Daltons or more, or a compound with a molecular weight of 100000 Daltons or more. In this application, a compound can also refer to a compound connected by chemical bonds, for example, a compound in which one or more molecules with a molecular weight of 1000 Daltons or less are connected to a biomacromolecule by chemical bonds, and the biomacromolecule can be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound of the present application can include a compound in which a protein is connected to one or more molecules with a molecular weight of 1000 Daltons or less, a compound in which a protein is connected to one or more molecules with a molecular weight of 10000 Daltons or less, or a compound in which a protein is connected to one or more molecules with a molecular weight of 100000 Daltons or less.

[0149] In this application, as known to those skilled in the art, terms such as "alkyl", "alkenyl", "cycloalkyl", etc. may be preceded by an identifier to indicate the number of atoms present in the group in a particular case, for example, C1-C4 alkyl, C3-C7 cycloalkyloxy, C1-C4 alkylcarbonylamino, etc., and the subscript number after "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group with three carbon atoms (e.g., n-propyl, isopropyl); C 1-10 In the examples, the members of the group may have any number of carbon atoms falling within the range of 1-10.

[0150] One or more hydrogen atoms in a group, for example up to 5, for example 1 to 3, hydrogen atoms, are independently replaced by a corresponding number of substituents. The substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0151] In the present application, the compound or ligand-drug conjugate of the present application includes its tautomer, mesomorph, racemate, enantiomer, and / or diastereomer. In the present application, the term "diastereomer" generally refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, for example, melting points, boiling points, spectral properties and reactivity. In the present application, the terms "tautomer" or "tautomeric form" are used interchangeably and generally refer to structural isomers of different energies that can be mutually converted by a low energy barrier. For example, proton tautomers (proton tautomers) (also referred to as prototropic tautomers) include mutual conversions performed by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers (valence tautomers) include mutual conversions performed by the reorganization of some bonding electrons. In this application, the term "meso" generally refers to a molecule containing an asymmetric atom but having symmetry factors such that the total optical rotation within the molecule is zero. The term "racemate" or "racemic mixture" refers to a composition composed of equimolar amounts of two enantiomeric species.

[0152] In this application, the term "isomer" of a compound or ligand-drug conjugate generally encompasses tautomers, mesomers, racemates, enantiomers, and diastereomers of the compound.

[0153] The term "ligand-drug conjugate" generally refers to a ligand linked to a biologically active cytotoxic drug via a stable linker. In this application, a "ligand-drug conjugate" may be an antibody-drug conjugate (ADC), which may be an antibody or antibody fragment linked to a biologically active drug (e.g., a cytotoxic drug) via a stable linker.

[0154] In this application, the term "ligand" generally refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The function of the ligand can be to present the drug to the target cell population bound to the ligand. These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells, receptors and / or antigens. In this application, the ligand can be expressed as Ab, and the ligand antigen forms a connection bond with the linker through the heteroatoms on the ligand. The ligand can be an antibody or an antigen-binding fragment thereof (Ab), and the antibody can be selected from a chimeric antibody, a humanized antibody, a fully human antibody or a mouse antibody; the antibody can be a monoclonal antibody.

[0155] The term "cytotoxic drug" generally refers to a toxic drug, which can be a chemical molecule that has a strong ability to disrupt the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations. The "cytotoxic drug" can include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 or radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, for example, can be toxic drugs, including but not limited to camptothecin derivatives, for example, can be the camptothecin derivative 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).

[0156] The term "linker unit" or "linker structure" generally refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end, and can also be connected to a cytotoxic drug after being connected to another linker. Direct or indirect connection to a ligand can refer to a group directly connecting to the ligand through a covalent bond, or it can be connected to the ligand through a linker structure. For example, a chemical structure fragment or bond comprising an acid-labile linker structure (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure can be used as a linker structure.

[0157] The term "optionally linked to other molecular moieties" generally means that the structure is not linked to any other chemical structure, or that the structure is linked (e.g., via a chemical bond or via a linker structure) to one or more other chemical structures (e.g., ligands described herein) that are different from the structure.

[0158] The term "drug loading" generally refers to the average number of cytotoxic drugs loaded per ligand and can also be expressed as the ratio of the cytotoxic drug to the antibody. The cytotoxic drug loading can range from 0 to 12, for example, 1 to 10, cytotoxic drugs per ligand (Ab). In embodiments of the present application, the drug loading is expressed as DAR, which can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The drug loading of each ADC molecule after the conjugation reaction can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0159] In the present application, some atoms of the compounds of the present application may appear in more than one isotopic form. For example, hydrogen may appear in the form of protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), carbon may exist in three different isotopes ( 12 C. 13 C and 14 C) naturally occurring. Examples of isotopes that can be incorporated into the compounds of the present application also 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. 125I, or similar isotopes. Therefore, the compounds of the present application may be enriched in one or more of these isotopes relative to the natural abundance of these isotopes. As known to those skilled in the art, such isotopically enriched compounds can be used for a variety of purposes. For example, using heavy isotopes such as deuterium ( 2 Deuterium (H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability. 2 The natural abundance of deuterium (H) is about 0.015%. Therefore, there is one deuterium atom for approximately every 6500 hydrogen atoms in nature. Therefore, the deuterium-containing compounds of the present application have a deuterium abundance greater than 0.015% at one or more positions (as the case may be). Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds whose remaining portions are consistent with the present structure except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon 13 or carbon 14, are within the scope of the present application.

[0160] The term "pharmaceutical composition" generally refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. Conventional pharmaceutical composition preparations can be found in the Chinese Pharmacopoeia. Pharmaceutical compositions can be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable preparations can also be sterile injectable solutions or suspensions prepared in a non-toxic, parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, sterile fixed oils can conveniently be used as solvents or suspending media. For example, any blended fixed oil, including synthetic mono- or di-glycerides, can be used. Fatty acids, such as oleic acid, can also be used to prepare injectable formulations.

[0161] The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" generally refers to a salt of a compound or ligand-drug conjugate of the present application, or a salt of a compound described in the present application. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The antibody-drug conjugate of the present application may 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, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0162] The term "pharmaceutically acceptable carrier" generally refers to a carrier or vehicle for administering therapeutic agents, such as antibodies or polypeptides, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and that can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, etc., may also be present in these carriers.

[0163] The terms "treatment" and "treating" generally refer to an approach to obtaining a beneficial or desired effect, including, but not limited to, a therapeutic benefit. A therapeutic benefit includes, but is not limited to, eradication, inhibition, reduction, or amelioration of the underlying disease being treated. Additionally, a therapeutic benefit is achieved by eradication, inhibition, reduction, or amelioration of one or more physiological symptoms associated with the underlying disease, such that an improvement is observed in the patient, but the patient may still be suffering from the underlying disease.

[0164] The term "B7H3-associated disease" refers to any disease or condition caused by, exacerbated by, or otherwise associated with increased or decreased (generally increased) expression or activity of B7H3 (eg, human B7H3).

[0165] The terms "prevention" and "preventing" generally refer to an approach to obtaining a beneficial or desired effect, including, but not limited to, a prophylactic benefit. For prophylactic benefit, a pharmaceutical composition can be administered to a patient at risk for developing a particular disease or to a patient reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

[0166] The term "subject" or "patient" generally refers to humans (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and / or turkeys.

[0167] The terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of a ligand-drug conjugate of the invention, alone or in combination with other therapeutic agents, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions, or the progression of such diseases or conditions, when administered to a cell, tissue, or subject. A therapeutically effective dose also refers to a dose sufficient to result in amelioration of symptoms, e.g., an amount that treats, cures, prevents, or ameliorates a related medical condition, or increases the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to a subject, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients, whether administered in combination, sequentially, or simultaneously, that results in a therapeutic effect. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0168] The term "cancer" is used herein to refer to a group of cells that exhibit an abnormally high level of proliferation and growth. Cancer may be benign (also known as a benign tumor), pre-malignant, or malignant. Cancer cells may be solid cancer cells or blood cancer cells. The term "tumor" as used herein refers to one or more cells comprising a cancer. The term "tumor growth" is used herein to refer to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.

[0169] Anti-B7H3 antibodies

[0170] 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 their ECD domains. The term "antibody" as used herein is defined above and includes full-length immunoglobulins and antibody portions thereof that bind to the same antigen. The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, or a single-chain antibody. In some embodiments, the antibody portion is a Fab fragment or a F(ab')2 fragment. In some embodiments, the antibody portion retains the ability to specifically bind to B7H3.

[0171] Recombinant anti-B7H3 antibodies, such as chimeric antibodies and humanized monoclonal antibodies, including human and non-human portions, can be prepared using standard recombinant DNA techniques and are within the scope of the present invention. Such chimeric 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, reverse mutation, and removal of post-translational modification sites.

[0172] Preferably, the antibodies or antigen-binding portions thereof as disclosed herein can bind to human 4IgB7H3 with high affinity. The antibodies or antigen-binding portions thereof as disclosed herein can bind to human 2IgB7H3 with much lower affinity. The antibodies or antigen-binding portions thereof as disclosed herein can bind to human 4IgB7H3 with high affinity, but bind to human 2IgB7H3 with much lower affinity.

[0173] The binding of the antibodies of the invention to B7H3 can be assessed using one or more techniques recognized in the art, such as ELISA or flow cytometry. In some embodiments, the antibodies can be tested by flow cytometry analysis, wherein the antibodies react with a cell line expressing human B7H3, such as MCF-7 cancer cells or CHOK1 cells that have been transfected to express B7H3 on their cell surface. In addition, the binding of the antibodies can also be tested in a binuclear binding assay, including binding kinetics (e.g., KD values). In some other embodiments, the antibodies are tested by ELISA, wherein the antibodies react with soluble B7H3 protein.

[0174] The present invention provides an antibody that binds to human 4IgB7H3 protein, and its K D 1×10 -8 M or smaller, K D 1×10 -9 M or smaller, K D 5×10 -10 M or smaller, K D 1×10 -10 M or smaller, K D 9×10 -11 M or smaller, K D 8×10 -11 M or smaller or K D 7×10 -11 M or less. The antibodies disclosed herein can also bind to human 2IgB7H3 protein as measured by surface plasmon resonance (SPR), with a K D 5×10 -9 M or above, K D 1×10 -8 M or above, K D 2×10 -8 M or above, K D 3×10 -8 M or above or KD is 4×10 -8 M or more. In some embodiments, the antibodies herein bind to human 4IgB7H3, and their K D The K value of binding to human 2IgB7H3 DThe value is smaller than 500 times, smaller than 100 times, or smaller than 50 times. D The value can be measured by surface plasmon resonance (SPR).

[0175] In some embodiments, the antibodies of the invention are capable of inhibiting the expression of antibodies with an EC of less than 5 nM, less than 4 nM, less than 3 nM, or less than 2 nM. 50 Binding to human or cynomolgus monkey B7H3 expressing cell lines was determined by FACS.

[0176] In some embodiments, the antibody or its antigen-binding portion is a chimeric or murine antibody that specifically binds to B7H3, preferably human 4IgB7H3. In some further embodiments, the antibody or its antigen-binding portion is a humanized antibody that specifically binds to B7H3, preferably human 4IgB7H3. In some further embodiments, the humanized antibody or its antigen-binding portion comprises one or more back mutations in the framework region. In some further embodiments, the humanized antibody or its antigen-binding portion comprises one or more modifications at potential post-translational modification (PTM) sites, for example, to remove any amino acid other than NG, NS, and DG in CDRs, NxS, and NxT (X can be other than P in the full length).

[0177] In some embodiments, an antibody, or antigen binding portion thereof, as disclosed herein comprises one or more heavy chain CDRs (HCDRs) selected from at least one of the group consisting of:

[0178] (i) HCDR1 comprising SEQ ID NO: 1;

[0179] (ii) HCDR2 comprising SEQ ID NO: 2; and

[0180] (iii) HCDR3 comprising SEQ ID NO: 3; and / or

[0181] One or more light chain CDRs (LCDRs) are selected from at least one of the following groups:

[0182] (i) LCDR1 comprising SEQ ID NO: 4, 7, or 18;

[0183] (ii) LCDR2 comprising SEQ ID NO: 5; and

[0184] (iii) LCDR3 comprising SEQ ID NO:6.

[0185] In some embodiments, the antibodies or antigen-binding portions thereof described above may include: a HCDR1 comprising or consisting of SEQ ID NO: 1; a HCDR2 comprising or consisting of SEQ ID NO: 2; a HCDR3 comprising or consisting of SEQ ID NO: 3; a LCDR1 comprising or consisting of SEQ ID NO: 7; a LCDR2 comprising or consisting of SEQ ID NO: 5; and a LCDR3 comprising or consisting of SEQ ID NO: 6. Specifically, the antibodies may further comprise one or more modifications in the CDRs to remove potential PTM sites. In some embodiments, the antibodies comprise one or more modifications in the LCDR1 sequence of SEQ ID NO: 7 ("KSSQSLLNSSNQKNYLA") to remove "NS," which is a potential PTM site in the sequence. In some preferred embodiments, the antibodies comprise a substitution at position 8 or 9 of the LCDR1 amino acid sequence of SEQ ID NO: 7 ("KSSQSLLNSSNQKNYLA"). In some more preferred embodiments, the antibody comprises an N to Q substitution at position 8 of the amino acid sequence of LCDR1 (i.e., SEQ ID NO: 4, "KSSQSLLQSSNQKNYLA"). In some other embodiments, the antibody comprises an S to P substitution at position 9 of the amino acid sequence of LCDR1 (i.e., SEQ ID NO: 18, "KSSQSLLNPSNQKNYLA"). It will be understood by those skilled in the art that other types of substitutions may also be selected as long as the binding affinity for B7H3 is substantially retained.

[0186] In some embodiments, the antibodies or antigen-binding portions thereof described above may include: HCDR1 comprising or consisting of SEQ ID NO: 1; HCDR2 comprising or consisting of SEQ ID NO: 2; HCDR3 comprising or consisting of SEQ ID NO: 3; LCDR1 comprising or consisting of SEQ ID NO: 4; LCDR2 comprising or consisting of SEQ ID NO: 5; LCDR3 comprising or consisting of SEQ ID NO: 6.

[0187] 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 comprise HCDR1-3 and LCDR1-3, respectively, as described above.

[0188] In some embodiments, the heavy chain variable region of the antibody, or antigen binding portion thereof, comprises:

[0189] (i) an 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 that has 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: 8 or 10.

[0190] In some embodiments, amino acid substitutions may be conservative substitutions.

[0191] In some embodiments, the light chain variable region of the antibody, or antigen binding portion thereof, comprises:

[0192] (i) an amino acid sequence of SEQ ID NO: 9 or 11; (ii) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 9 or 11; (iii) an amino acid sequence that has 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.

[0193] In some embodiments, amino acid substitutions may be conservative substitutions.

[0194] 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 VH comprises:

[0195] (i) the amino acid sequence of SEQ ID NO: 8 or 10; (ii) an amino acid sequence comprising one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid substitutions in the framework regions compared to SEQ ID NO: 8 or 10;

[0196] and / or VL including:

[0197] (i) the amino acid sequence of SEQ ID NO: 9 or 11; or (ii) an amino acid sequence comprising one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid substitutions in the framework regions compared to SEQ ID NO: 9 or 11.

[0198] In some embodiments, amino acid substitutions may be conservative substitutions.

[0199] In some embodiments, the antibody or antigen binding portion comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region (VH) of SEQ ID No: 8 or 10, and LCDR1, LCDR2 and LCDR3 of the light chain variable region (VL) of SEQ ID No: 9 or 11.

[0200] As will be appreciated by those skilled in the art, the exact numbering and placement of CDRs may vary in different numbering systems. However, it should be understood that disclosure of the variable heavy chain and / or variable light chain sequences includes disclosure of the associated (intrinsic) CDRs. Thus, disclosure of each heavy chain variable region is disclosure of the heavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR3), and disclosure of each light chain variable region is disclosure of the light chain CDRs (e.g., LCDR1, LCDR2, and LCDR3). The assignment of amino acids to each CDR may be based on one or a combination of numbering schemes, all of which are well known in the art. Below is a comparison of CDR numbering, see Lafranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003):

[0201] In some embodiments, the CDR amino acid sequences may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequences. In some embodiments, the variable region amino acid sequences may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequences.

[0202] As described above, the addition, deletion and / or substitution of at least one amino acid in the VH or VL region may not be in any CDR sequence, but rather in the framework (FR) sequence. For example, an isolated antibody or antigen-binding portion thereof may comprise one or more replacements of amino acids in a framework sequence, such as FR1, FR2, FR3 and / or FR4 of the VH or VL region. In some embodiments, the framework region of the VH region comprises one or more of the following substitutions: S025T, V071R, Y091F, V089T.

[0203] In some embodiments, the changes in the 6 CDRs can have a total of 0, 1, 2, 3, 4 or 5 amino acid modifications (preferably amino acid substitutions), as well as changes in the framework regions of the heavy and light chain variable regions, as long as the framework (excluding the CDRs) maintains at least about 80, 85 or 90% identity with the parent antibody (e.g., W301088-1.145.16). Thus, the same CDRs as described herein can be combined with different framework sequences from human germline sequences, as long as the framework regions maintain at least 80, 85 or 90% identity with the human germline sequences.

[0204] In certain embodiments, the isolated antibodies or antigen-binding portions thereof provided herein comprise any suitable framework region (FR) sequences, so long as the antigen-binding domain can specifically bind to B7H3, preferably human Ig4B7H3.

[0205] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0206] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0207] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous 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, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran 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.

[0208] Any suitable method for producing antibodies can be used to produce the antibodies of the present invention. Any suitable form of B7H3 can be used as an immunogen (antigen) for producing antibodies. By way of example and not limitation, any B7H3 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing murine monoclonal anti-human B7H3 antibodies can be produced by methods known in the art. Antibodies derived from rodents (e.g., mice) can cause unwanted antibody immunogenicity when used as therapeutic agents in vivo. Repeated use can lead to an immune response against the therapeutic antibody in the human body, which can at least result in a loss of therapeutic efficacy and, in severe cases, potentially lethal allergic reactions. One method of reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies in which mouse variable regions are fused to human constant regions (Liu et al. (1987), Proc. Natl. Acad. Sci. USA 84: 3439-3443). However, the retention of intact rodent variable regions in chimeric antibodies can still cause adverse immunogenicity in patients. Grafting the complementarity determining region (CDR) loops of rodent variable domains onto human frameworks (i.e., humanization) has been used to further minimize rodent sequence (Jones et al. (1986), Nature 321:522; Verhoeyen et al. (1988), Science 239:1534).

[0209] In some embodiments, chimeric or humanized antibodies of the present invention can be prepared based on the sequences of prepared mouse monoclonal hybridoma antibodies. DNA encoding heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0210] In some embodiments, the chimeric B7H3 antibodies of the present invention can be prepared by operatively linking hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy and light chains. In some embodiments, the constant region comprised by the chimeric antibodies of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, and IgG4, preferably IgG1.

[0211] In some embodiments, the chimeric B7H3 antibodies of the present invention can be obtained by transfecting expression cells with "mixed and matched" chimeric light chain and chimeric heavy chain expression plasmids, and the binding of such "mixed and matched" antibodies to B7H3 can be tested using the above-mentioned binding assays and other conventional binding assays (e.g., ELISA).

[0212] The humanized antibodies of the present invention can be prepared by inserting murine CDR regions into human germline framework regions using methods known in the art, such as those described in U.S. Patent Nos. 5,225,539 to Winter et al. and 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0213] In some embodiments, the CDR sequence of the antibody used in the drug conjugate, composition, use or method of the present invention includes the CDR sequence of the antibody W301088-1.145.16-z3-p1-uIgG1KV320 (abbreviated as WBP301088). In some embodiments, the CDR sequence of the antibody used in the drug conjugate, composition, use or method of the present invention includes the CDR sequence of the antibody W301088-1.145.16-xIgG1KV320. In some embodiments, the variable region sequence of the antibody used in the drug conjugate, composition or use of the present invention includes the variable region sequence from the antibody W301088-1.145.16-z3-p1-uIgG1KV320 or W301088-1.145.16-xIgG1KV320. In some embodiments, the amino acid sequence of the antibody used in the drug conjugate, composition, use or method of the present invention includes the full-length amino acid sequence from the antibody W301088-1.145.16-z3-p1-uIgG1KV320 or W301088-1.145.16-xIgG1KV320.

[0214] The anti-B7H3 antibody W301088-1.145.16-xIgG1KV320 or its antigen-binding fragment described herein is prepared as described in the Examples. In some embodiments, the CDR sequences of the antibody used in the drug conjugate, composition, use, or method of the present invention comprise HCDR1, HCDR2, and HCDR3 with 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 with 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 antibody used in the drug conjugate, composition, or use of the present invention comprise a heavy chain variable region with amino acid sequence set forth in SEQ ID NO: 8, and a light chain variable region with amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the antibody used in the drug conjugate, composition, use, or method of the present invention comprises a heavy chain with amino acid sequence set forth in SEQ ID NO: 14, and a light chain with amino acid sequence set forth in SEQ ID NO: 15.

[0215] The anti-B7H3 antibody W301088-1.145.16-z3-p1-uIgG1KV320 (abbreviated as WBP301088) or its antigen-binding fragment described herein is prepared as described in the Examples. In some embodiments, the CDR sequences of the antibody used in the drug conjugates, compositions, uses, or methods of the present invention comprise HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In some embodiments, the variable region sequences of the antibody used in the drug conjugates, compositions, or uses of the present invention comprise the heavy chain variable region as shown in SEQ ID NO: 10, and the light chain variable region as shown in SEQ ID NO: 11. In some embodiments, the antibody used in the drug conjugate, composition, use or method of the present invention comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 12, and a light chain having an amino acid sequence as shown in SEQ ID NO: 13.

[0216] The amino acid sequences of the chimeric antibody W301088-1.145.16-xIgG1KV320 and the humanized antibody W301088-1.145.16-z3-pl-ulgG1KV320 of the present invention are shown below. In some embodiments, the CDR numbering method of the antibody of the present invention is: Kabat + Chothia combined numbering.

[0217] W301088-1.145.16-xlgGlKV320 amino acid sequence (the underlined part is CDR)

[0218] Heavy chain variable region VH SEQ ID NO: 8

[0219] HCDR1: DYSITGDYAWN (SEQ ID NO: 1)

[0220] HCDR2: YISYSGSTSYNPSLQS (SEQ ID NO: 2)

[0221] HCDR3: SLGRRWYFVV (SEQ ID NO: 3)

[0222] Light chain variable region VL SEQ ID NO: 9

[0223] LCDR1: KSSQSLLNSSNQKNYLA (SEQ ID NO: 7)

[0224] LCDR2: FASTRES (SEQ ID NO: 5)

[0225] LCDR3: QQHYSAPWT (SEQ ID NO: 6)

[0226] Heavy chain SEQ ID NO: 14

[0227] Light chain SEQ ID NO: 15

[0228] W301088-1.145.16-z3-p1-ulgG1KV320 amino acid sequence (the underlined part is the CDR)

[0229] Heavy chain variable region VH SEQ ID NO: 10

[0230] HCDR1: DYSITGDYAWN (SEQ ID NO: 1)

[0231] HCDR2: YISYSGSTSYNPSLQS (SEQ ID NO: 2)

[0232] HCDR3: SLGRRWYFVV (SEQ ID NO: 3)

[0233] Light chain variable region VL SEQ ID NO: 11

[0234] LCDR1: KSSQSLLQSSNQKNYLA (SEQ ID NO: 4)

[0235] LCDR2: FASTRES (SEQ ID NO: 5)

[0236] LCDR3: QQHYSAPWT (SEQ ID NO: 6)

[0237] Heavy chain SEQ ID NO: 12

[0238] Light chain SEQ ID NO: 13

[0239] The antibodies or antigen-binding portions thereof disclosed herein have specific functional characteristics or properties. In some embodiments, the antibodies (including chimeric antibodies and humanized antibodies) have one or more of the following properties:

[0240] (a) specifically binds to human 4IgB7H3-expressing cells with high affinity (e.g., less than 2 nM as measured by FACS);

[0241] (b) The affinity for binding to human 2IgB7H3 was significantly reduced compared to human 4IgB7H3, and the difference in the affinity of the antibody binding to 2IgB7H3 and 4IgB7H3 was significant;

[0242] (c) specifically binds to cyno B7H3 with high affinity (less than 5 nM as measured by FACS) that is superior to a benchmark antibody; and

[0243] (d) Cancer cells expressing B7H3 showed good internalization ability of the antibody.

[0244] Pharmaceutical compositions and pharmaceutical preparations

[0245] In yet another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate as described herein, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0246] It should be understood that the antibody-drug conjugates provided by the present invention, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof can be integrated with suitable carriers, excipients, and other agents in the formulation for co-administration, thereby providing improved transfer, delivery, tolerance, and the like.

[0247] The term "pharmaceutical composition" refers to a formulation that allows the active ingredient contained therein to exist in biologically effective form, and that contains no additional ingredients that are unacceptably toxic to a subject to which the formulation would be administered.

[0248] Pharmaceutical formulations containing the anti-B7H3 antibodies described herein can be prepared by mixing the anti-B7H3 antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt thereof having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of an aqueous solution or a lyophilized preparation.

[0249] The pharmaceutical composition or formulation of the present invention may also include one or more other active ingredients, which are required for the specific indication being treated, preferably those active ingredients that do not adversely affect the complementary activities of each other. In some embodiments, the other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, cell growth inhibitors, antibiotics or various known anti-tumor or anti-cancer agents, and the active ingredients are suitably combined in an amount effective for the intended use. In some embodiments, the pharmaceutical composition of the present invention further comprises a composition of a polynucleotide encoding an anti-B7H3 antibody.

[0250] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody-drug conjugate as described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0251] In yet another aspect, the present invention provides a kit comprising the antibody-drug conjugate as described herein, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described herein, preferably further comprising a drug delivery device.

[0252] Medical uses

[0253] In another aspect, the present invention provides use of an antibody-drug conjugate as described herein, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein in the preparation of a medicament for treating and / or preventing a disease or condition mediated by B7H3, preferably, the disease or condition is cancer.

[0254] In another aspect, the present invention provides use of an antibody-drug conjugate as described herein, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein for treating and / or preventing a disease or condition mediated by B7H3, preferably, the disease or condition is cancer.

[0255] In another aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by B7H3, comprising administering to a subject in need thereof an antibody-drug conjugate as described herein, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein, preferably, the disease or condition is cancer.

[0256] In yet another aspect, the present invention provides an antibody-drug conjugate, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein for use in treating and / or preventing a disease or condition mediated by B7H3, preferably, the disease or condition is cancer.

[0257] In some embodiments, the cancer of the present invention is selected from 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 carcinoma, lymphoma (such as non-Hodgkin's lymphoma), chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma.

[0258] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody-drug conjugate as described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0259] In yet another aspect, the present invention provides a kit comprising the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein.

[0260] In some embodiments, the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration.

[0261] In some embodiments, the present invention provides for administering to a subject a therapeutically effective amount of one or more therapies (eg, treatment modalities and / or other therapeutic agents) in combination. In some embodiments, the therapies include surgery and / or radiation therapy.

[0262] In some embodiments, the methods or uses provided herein further comprise administering one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to an individual. The antibody-drug conjugates of the present invention, or pharmaceutically acceptable salts thereof, may be administered alone or in combination with other therapeutic agents in the therapy. For example, they may be co-administered with at least one additional therapeutic agent.

[0263] The antibody-drug conjugates described herein may have an inhibitory activity on the in vitro proliferation of tumor cells. The inhibitory activity may be that when the drug conjugate of the present application is added to the culture medium of tumor cells, the proliferation ability of the tumor cells decreases by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, more than 10%, more than 15%, more than 18%, more than 20%, more than 25%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% compared to the addition of a negative control or a control drug. For example, the inhibitory activity may be such that the IC50 value (nM) against tumor cells may be 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, 97nM or less, 95nM or less, 90nM or less, 80nM or less, 75nM or less, 70nM or less, 65nM or less, 62nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 25nM or less, 23nM or less, 22nM or less, 20nM or less, 19nM or less, 18nM or less, 18.5nM or less, 17nM or less, 15nM or less, 12nM or less Below, 10nM below, 9nM below, 8.5nM below, 7nM below, 6.7nM below, 6nM below, 5.9nM below, 5.5nM below, 5.0nM below, 4.8nM below, 4.5nM below, 4.4nM below, 4nM below, 3.5nM below, 3nM below, 2.5nM below, 2nM below, 1.5nM below, 1.0nM below, 0.5nM below, 0.3nM below, 0. 0.29nM or less, 0.25nM or less, 0.21nM or less, 0.20nM or less, 0.18nM or less, 0.17nM or less, 0.15nM or less, 0.12nM or less, 0.10nM or less, 0.09nM or less, 0.08nM or less, 0.07nM or less, 0.06nM or less, 0.05nM or less, 0.04nM or less, 0.03nM or less, 0.02nM or less, or 0.01nM or less. For example, the tumor cells may include but are not limited to solid tumor cells, for example, the tumor cells include but are not limited to lung cancer cells, for example, the tumor cells may include but are not limited to lung cancer cells Calu-6.

[0264] The antibody-drug conjugates described herein may have an in vivo tumor-suppressing effect. The tumor-suppressing effect may be that the antibody-drug conjugates of the present application are administered to an animal, and compared to the addition of a negative control or control drug, the volume of the animal's tumor is reduced by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, more than 10%, more than 15%, more than 18%, more than 20%, more than 25%, more than 40%, more than 50%, more than 55%, more than 60%, more than 70%, more than 73%, more than 74%, more than 76%, more than 77%, more than 78%, more than 79%, more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 10 ... The invention provides a method for treating a tumor of an animal by reducing the volume of the tumor by more than 1.1 times, more than 1.3 times, more than 1.5 times, more than two times, more than three times, more than five times, more than ten times, more than twenty times, more than twenty-two times, more than thirty times, more than fifty times, more than one hundred times, more than five hundred times, more than one thousand times, or more than one thousand five hundred times, compared to the administration of a negative control or a control drug on day 1, 3, 5, 7, 14, 20, 21, or 30. The animal may include, but is not limited to, a mammal, for example, the animal may include, but is not limited to, a cat, a dog, a horse, a pig, a cow, a sheep, a rabbit, a mouse, a rat, a monkey, or a human. The administration may include, but is not limited to, oral administration, intravenous injection, intravenous drip, intraperitoneal injection, or local administration. For example, the tumor cells may include but are not limited to solid tumor cells, for example, the tumor cells include but are not limited to lung cancer cells, melanoma cells, brain cancer cells, prostate cancer cells, for example, the tumor cells may include but are not limited to lung cancer cells Calu-6, melanoma cells A375, brain cancer cells U87, prostate cancer cells PC-3.

[0265] The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0266] Example

[0267] The following examples are provided to demonstrate and further explain some preferred embodiments and aspects of the present invention and should not be construed as limiting the scope thereof.

[0268] Materials and methods:

[0269] Table 1. Information of commercially available materials

[0270] Sample testing method

[0271] 1. ADC DAR value analysis method - HIC-HPLC (hydrophobic chromatography)

[0272] High performance liquid chromatography: Waters e2965 high performance liquid chromatography system.

[0273] Column: MabPac TM HIC-Butyl 5μm 4.6×100mm (Manufacturer: Thermo);

[0274] Mobile phase A: 1.5 M (NH 4 ) 2 SO 4 + 50 mM K 2 HPO 4 (pH 7.0);

[0275] Mobile phase B: 50 mM K2HPO4 (pH 7.0) / isopropanol (75:25 V / V);

[0276] Elution was performed according to the following elution procedure:

[0277] Detection conditions: Set the mobile phase flow rate to 1 ml / min, the detection wavelength to 280 nm, and the column temperature to 30 °C.

[0278] 2.SEC Purity Analysis -SEC-HPLC (Size Exclusion Chromatography)

[0279] High performance liquid chromatograph: Agilent 1260 liquid chromatograph.

[0280] Chromatographic column: Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm)

[0281] Mobile phase: 50 mM NaH2PO4 + 200 mM arginine (pH 6.80) + 10% isopropanol

[0282] Detection conditions: Set the mobile phase flow rate to 0.5 ml / min, the detection wavelength to 280 nm, and the column temperature to 30 °C.

[0283] Example 1. Generation and characterization of anti-B7H3 antibodies

[0284] 1.1 Preparation of antigens, reference antibodies, and cell lines

[0285] 1.1.1 Antigen production

[0286] 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 mammalian expression and then synthesized by Sangon Biotech (Shanghai, China). The DNA fragment was subcloned into a pcDNA3.3 expression vector with 6xHis at the C-terminus and then transfected into Expi293F cells (Invitrogen, A14635). After five days of incubation, the supernatant was purified using a Ni column (Cytiva, 173712). The eluted protein was dialyzed into PBS via a dialysis bag (Spectrum-888-10987, MWCO 3.5 kDa). The protein concentration was determined by absorbance at 280 nm on a NanoDrop device. 2 μg of purified protein was electrophoresed on an SDS-PAGE gel (Invitrogen) with and without a reducing agent. The purity of the purified protein was quantified by HPLC-SEC using a TSKgel G3000SWXL size exclusion chromatography column (Tosoh, 008541). The purified protein was stored at -80°C. Human 2IgB7H3 was purchased from ACRO (Catalog No. B73-H52E2); cynomolgus monkey B7H3 was purchased from ACRO (Catalog No. B73-C52Ha).

[0287] 1.1.2 Generation of benchmark antibodies

[0288] The anti-B7H3 antibody Enoblituzumab (MacroGenics, whose light and heavy chain amino acid sequences are SEQ ID NOs: 117, 119 in US20120294796A1) was used as a 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). The DNA fragment was then subcloned into a modified pcDNA3.4 expression vector with the constant region of human IgG1. Plasmids containing the VH and VL genes were co-transfected into Expi293F cells (Invitrogen, A14635). After five days of incubation, the supernatant was purified using a protein A column (Cytiva, 175438). The eluted protein was dialyzed into PBS via a dialysis bag (Spectrum, 888-10987). Protein concentration was determined by absorbance at 280 nm on a NanoDrop device. 2 μg of purified protein was electrophoresed on SDS-PAGE gel (Invitrogen) with and without reducing agent. The purity of the purified protein was quantified by HPLC-SEC using a TSKgel G3000SWXL size exclusion chromatography column (Tosoh, 008541). The purified antibody was stored at -80°C.

[0289] 1.1.3 Establishment of stable cell lines / cell pools

[0290] Generate a cell line expressing human 4IgB7H3. Briefly, CHOK1 cells were transfected with a pcDNA3.3 expression vector containing full-length 4IgB7H3 using the Lipofectamine 2000 transfection kit (Invitrogen, 11668027) according to the manufacturer's protocol. 48 hours after transfection, cells were subcultured into a T75 flask containing selective medium (F12-K with 10% FBS and 15 μg / mL blasticidin). After two or three passages, a cell line (W3XX088-CHOK1.hPro1.2A5) expressing human 4IgB7H3 stably was obtained by blasticidin selection and limiting dilution, and expression levels were determined by FACS using an anti-B7H3 antibody.

[0291] Generate a pool of cells expressing cynomolgus monkey B7H3. Briefly, FlipinCHO cells were transfected with the pcDNA5 / pOG44 expression vector containing full-length cynomolgus monkey B7H3 using the Lipofectamine 2000 transfection kit (Invitrogen, 11668027) according to the manufacturer's protocol. 48 hours after transfection, cells were subcultured into selective medium (F12 containing 10% FBS and 600 μg / mL hygromycin B) in T75 flasks. After two or three passages, cells were selected by hygromycin B selection and BD FACSMelody TM Cell sorting was used to obtain a cell pool (WBP3XX088-FlpinCHO.cPro1.pool) that stably and highly expressed cynomolgus monkey B7H3.

[0292] 1.2 Antibody Production

[0293] 1.2.1 Animal immunization

[0294] Four Balb / c mice aged 6 to 8 weeks were immunized with the DNA plasmid encoding the full-length human 4IgB7H3 of Example 1.1.3, with 200 μg to 400 μg each. The adjuvant mixture included Adju-Phos, CpG-ODN, and GM-CSF. Animals were injected subcutaneously, intramuscularly, and hydrodynamically into the tail vein once every other week. Blood was drawn from the mice after the third injection (first bleed) and after the fifth injection (second bleed). Serum titers were measured by ELISA and FACS.

[0295] ELISA assay was used to measure serum antibody titers against the 4IgB7H3 antigen. Plates were coated with 100 μL of His tag at 0.5 μg / mL (Nunc) overnight at 4°C and then blocked with blocking buffer (2% BSA in PBS) at ambient temperature for 1 hour. The plates were then washed and incubated with 1 μg / mL of antigen for 1 hour at ambient temperature. After washing, mouse serum was serially diluted 1:3 in blocking buffer starting from a 1:100 dilution, added to the culture plates, and incubated at ambient temperature for 2 hours. The plates were then washed and subsequently incubated with the secondary antibody, i.e., goat anti-mouse IgG-Fc-HRP (Bethyl, A90-231P), for 1 hour. After washing, TMB substrate was added and the interaction was terminated with 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device). Serum titers were measured under 2 times the background.

[0296] Table 2. Serum titer of mouse sera binding to human 4IgB7H3 by ELISA

[0297] FACS assay was used to measure serum antibody titers against 4IgB7H3 antigen. Briefly, the engineered B7H3-expressing cells of Example 1.1.3 were cultured at 1×10 5 The cells were seeded into 96-well U-bottom plates at a density of 10 cells / well, centrifuged at 1500 rpm for 4 minutes at 4°C, and the supernatant was removed. Mouse serum was serially diluted 1:3 in 1×PBS / 1% BSA starting from a 1:100 dilution to resuspend the cells and incubated at 4°C for 1 hour. The cells were washed twice with 180 μL 1×PBS / 1% BSA. The secondary antibody goat anti-mouse IgG-Fc Alexa 647 (Jackson, 109-605-098) was added to the resuspended cells and incubated in the dark for 30 minutes at 4°C, then washed with 180 μL 1×PBS / 1% BSA. Finally, the cells were resuspended in 100 μL 1×PBS / 1% BSA, and the fluorescence intensity was measured by FACS (BD Canto II) and analyzed by FlowJo version software. Serum titers were determined under 2 times the background.

[0298] Table 3. Serum titer testing by FACS for binding to cell lines expressing human 4IgB7H3

[0299] When the serum titer was high enough, 400 μg of the DNA plasmid encoding human full-length 4IgB7H3 of Example 1.1.3 was used for transfection via the tail vein hydrodynamics and 4×10 6 MCF-7 cells were administered as a final booster via footpad and subcutaneous administration. Lymph nodes and spleens were harvested for cell fusion.

[0300] 1.2.2 Hybridoma Production

[0301] The lymph nodes and spleens of the immunized mice from Example 1.2.1 were homogenized and filtered to remove blood clots and cell debris. The Sp2 / 0 myeloma cells growing exponentially were collected and centrifuged. According to the conventional electrofusion procedure, the B cells obtained by homogenizing the lymph nodes and spleens of the immunized mice were fused with the Sp2 / 0 myeloma cells at a ratio of 1:1 in the electrofusion solution. The fused cells were suspended in DMEM culture medium supplemented with 20% FBS and 1X 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 to 14 days.

[0302] 1.2.3 Antibody Screening

[0303] First screening: A cell-based ELISA assay was used as the first screening to test the binding of hybridoma supernatants to human 4IgB7H3. Briefly, CHOK1 cells transfected with human 4IgB7H3 from Example 1.1.3 were cultured at 5×10 3 Cells were seeded at a density of 10 cells / well in a 96-well plate (CORNING) and then maintained in an incubator set to 37°C, 5% CO2 for 2 days. The plate was then washed and incubated with hybridoma supernatant for 1 hour at ambient temperature. The plate was then washed and incubated with the secondary antibody goat anti-mouse IgG-Fc-HRP (Bethyl, A90-231P) for 1 hour. After washing, TMB substrate was added and the interaction was terminated with 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0304] Secondary screening: To confirm the binding of hybridoma supernatants to human and cynomolgus monkey antigens, FACS was performed. The parental CHOK1 cell line and the CHOK1 cell line transfected with 4IgB7H3 from Example 1.1.3 were stained with CellTrace dyes (Violet and FarRed, respectively), while the FlipinCHO cell pool transfected with B7H3 from Example 1.1.3 was prepared without dye. After incubation at ambient temperature for 15 minutes and washing, the three cell types were mixed in equal amounts to 1×10 6 cells / mL. The three cell mixtures were diluted to 1×10 5 Cells were transferred to a 96-well U-bottom plate (BD) at a density of 10 cells / well. Hybridoma supernatants were then transferred to the plate and incubated at 4°C for 1 hour. After washing, the secondary antibody, goat anti-mouse IgG Fc PE (Jackson, 115-115-164), was added and incubated with the cells at 4°C in the dark for 0.5 hours. The cells were then washed and resuspended in 1×PBS / 1% BSA before analysis by flow cytometry.

[0305] To test the binding of hybridoma supernatants to human 2IgB7H3, a conventional ELISA assay was used. Plates were pre-coated with 100 μL / well of human 2IgB7H3 (0.2 μg / mL) at ambient temperature for 1 hour. After blocking with 200 μL / well of 1×PBS / 2% BSA for 1 hour, the plates were washed 3 times with 1×PBST. Hybridoma supernatants were added to the plates at a volume of 100 μL / well and incubated at ambient temperature for 2 hours. After washing the plates 3 times with 1×PBST, 100 μL / well of goat anti-mouse IgG-Fc HRP (Bethyl, A90-231P) was added to the plates and incubated at ambient temperature for 1 hour. After washing 6 times, color was developed by adding TMB substrate to the plates at 100 μL / well for 5-10 minutes, and the reaction was stopped by adding 100 μL / well of 2M HCl. Read the absorbance at 450 nm using a microplate reader.

[0306] Selected positive cell lines screened by FACS were further confirmed by SPR. SPR allows for real-time, label-free detection of biomolecular interactions. SPR occurs when polarized light is incident on a conductive surface at the interface between two media. This generates an electron charge density wave called a plasmon, which reduces the intensity of the reflected light at a specific angle (called the resonance angle) that is proportional to the mass of the sensor surface. The binding of the supernatant to human 4IgB7H3 was graded using Biacore 8K. The activator was prepared by mixing 400mM EDC and 100mM NHS (GE) immediately before injection into the channel. The CM5 sensor chip was activated with the activator for 420 seconds. Goat anti-mouse Fc IgG (30μg / mL in 10mM NaAc, pH 4.5) was then injected into the channel at a flow rate of 10μL / min and held for 420 seconds. The chip was deactivated with 1M ethanolamine-HCl. The supernatant was injected into the channel at a flow rate of 10μL / min and held for 30 seconds. 200 nM human 4IgB7H3 analyte was injected into the channel at a flow rate of 30 μL / min, with a 120-second association period followed by a 300-second dissociation period. Following the dissociation period, glycine (10 mM, pH 1.5) was injected as a regeneration buffer. The sensorgram was subtracted from the reference and buffer channels. The data were then fitted with a 1:1 Langmuir binding model.

[0307] pHrodo iFL dyes can be conjugated to biomolecules such as antibodies and become highly fluorescent only when present in acidic environments such as lysosomes and endosomes. This enables 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 pHrodo iFL Red STP ester amine reactive dye and purified through Zeba Spin desalting columns according to the manufacturer's instructions. MCF-7 cells (2×10 4 Cells were plated in 96-well clear bottom black plates pre-coated with 8 μg / mL poly-D-lysine (cells / well). The cells were grown overnight in an incubator set at 37°C, 5% CO2. The next day, WBP301088-1 positive cell line supernatant was added to the corresponding wells at a volume of 100 μL. The cells were incubated in an incubator set at 37°C, 5% CO2 for 0.5 hours. Human and mouse IgG1 isotype antibodies were used as negative controls. 50 nM pHrodo iFL Red / F(ab')2 goat anti-mouse or anti-human IgG Fcγ conjugate (diluted in complete medium) was added at a volume of 100 μL / well. The plate was then incubated in the dark in an incubator set at 37°C, 5% CO2 for 5 hours. After incubation, the supernatant was discarded. The plate was washed with complete medium. A mixed dye solution (1 μg / mL Hoechst 33342 and 0.5 μg / mL Calcein AM diluted in DPBS) was added to the plate at a volume of 100 μL / well and maintained at ambient temperature for 15 minutes. After incubation, the dye solution was discarded and the plate was washed once with 1×PBS / 1% BSA. The fluorescence intensity of the MCF-7 cells was read and analyzed by the Perkin Elmer Operetta CLS high-content analysis system.

[0308] A total of 8460 wells were tested in the primary screening, and 279 hybridomas were selected and expanded in 24-well plates. In a secondary screening, supernatants from the 24-well plates were used to confirm strong binding to human 4IgB7H3 and cynomolgus monkey B7H3, weak binding to human 2IgB7H3, and strong internalization activity against MCF-7. Based on the results of the secondary screening, the WBP301088-1.145 hybridoma was selected for subcloning.

[0309] 1.2.4 Antibody Subcloning

[0310] The positive hybridoma cells selected in Example 1.2.3 were subcloned. Cells growing in logarithmic numbers were counted and added to 1.5 mL of semi-solid HAT culture medium. The cells were gently mixed on a vortex shaker for 5-10 seconds and then seeded in a 6-well plate (CORNING). The plate was kept in an incubator set to 37°C, 5% CO2 for 7 to 8 days. Each visible single colony was picked into a 96-well plate (CORNING), which had DMEM culture medium supplemented with 10% FBS. After 2 to 3 days, the cell supernatant was collected and screened by cell-based ELISA (human 4IgB7H3) and conventional ELISA (human 2IgB7H3) using the antibody screening scheme as described above. After confirmatory screening of the antibody subclones using cells expressing human 4IgB7H3 and cynomolgus monkey B7H3 by FACS, the culture supernatant of the selected single-positive clone was collected, and the antibody was purified for further characterization. After subclone screening and confirmatory screening, the W301088-1.145.16 hybridoma clone was obtained.

[0311] 1.2.5 Antibody typing

[0312] ELISA typing was performed in 96-well high binding plates (Nunc). Each well was coated with 2 μg / mL capture antibodies goat anti-mouse IgG1 (Bethyl, A90-205A), goat anti-mouse IgG2a (Bethyl, A90-207A), goat anti-mouse IgG2b (Bethyl, A90-209A), and goat anti-mouse IgG3 (Bethyl, A90-211A), respectively, overnight at 4°C, 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 peroxidase-conjugated goat anti-mouse κ / λ light chain antibody. The HRP signal was detected by adding TMB substrate, and the reaction was terminated with 2M HCl after 12 minutes. All incubation steps were performed at ambient temperature, and the plates were washed with PBS between each step. The absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0313] The typing results of the W301088-1.145.16 clone are shown in the following table (Table 4).

[0314] Table 4. Typing results of W301088-1.145.16 clone

[0315] 1.2.6 Antibody Production

[0316] When a small amount of purified antibody is needed, T75 flasks (CORNING) are usually used to expand the scale of hybridoma cell culture. Monoclonal hybridoma cells are cultured at a rate of 5×10 6 to 1×10 7 Inoculate 100 cells / T75 flask into the flask. Grow the cells continuously for approximately 7-10 days, until cell viability reaches approximately 30%-40%. Harvest the culture supernatant and remove cell debris by centrifugation. Sterile filter the supernatant and save for antibody purification.

[0317] 1.2.7 Hybridoma Sequencing

[0318] Total RNA from a W-301088-1.145.16 hybridoma cell sample was first extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit. RNA was then converted to cDNA using the SMART RACE cDNA Amplification Kit from Clonetech. VH and VL domain DNA sequences were then amplified from the cDNA using 30 cycles of PCR, with each cycle consisting of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The PCR products were then subcloned into a TA cloning vector and sent to Biosune Biotech (Shanghai, China) for sequencing.

[0319] Once the sequencing data confirmed the monoclonality of the hybridoma sample, the DNA sequences of the VH and VL domains were amplified by PCR. Primers were synthesized by Sangon Biotech (Shanghai, China). The DNA fragments were then subcloned into the pcDNA3.4 expression vector for the human IgG1 constant region with the LALA mutation and sequenced by Tsingke Biotechnology (Beijing, China).

[0320] 1.2.8 IgG conversion

[0321] Once sequencing data confirmed the monoclonality of the hybridoma cell sample, the nucleotide sequences encoding the amino acid sequences of the VH and VL domains were codon-optimized for mammalian expression and synthesized by Sangon Biotech (Shanghai, China). The DNA fragments were then subcloned into the pcDNA3.4 expression vector for the human IgG1 constant region with the L234A / L235A (LALA) mutation.

[0322] In this way, the antibody produced by clone W301088-1.145.16 was converted into a human IgG1 antibody with the LALA mutation, thereby obtaining a chimeric antibody designated as antibody W301088-1.145.16-xIgG1KV320. After protein A purification and buffer exchange, the protein was characterized by SDS-PAGE and SEC-HPLC. Under reducing conditions, the protein migrated on SDS-PAGE with apparent molecular weights of 50 kDa and 25 kDa, corresponding to the IgG heavy and light chains. The purity was greater than 99% (SEC-HPLC).

[0323] Table 5. Chimeric Antibodies Generated

[0324] 1.2.9 Humanization of Antibodies

[0325] A local copy of the open source software ANARCI (Dunbar J and Deane CM, "ANARCI: Antigen receptor numbering and receptor classification", Bioinformatics, 2015; Vol. 32, No. 5: pp. 298-300) was run to assign Kabat numbers to the W301088-1.145.16 antibody. CDRs were identified according to the definition on Dr. Martin's website (Martin, 2018, supra) and are shown in Table 6.

[0326] Table 6. CDR ranges defined according to Dr. Martin's antibody informatics website

[0327] Table 7. VH and VL amino acid sequences of the W301088-1.145.16 antibody, with CDR sequences underlined

[0328] Table 8. Identified heavy and light chain CDR sequences of the W301088-1.145.16 antibody

[0329] The VH and VL domain sequences of the murine antibody W301088-1.145.16 were aligned with the human germline sequence library of VH and VL domains from IMGT, respectively. Human germline sequences with the minimum number of amino acid differences in the framework relative to the VH / VL domain sequences of the W301088-1.145.16 antibody were selected as templates for humanization of the VH / VL domains. The binding of IGHV4-31*02 to IGHJ6*01 and the binding of IGKV4-1*01 to IGKJ4*01 are the human germline sequences most homologous to the VH and VL domain sequences of the W301088-1.145.16 antibody, respectively. The CDRs of the W301088-1.145.16 antibody were grafted into the frameworks of these two human germline templates to form the germline sequences.

[0330] Several "back mutation" positions in the framework were empirically selected to convert the amino acids in the germline sequence to their amino acid counterparts in the original mouse sequence. A group of humanized variants were empirically designed to study the different combinations of these selected back mutation sites. At 16 positions in the VL domain and 20 positions in the VH domain, the germline sequence differs from the original mouse W301088-1.145.16 antibody sequence. For back mutation, 14 sites in a total of 36 different sites were considered, namely VH: Q001D, S025T, H040F, K043N, G044K, I048M, V067I, V071R, A085E, V089T, Y091F, VL: I021M, P043S, Y087F. A group of humanized variants were empirically designed to test the different combinations of these three back mutations.

[0331] 1.2.10 PTM Removal

[0332] The VH and VL domain sequences of the humanized variants were scanned for several types of key post-translational modification (PTM) sites: asparagine deamination (NG and NS) in the CDRs, aspartate isomerization (DG) in the CDRs, unpaired Cs over the entire length, and N-linked glycosylation sites (NXS / T, where X can be any amino acid except P) over the entire length. Point mutations were empirically designed to remove these key PTM sites in the humanized variants to avoid the potential risks of PTM modifications. Finally, PTM-removed antibodies were selected that did not affect expression, binding, and thermal stability compared to the parent antibody.

[0333] Considering that the VL domain sequence of the murine antibody W301088-1.145.16 contains NSS N-linked glycosylation sites, three point mutations, VL: N027dQ, VL: S027eP, and VL: S027fA (according to Kabat numbering), were designed to remove the key PTM sites. Together with the humanized variants, they were expressed in the presence of NSS N-linked glycosylation sites. off Test in stages.

[0334] 1.2.11 k by SPR off Grading

[0335] k of the filtered supernatant was performed on a Biacore 8K surface plasmon resonance (SPR) instrument. off Grading. First, the CM5 sensor chip was activated with 400mM EDC and 100mM NHS (GE) at a flow rate of 10μL / min for 420 seconds. Then, 30μg / mL anti-human Fc IgG (Jackson) in 10mM NaAc (pH 4.5) was injected into channels 1 to 8 at a flow rate of 10μL / min for 420 seconds. The chip was then deactivated with 1M ethanolamine-HCl (GE) at a flow rate of 10μL / min for 420 seconds. The diluted supernatant of each antibody was injected into the Fc2 of the channel at a flow rate of 10μL / min. The supernatant of the cell culture without plasmid transfection was used as a negative control and was injected into the Fc1 of the channel at a flow rate of 10μL / min. 100 nM human 4IgB7H3 and running buffer were sequentially injected into the Fc1-Fc2 channels at a flow rate of 30 μL / min, with an association period of 180 seconds, followed by a dissociation period of 3600 seconds. After each run, 10 mM glycine (pH 1.5) was injected to regenerate the chip. Sensorgrams were analyzed using the software included with the Biacore 8K instrument. The sensorgrams of the sample were subtracted from those of the reference and buffer channels. The data were then fitted with a 1:1 Langmuir binding model.

[0336] 1.2.12 Combination of PTM-removing mutations and humanized variants

[0337] Example 1.2.11 shows the best k off The rate of PTM removal point mutations is combined to have the optimal k off The humanized variants were subjected to rate-stepwise mutagenesis to compose the final construct for affinity validation.

[0338] According to k offThe final antibody was constructed by combining the PTM removal mutation of VL: N027dQ with four selected humanized mutant amino acids: S025T, V071R, Y091F, and V089T. The final antibody was named W301088-1.145.16-z3-p1-uIgG1KV320.

[0339] Table 9. Heavy and light chain CDR sequences of W301088-1.145.16-z3-p1-uIgG1KV320 antibody

[0340] Figures 12A-12B show the amino acid sequence alignment results of the VH and VL regions of antibodies W301088-1.145.16-xIgG1KV320 and W301088-1.145.16-z3-p1-uIgG1KV320. The boxed amino acid segments in the figures represent the CDR regions in the VH and VL regions.

[0341] 1.2.13 Production-Scale Transient Transfection and Purification

[0342] Using ExpiFectamine TM Transient transfection kit was used to transiently transfect 20 mL of Expi293F cells with a final concentration of 1 μg / mL of plasmid at a cell density of 3.0 × 10 6 Cells were grown in a humidified horizontal shaker at 150 rpm, with a temperature of 37°C and a CO2 level of 8%.

[0343] After 5 days of cell culture incubation, the supernatant expressing the target antibody was collected and filtered, and purified using a GE MabSelect SuRE protein A column (Cytiva-175438). The eluted antibody was dialyzed into PBS via a D-Tube dialyzer Maxi (EMD Millipore 71508, MWCO 3.5 kDa). The antibody concentration was determined by absorbance at 280 nm on a NanoDrop device. 2 μg of the purified antibody sample was run on an SDS-PAGE gel (Invitrogen NuPAGE) with and without a reducing agent. TM The purified antibody samples were electrophoresed on a 4%-12% Bis-Tris protein gel. The purity of the purified antibody samples was quantified by HPLC-SEC using a TSKgel G3000SWXL size exclusion chromatography column (Tosoh 008541). The purified antibodies were stored at -80°C.

[0344] 1.3 In vitro characterization of antibodies

[0345] 1.3.1 Purity testing by SDS-PAGE and SEC-HPLC

[0346] The purified antibody sample of Example 1.2 was mixed with the loading buffer and heated at 75°C for 10 minutes using a drying bath. After the sample was loaded, SDS-PAGE was performed at a constant voltage (200 V) for 35 minutes. The gel was stained and stained with eStain. TM L1 was destained. SDS-PAGE results were obtained using a BIO-RAD imaging system.

[0347] The results are summarized below in Table 10. Under reducing conditions, the protein migrated on SDS-PAGE with apparent molecular weights of 50 kDa and 25 kDa, corresponding to the IgG heavy and light chains, whereas under non-reducing conditions, it migrated as the expected band of approximately 150 kDa (Figure 1).

[0348] 1.3.2 Purity testing by SEC-HPLC

[0349] The HPLC was carried out using an Agilent 1260 Infinity II system (Agilent Technologics) equipped with a TSKgel G3000SWXL column (Tosoh Bioscience-0008541). TM ) The purity of the antibody of Example 1.2 was detected. An appropriate volume of antibody sample (5 μL-100 μL) was injected into the column and separated at a flow rate of 1 mL / min for 20 minutes. 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 all peak areas 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).

[0350] The results are summarized in the following Table 10. The purity of the W301088-z3-p1 antibody was higher than 99% as determined by SEC-HPLC ( FIG. 2 ).

[0351] 1.3.3 Thermal stability (T m )

[0352] use 7Flex Real-Time PCR System (Applied Biosystems) was used to study the T m(Melting temperature). 19 μL of the antibody solution of Example 1.2 was mixed with 1 μL of SYPRO Orange solution (Invitrogen) and transferred to a 96-well plate (Applied Biosystems). The plate was sealed with an optical adhesive film (Applied Biosystems) and centrifuged at 3,000 rpm for 5 minutes to remove any bubbles. The plate was heated from 26°C to 95°C at a rate of 0.9°C / min, and the resulting fluorescence data were collected. The negative derivative of the fluorescence as a function of temperature was calculated, and the maximum value was defined as the melting temperature T. m If a protein has multiple unfolding transitions, the first T is reported. m , named it T m 1. Pass Real-time PCR software (v1.3) automatically collected data and T m calculate.

[0353] The W301088-1.145.16-z3-p1-uIgG1KV320 antibody was m The value is defined as the maximum value of the negative derivative of fluorescence with different temperatures, as listed in Table 10 below. The data distribution is shown in Figure 3, where the dotted line represents T m The position of the 1 value in the fluorescence curve. T of W301088-1.145.16-z3-p1-uIgG1KV320 antibody m 1 is 70.1℃, which shows that it has good thermal stability.

[0354] 1.3.4 Hydrophobicity detection by HIC-HPLC

[0355] The results were analyzed by HPLC 1260 infinity II system (Agilent Technologics TM ) The hydrophobic properties of the antibody of Example 1.2 were detected using a TSKgel butyl-NPR column (Tosoh-0042168). The antibody sample was diluted to 0.5 mg / mL with PBS buffer, 20 μL of the diluted sample was injected into the column, and separated at a flow rate of 0.5 mL / min for 61 minutes. The running buffer was 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% buffer D. Peak retention was detected using UV light at wavelengths of 280 nm and 230 nm. Retention times were analyzed by integrating all peak areas from 20 minutes to 40 minutes using the HIC-HPLC analysis method. The operating analysis software was OpenLab CDS workstation (v2.6.0.691).

[0356] The HIC retention time of the W301088-1.145.16-z3-p1-uIgG1KV320 antibody was 23.52 minutes (Table 10 and Figure 4), indicating that it is an antibody with good hydrophilicity.

[0357] 1.3.5 Determination of diffusion interaction parameter (k) by DLS D )

[0358] DLS-k was studied using DynaPro plate reader III (Wyatt Technology). D Measurement results. During the antibody sample preparation process, the appearance of the sample was recorded when thawing, filtering and concentrating. The antibody sample was concentrated to more than 20 mg / mL and then diluted with PBS buffer to a final concentration of 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL and 20 mg / mL. 7.5 μL sample solution was then added to a 1536-well microplate (Aurora-ABI1-00110A). The plate was sealed with ClearSeal film (Hampton Research-HR4-521) and centrifuged at 3,000 rpm for 5 minutes to allow the sample to fall to the bottom of the well. Each sample was tested in duplicate in the well. The plate was placed in the corresponding position and data collection was performed using DYNAMICS operating software (v7.8.1.3). 5 acquisitions of each protein sample were collected, with each acquisition time being 5 s. For each measurement, the diffusion coefficient was determined and mapped for protein concentration.

[0359] k D The values ​​were automatically calculated by DYNAMICS operating software (v7.8.1.3) and the results are shown in Table 10. The data distribution is shown in Figure 5. K values ​​of W301088-1.145.16-z3-p1-uIgG1KV320 antibody in PBS D The solubility of the antibody was -5.17 mL / g, indicating a high solubility. During the assay, the appearance of the antibody protein was recorded, and some particles were observed after thawing and gentle shaking. However, no particles were observed when the buffer was changed to 20 mM His, 8% sucrose, 0.02% PS80, pH 6.5.

[0360] The above SDS-PAGE, SEC-HPLC, HIC-HPLC, DSF and DLS results are summarized in Table 10.

[0361] Table 10. Summary of analytical data for the W301088-1.145.16-z3-p1-uIgG1KV320 antibody

[0362] 1.3.6 Binding to human B7H3 (FACS)

[0363] FACS was used to detect the binding of antibodies to human B7H3. This method can quantitatively analyze and identify specific molecules expressed on the surface of living cells. Unlabeled cells were used as a control to set the threshold before detection, and the percentage change of each group exceeding the fluorescence intensity threshold was analyzed. MCF-7 cells expressing human full-length B7H3 (1×10 5 Cells / well) were incubated with various concentrations of antibodies (serial dilutions 3.16-fold from 50 nM to 0.0005 nM with 1× DPBS / 1% BSA) at a volume of 100 μL / well in a refrigerator set at 4°C for 1 hour. The anti-human B7H3 reference antibody enoxicam (MacroGeneics) was used as a positive control. 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 1:500 in 1× DPBS / 1% BSA) was added. The cells were incubated in the dark for 0.5 hours in a refrigerator set at 4°C. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed with FlowJo. EC was calculated by four-parameter nonlinear regression analysis using GraphPad Prism 7 software. 50 value.

[0364] The W301088-1.145.16-z3-p1-uIgG1KV320 antibody showed good binding to MCF-7 cells expressing B7H3, EC 50 The MFI was 1.08 nM and the maximum MFI was 19900, which was comparable to the chimeric antibody W301088-1.145.16-xIgG1KV320 and superior to the reference antibody enoxaparin (MacroGenics) (Table 11, Figure 6).

[0365] Table 11. FACS binding data for MCF-7 cells expressing human B7H3

[0366] Note: NA means not applicable.

[0367] 1.3.7 Binding to cynomolgus monkey B7H3 (FACS)

[0368] FACS was used to detect the binding of antibodies to cynomolgus monkey B7H3. Cynomolgus monkey B7H3 (W3XX088-FlpinCHO.cPro1.pool) (1×10 5FlpinCHO cells were transfected with 1x DPBS / 1% BSA (cells / well) and the transfected cells were incubated with various concentrations of antibodies (serial dilutions 3.16-fold from 50 nM to 0.0005 nM with 1× DPBS / 1% BSA) at a volume of 100 μL / well in a refrigerator set at 4°C for 1 hour. The anti-human B7H3 reference antibody enoxicam (MacroGeneics) was used as a positive control. Human IgG1 isotype antibody was used as a negative control. After washing the cells with 1× DPBS / 1% BSA, Alexa647 goat anti-human antibody (diluted 1:500 in 1× DPBS / 1% BSA) was added. The cells were incubated in the dark for 0.5 hours in a refrigerator set at 4°C. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed with FlowJo. EC was calculated by four-parameter nonlinear regression analysis using GraphPad Prism 7 software. 50 value.

[0369] The W301088-1.145.16-z3-p1-uIgG1KV320 antibody showed an EC of 4.68 nM. 50 Good binding to cynomolgus monkey-transfected cells was comparable to the chimeric antibody W301088-1.145.16-xIgG1KV320 and superior to enoxaparin (MacroGenics) (Table 12, Figure 7).

[0370] Table 12. FACS binding data for cynomolgus monkey B7H3 transfected stable cell pools

[0371] Note: NA means not applicable.

[0372] 1.3.8 Binding affinity to human 4IgB7H3 (SPR)

[0373] Surface plasmon resonance (SPR) was used to determine the affinity of the antibody for human 4IgB7H3. The affinity of the antibody for human 4IgB7H3 was determined by Biacore 8K. The activator was prepared by mixing 400mM EDC and 100mM NHS (GE) immediately before injection into the channel. The CM5 sensor chip was activated with the activator and kept for 420 seconds. Goat anti-human Fc IgG (30μg / mL, in 10mM NaAc, pH 4.5) was then injected into the channel at a flow rate of 10μL / min and kept for 420 seconds. The chip was deactivated with 1M ethanolamine hydrochloric acid. The antibody diluted in the flow buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10μL / min and kept for 30 seconds. Seven concentrations of analyte human 4IgB7H3 (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 2.125 nM, and 1.563 nM) were sequentially injected into the channel at a flow rate of 30 μL / min, with an association period of 180 seconds followed by a dissociation period of 3600 seconds. Glycine (10 mM, pH 1.5) was injected after the dissociation period as a regeneration buffer.

[0374] The sensorgrams of the reference channel and the buffer channel were subtracted from the test sensorgram. The experimental data were fitted with a 1:1 binding model. The molecular weight of human 4IgB7H3 used for calculation was 47.8 kDa.

[0375] The experimental data were fitted with a 1:1 binding model included with the 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 to human 4IgB7H3. D The value was 7.08E-11M, which was superior to enoxaparin (MacroGenics).

[0376] Table 13. Antibody affinity results for binding to human 4IgB7H3

[0377] 1.3.9 Binding affinity to human 2IgB7H3 (SPR)

[0378] Surface plasmon resonance (SPR) was used to determine the affinity of the antibody for human 2IgB7H3. The affinity of the antibody for human 2IgB7H3 was determined by Biacore 8K. The activator was prepared by mixing 400mM EDC and 100mM NHS (GE) immediately before injection into the channel. The CM5 sensor chip was activated with the activator and kept for 420 seconds. Goat anti-human Fc IgG (30μg / mL, in 10mM NaAc, pH 4.5) was then injected into the channel at a flow rate of 10μL / min and kept for 420 seconds. The chip was deactivated with 1M ethanolamine hydrochloric acid. The antibody diluted in the flow buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10μL / min and kept for 30 seconds. Seven concentrations of analyte human 2IgB7H3 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, and 7.813 nM) were sequentially injected into the channel at a flow rate of 30 μL / min, with a 120-second association period followed by a 300-second dissociation period. Glycine (10 mM, pH 1.5) was injected after the dissociation period as a regeneration buffer.

[0379] The sensorgrams of the reference channel and the buffer channel were subtracted from the test sensorgram. The experimental data were fitted with a 1:1 binding model. The molecular weight of human 2IgB7H3 used for calculation was 25.2 kDa.

[0380] The experimental data were fitted with a 1:1 binding model included 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 weaker binding affinity for human 2IgB7H3 than for human 4IgB7H3. D The value is 4.64E-08M.

[0381] Table 14. Antibody affinity results for binding to human 2IgB7H3

[0382] 1.3.10 Binding affinity to cynomolgus monkey B7H3 (SPR)

[0383] Surface plasmon resonance (SPR) was used to determine the affinity of the antibody for cynomolgus monkey B7H3. The affinity of the antibody for cynomolgus monkey B7H3 was determined by Biacore 8K. The activator was prepared by mixing 400mM EDC and 100mM NHS (GE) immediately before injection into the channel. The CM5 sensor chip was activated with the activator and kept for 420 seconds. Goat anti-human Fc IgG (30μg / mL, in 10mM NaAc, pH 4.5) was then injected into the channel at a flow rate of 10μL / min for 420 seconds. The chip was deactivated with 1M ethanolamine hydrochloric acid. The antibody diluted in flow buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10μL / min for 30 seconds. The analyte cynomolgus monkey B7H3 at 9 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) was sequentially injected into the channel at a flow rate of 30 μL / min, with an association period of 180 seconds followed by a dissociation period of 3600 seconds. Glycine (10 mM, pH 1.5) was injected after the dissociation period as a regeneration buffer.

[0384] The sensorgrams of the reference and buffer channels were subtracted from the test sensorgrams. Experimental data at 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 for calculations was 49 kDa.

[0385] The experimental data were fitted with a 1:1 binding model included 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 comparable to its binding to human 4IgB7H3. D The value was 5.74E-11M, which was superior to enoxaparin (MacroGenics).

[0386] Table 15. Antibody affinity results for binding to cynomolgus monkey B7H3

[0387] 1.3.11 Internalization Test (HCS)

[0388] Antibody internalization was determined by operetta CLS (PerkinElmer), a high-content imaging and analysis system that can collect and analyze sample images at high speed and high sensitivity. MCF-7 cells (1.5×10 4Cells were seeded into 96-well clear-bottom black plates pre-coated with 8 μg / mL poly-D-lysine (100 cells / well). After overnight incubation in a 37°C, 5% CO2 incubator, various concentrations of antibodies (serialized 3.16-fold from 50 nM to 0.0005 nM with 1×DPBS / 1% BSA) were incubated at a volume of 100 μL / well in a refrigerator set at 4°C for 2 hours. The anti-human B7H3 reference antibody, enoxaparin (MacroGenetic S), 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 1:500 in 1×DPBS / 1% BSA) was added. The cells were incubated in the dark in a refrigerator set at 4°C for 1 hour. The cells were then washed once and resuspended in 1×DPBS / 1% BSA at 37°C for 2 hours. After incubation, the supernatant was discarded, 100 μL / well Hoechst 33342 (diluted 1:5000 in 1×DPBS) was added and incubated at ambient temperature for 15 minutes. After washing the cells with 1×DPBS, 100 μL / well acid wash buffer (100 mM glycine, 150 mM NaCl, pH 2.5) was added and incubated in a refrigerator set at 4°C for 5 minutes. The cells were washed once with 1×DPBS and fixed with 60 μL 4% PFA. The mean fluorescence intensity (MFI) of the cells was measured and analyzed by operetta CLS. EC was calculated by four-parameter nonlinear regression analysis using GraphPad Prism 7 software. 50 value.

[0389] The W301088-1.145.16-z3-p1-uIgG1KV320 antibody showed an EC of 0.51 nM. 50 Internalization by B7H3-expressing MCF-7 cells demonstrated good internalization capacity, which was comparable to that of the chimeric antibody W301088-1.145.16-xIgG1KV320 and superior to the reference antibody enoxaparin (MacroGenics) (Table 16, Figure 11).

[0390] Table 16. Internalization of antibodies by MCF-7 cells expressing human B7H3

[0391] Note: NA means not applicable.

[0392] Example 2. Preparation of Antibody-Drug Conjugates (ADCs) and Their Binding Activity to B7H3 Antigen

[0393] The anti-B7H3 antibody used in the antibody-drug conjugate of this example is W301088-1.145.16-z3-p1-uIgG1KV320 (abbreviated as WBP301088) or W301088-1.145.16-xIgG1KV320. The heavy chain amino acid sequence of W301088-1.145.16-z3-p1-uIgG1KV320 (WBP301088) is shown in SEQ ID NO: 12, and the light chain amino acid sequence is shown in SEQ ID NO: 13.

[0394] 2.1 Preparation of linker-payload

[0395] 2.1.1 Preparation of Linker-Cytotoxin X1:

[0396] The preparation scheme of linker-cytotoxin X1 is as follows:

[0397] first step

[0398] To a solution of 27a (5.00 g, 43.0 mmol) and NaHCO₃ (10.9 g, 129 mmol) in DMF (50 mL) was added benzyl bromide (11.0 g, 64.6 mmol) dropwise under nitrogen. The mixture was allowed to react at 25°C for 17 hours. After completion of the reaction as indicated by TLC (PE / EA = 2 / 1) (PE is petroleum ether; EA is ethyl acetate), the reaction solution was added to 500 mL of water and extracted twice with EA (250 mL). After separation, the product was washed with saturated aqueous sodium chloride (500 mL), dried over anhydrous Na₂SO₄, and concentrated through a column chromatography (PE:EA = 3:2) to afford 5.1 g of a colorless liquid (yield: 57.1%).

[0399] Step 2

[0400] Under nitrogen, 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. The mixture was allowed to react at 25°C for 2 hours. After TLC (PE / EA = 1 / 2) showed the reaction was complete, the reaction solution was added to 200 mL of water and extracted twice with EA (200 mL). The mixture was dried over anhydrous Na2SO4 and concentrated by column chromatography (PE / EA = 3 / 2) to afford 1.56 g of a white solid (yield: 26%).

[0401] Step 3

[0402] To a solution of 27c (800 mg, 1.55 mmol) in EtOH (8 mL) and EA (8 mL) was added Pd / C (80 mg) under hydrogen atmosphere at 0°C and stirred at 0°C for 2.5 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered through celite, washed with EA (200 mL), concentrated, and dissolved in THF (20 mL) and dried to afford 600 mg of a white solid (yield: 91%).

[0403] Step 4

[0404] Under nitrogen, 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. The mixture was allowed to react for 2 hours at 0°C. LCMS indicated the reaction was complete. The reaction solution was added to aqueous citric acid (pH = 4) (150 mL), filtered, and the filter cake was washed with 175 mL of water, filtered, and pulled dry using an oil pump to afford 260 mg of a brown solid (yield: 66%).

[0405] Step 5

[0406] Under nitrogen, diethylamine (8 mL) was added dropwise to a solution of 27e (260 mg, 0.309 mmol) in dichloromethane (DCM) (30 mL) at 0°C. The mixture was allowed to react for 3 hours at 0°C. LCMS indicated the reaction was complete. The reaction solution was added to 600 mL of petroleum ether at 0°C, resulting in the precipitation of a solid. After the solid was allowed to settle at the bottom of the flask, the solution was decanted and pumped dry with an oil pump to afford 90 mg of a brown solid (47.1% yield).

[0407] Step 6

[0408] Under nitrogen, 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 and allowed to react for 2 hours. LCMS indicated the reaction was complete. The reaction solution was added to a pH 4 aqueous citric acid solution (30 mL) at 0°C. A flocculent solid precipitated, which was filtered and purified by preparative chromatography (DCM / MecOH = 10 / 1) to afford 9.2 mg of a light yellow solid, X1, in a 6% yield.

[0409] MS m / z(ESI):1074[M+1]

[0410] H-NMR (400MHz, 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)

[0411] 2.1.2 Preparation of Linker-Cytotoxin X2

[0412] The preparation scheme of linker-cytotoxin X2 is as follows:

[0413] first step

[0414] 34a (5 g, 48.0 mmol) and K2CO3 (19.9 g, 144.0 mmol) were dissolved in DMF (20 mL), and benzyl bromide (12.3 g, 72.0 mmol) was added dropwise. The mixture was allowed to react at 25°C for 17 hours. TLC (PE / EA = 3 / 1) confirmed the complete reaction. The reaction solution was added to water (200 mL) and extracted with EA (250 mL). The mixture was washed with saturated NaCl, dried over anhydrous Na2SO4, and concentrated to give 8.7 g of 34b as a colorless liquid (93% yield). MS-ESI: m / z 195.1 [M+H]+.

[0415] Step 2

[0416] 43c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) were dissolved in THF (20 mL) under nitrogen and cooled to 0°C. A solution of 34b (7.7 g, 39.6 mmol) in THF (10 mL) was added dropwise. After complete addition, the mixture was allowed to react at 0°C for 2 hours. TLC (PE / EA = 2 / 1) showed that most of the starting material had reacted. The reaction solution was poured into 100 mL of water and extracted with DCM (100 mL). The mixture was separated and washed with saturated NaCl, dried over anhydrous Na2SO4, and filtered through a column (PE / EA = 1 / 1) to give 34d as a colorless viscous product (39% yield). MS-ESI: m / z 503.3 [M+H]+.

[0417] Step 3

[0418] To a solution of 34d (1.9 g, 3.78 mmol) in EtOH (100 mL) and EA (100 mL) was added Pd / C (1 g, 10 wt.%) under hydrogen at 0°C for 3 hours. TLC (PE / EA = 2 / 1) indicated the reaction was complete. The reaction mixture was filtered through celite, and the filter cake was washed with EA / EtOH (1:1, 100 mL x 3). The filtrate was concentrated and dissolved in THF (50 mL x 3) and the mixture was dried by spin drying. This was repeated three times to afford 1 g of a gray solid 34e in a 64% yield. MS-ESI: m / z 435.2 [M+Na]+.

[0419] Step 4

[0420] Under nitrogen, 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. The mixture was allowed to react at 0°C for 2 hours. LCMS indicated the reaction was complete. The reaction solution was added dropwise to 300 mL of water, stirred, allowed to stand for 5 minutes, and filtered. The filter cake was dissolved in DCM / MeOH (10:1, 100 mL), dried, and then spin-dried. The sample was purified by column chromatography (EA:MeOH = 30:1) to afford 600 mg of 34f as a yellow solid, in a 77% yield. MS-ESI: m / z 830.3 [M+H]+.

[0421] Step 5

[0422] Under nitrogen, diethylamine (5 mL) was added dropwise to a solution of 34f (150 mg, 0.18 mmol) in DCM (5 mL) at 0°C. The mixture was allowed to react for 2 hours at 0°C. LCMS indicated the reaction was complete. Petroleum ether solution (100 mL x 6) was added to the reaction mixture, causing solid precipitation. After standing for solid precipitation, the solution was decanted and pumped dry using an oil pump to yield 120 mg of a white powder (34 g). LCMS indicated a product content of 70%, for a yield of 76%. MS-ESI: m / z 608.3 [M+H]+.

[0423] Step 6

[0424] Under nitrogen, a solution of HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to a solution of 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) at 0°C. The mixture was allowed to react at 0°C for 2 hours. LCMS indicated complete reaction of the starting materials. The reaction solution was directly purified by a reverse phase column using an eluent (MeCN / MeOH = 1 / 1): HO = 60%:40%) to afford 14.8 mg of a yellow solid, X2, in a 14% yield.

[0425] MS-ESI: m / z 1062.4[M+H]+.

[0426] 1H NMR (400MHz, Methanol-d4) δ7.69-7.61 (m, 2H), 7.22-7.16 (m, 2H), 7.16-7.09 (m, 3H), 6.76 (s, 2H), 5.70-5.64 (m, 1H), 5.6 0 (d, J=16.4Hz, 1H), 5.40-5.31 (m, 2H), 5.26 (d, J=19.0Hz, 1H), 4.65-4.50 (m, 7H), 4.25-4.16 (m, 1H), 3.87 (d, J=16.7Hz, 1 H), 3.83-3.76 (m, 3H), 3.72 (d, J=17.0Hz, 2H), 3.44 (t, J=7.1Hz, 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=7.3Hz, 3H).

[0427] 2.1.3 Preparation of Linker-Cytotoxin X3

[0428] The preparation scheme of linker-cytotoxin X3 is as follows:

[0429] first step

[0430] To a solution of 32a (2.00 g, 6.6 mmol) and KCO (1.82 g, 13.2 mmol) in MeCN (20 mL) was added allyl bromide (960 mg, 7.92 mmol), and the mixture was stirred at 20°C for 5 hours. TLC (PE / EA = 1 / 2) indicated the reaction was complete. The reaction solution was poured into 100 mL of water, the pH was adjusted to 5, and the mixture was extracted three times with EA (100 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (PE / EA = 2 / 1) to afford 1.83 g of 32b as a white solid (yield: 81%).

[0431] Step 2

[0432] To a solution of 32b (1.38 g, 4.02 mmol) in DCM (10 mL) was added TFA (10 mL) and stirred at 25°C for 17 hours. TLC (PE / EA = 1 / 3) indicated the reaction was complete. The reaction solution was dried to afford 0.91 g of a yellow viscous product 32c. The yield was negligible.

[0433] Step 3

[0434] To a solution of 32c (910 mg, 4.87 mmol) and NaHCO₃ (613 mg, 7.3 mmol) in DME / H₂O (20 mL / 10 mL) was added 41d (1.92 g, 4.87 mmol) and stirred at 25°C for 3 hours. TLC (DCM / MeOH = 1 / 1) indicated the reaction was complete. The reaction solution was poured into 100 mL of water, the pH was adjusted to 5 with aq. HCl (1 N), and the mixture was extracted twice with EA (150 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (DCM / MeOH = 20 / 1) to afford 1.53 g of 32e as a white solid in a 67% yield. MS-ESI: m / z 467.4 [M+H]⁺.

[0435] Step 4

[0436] To a solution of 32f (3 g, 5.83 mmol) in MeOH (50 mL) was added Pd / C (600 mg) and stirred at 25°C under a hydrogen balloon for 5 hours. TLC (EA) indicated the reaction was complete. The reaction solution was filtered and dried to afford 1.9 g of a white solid (32g, yield: 77%).

[0437] Step 5

[0438] HATU (707 mg, 1.86 mmol) was added to 32 g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol), and triethylamine (342 mg, 3.38 mmol) in DMF (10 mL). The mixture was stirred at 0°C for 3.5 hours. TLC (EA) indicated the reaction was complete. The reaction solution was poured into H2O (80 mL), extracted twice with EA (100 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (EA) to afford 1.186 g of a white solid (32h, yield: 83%). MS-ESI: m / z 842.3 [M+H]+.

[0439] Step 6

[0440] A solution of 32h (1.186 g, 1.41 mmol) in DCM / diethylamine (20 mL, 20 / 1) was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 10 / 1) indicated the reaction was complete. The reaction solution was poured into petroleum ether (200 mL) and filtered to afford 768 mg of 32i as a white solid in an 88% yield. MS-ESI: m / z 620.3 [M+H]+.

[0441] Step 7

[0442] To a solution of 32i (676 mg, 1.09 mmol), 32e (508 mg, 1.09 mmol), and DIEA (423 mg, 3.27 mmol) in DMF (10 mL) was added HATU (414 mg, 1.09 mmol), and the mixture was stirred at 20°C for 17 hours. TLC (PE / EA = 1 / 5) indicated the reaction was complete. The reaction solution was poured into water (30 mL) and filtered. The filter cake was purified by column chromatography (DCM / MeOH = 50 / 1) to afford 511 mg of 32j as a white solid in a 44% yield. MS-ESI: m / z 1068.3 [M+H]+.

[0443] Step 8

[0444] A solution of 32j (482 mg, 0.451 mmol) in diethylamine / DCM (10 mL, 1 / 5) was stirred at 10°C for 17 hours. TLC (EA) indicated the reaction was complete. The reaction solution was poured into PE (300 mL) and filtered to afford 32k as a white solid (301 mg). The yield was negligible.

[0445] Step 9

[0446] To a solution of 32k (301 mg, 0.356 mmol) and Pd(PPh3)4 (82 mg, 0.071 mmol) in THF (5 mL) was added morpholine (93 mg, 1.07 mmol) and stirred at 25°C for 5 hours. LCMS indicated the reaction was complete. The reaction mixture afforded 108 mg of 321 as a white solid, yield: 38%. MS-ESI: m / z 806.3 [M+H]+.

[0447] Step 10

[0448] To a solution of 321 (108 mg, 0.134 mmol) and triethylamine (41 mg, 0.402 mmol) in THF (2 mL) and DMF (2 mL) was added bromoacetyl bromide (27 mg, 0.134 mmol), and the mixture was stirred at 0°C for 1 hour. TLC (DCM / MeOH = 10 / 1) indicated the reaction was complete. The reaction solution was directly used to obtain 15 mg of a white solid, X3, in a 12% yield.

[0449] MS-ESI: m / z 926.3[M+H]+.

[0450] 1H NMR (400MHz, DMSO-d6) δ12.11 (s, 1H), 8.54-8.42 (m, 3H), 8.27-8.16 (m, 2H), 7.78 (d, J=11.0Hz, 1H), 7.3 0(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=5.6Hz, 2H), 3.70 (d, J=5.9Hz, 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=7.3Hz, 3H).

[0451] 2.1.4 Preparation of Linker-Cytotoxin X4:

[0452] The preparation scheme of linker-cytotoxin X4 is as follows:

[0453] first step

[0454] To a solution of 33a (2.00 g, 2.58 mmol) in MeOH (20 mL) was added Pd / C (400 mg, 10 wt.%) and stirred at 20°C for 5 h. TLC (EA) indicated the reaction was complete. The reaction solution was filtered and dried to afford 1.3 g of 33b as a white solid (yield: 74%).

[0455] Step 2

[0456] To a solution of 33b (0.55 g, 0.802 mmol), KI4 (427 mg, 0.802 mmol), and DIPEA (310 mg, 2.40 mmol) in DMF (5 mL) was added HATU (305 mg, 0.802 mmol), and the mixture was stirred at 0°C for 2 hours. TLC (DCM / MeOH = 1 / 10) indicated the reaction was complete. The reaction solution was poured into water (40 mL) and filtered to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 20 / 1) to afford 33c as a yellow solid (360 mg, 41% yield).

[0457] Step 3

[0458] To a solution of 33c (360 mg, 0.326 mmol) in DCM (10 mL) was added diethylamine (2 mL). The mixture was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 5 / 1) indicated the reaction was complete. The reaction solution was poured into PE (100 mL) and filtered to afford 205 mg of 33d as a white solid in a 71% yield. MS-ESI: m / z 881.3 [M+H]+.

[0459] Step 4

[0460] To a solution of 33d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) and water (1 mL) was added a solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL), and stirred at 0°C for 1 hour. The reaction solution directly produced 15 mg of a white solid X4, with a yield of 6%.

[0461] MS-ESI: m / z 1001.2[M+H]+.

[0462] 1H NMR (400MHz, DMSO-d6) δ8.57-8.50 (m, 1H), 8.50-8.43 (m, 2H), 8.35-8.29 (m, 1H), 8.19-8.12 (m, 2H), 7 .80 (d, J=10.8Hz, 1H), 7.27-7.14 (m, 7H), 6.53 (s, 1H), 5.59-5.51 (m, 1H), 5.44-5.39 (m, 2H), 5.20-5.0 7(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), 2.83-2.7 4(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=7.3Hz, 3H).

[0463] 2.2 Preparation of anti-B7H3 antibody-drug conjugates

[0464] 2.2.1 Preparation of ADC-1 (Daiichi Sankyo, DS7300)

[0465] DS-7300 consists of a humanized anti-B7-H3 IgG1 monoclonal antibody coupled to the topoisomerase I inhibitor payload, the exatecan derivative deruxtecan, via a tetrapeptide-based cleavable linker.

[0466] The reducing agent and protective agent were prepared using ultrapure water as follows: 2 mg / ml TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo) aqueous solution and 100 mmol / L EDTA (disodium ethylenediaminetetraacetic acid, manufacturer: Sigma) aqueous solution.

[0467] Linker-payload (Deruxtecan) was dissolved in dry DMA (N,N-Dimethylacetamide, dimethylacetamide, manufacturer: Sinopharm Group) to prepare a 10 mg / mL linker-payload DMA solution.

[0468] 16 mg of a 7.4 mg / ml reference monoclonal antibody (the antibody sequence used the heavy chain shown in SEQ ID NO: 9 and the light chain shown in SEQ ID NO: 16 in patent "CN104755494B") was transfected into CHO cells and purified according to conventional antibody expression procedures, with an antibody purity of >95%). Placed in a 50 ml centrifuge tube, the antibody concentration was diluted to 5 mg / ml with 30 mM His-HAc, pH 5.5 buffer. 100 mM EDTA aqueous solution was added to 5% of the total volume of the reaction solution, and the reaction was mixed by vortexing. 2 mg / ml TCEP aqueous solution was added to reduce the antibody, with a molar ratio of TCEP to antibody of 2.7:1. The reaction was mixed by vortexing and the reaction was allowed to react on a refrigerated thermomixer at 37°C for 2 h. The aforementioned linker-payload DMA solution was added to a final drug-to-antibody molar ratio of 9:1. DMA was supplemented to 10% of the total volume of the reaction solution, and the reaction was allowed to react on a refrigerated thermomixer at 4°C for 1 h. The sample storage buffer was replaced using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). The sample was first ultrafiltered three times with 30 mM His-HAc, pH 5.5 buffer containing 10% DMSO, then ultrafiltered six times with 30 mM His-HAc, pH 5.5, without DMSO. Finally, the sample was sterilized by filtration using a 0.2 μm PES membrane to obtain 11 mg of the antibody-drug conjugate reference ADC-1 at a concentration of 5.825 mg / mL, with a yield of 68.75%.

[0469] After testing, the drug loading rate (DAR) of the antibody-drug conjugate reference ADC-1 was 4.59, and the SEC purity was 99.64%.

[0470] 2.2.2 Preparation of Antibody-Drug Conjugate WBP301088-X2

[0471] The reducing agent and protective agent were prepared using ultrapure water as follows: 2 mg / ml TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo) aqueous solution and 100 mmol / L EDTA (disodium ethylenediaminetetraacetic acid, manufacturer: Sigma) aqueous solution.

[0472] The linker-payload X2 prepared in Example 2.1.2 was dissolved in dry DMA (N,N-Dimethylacetamide, dimethylacetamide, manufacturer: Sinopharm Group) to prepare a 10 mg / mL linker-payload DMA solution.

[0473] 1) The antibody-drug conjugate WBP301088-X2 (DAR4) was prepared as follows:

[0474] 20 mg of 11.3 mg / ml WBP301088 mAb was placed in a 50 ml centrifuge tube. 30 mM His-HAc, pH 5.5 buffer was added to dilute the antibody concentration to 5 mg / ml. 100 mM EDTA aqueous solution was added to 5% of the total reaction volume. After vortexing, the antibody was reduced by adding 2 mg / ml TCEP aqueous solution at a 4.5:1 molar ratio of TCEP to antibody. After vortexing, the reaction was incubated on a refrigerated thermomixer at 37°C for 2 hours. The aforementioned linker-payload DMA solution was added to a final drug-to-antibody molar ratio of 12:1. DMA was added to 10% of the total reaction volume. After vortexing, the reaction was incubated on a refrigerated thermomixer at 4°C for 1 hour. The sample storage buffer was replaced using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). The sample was first ultrafiltered three times with 30 mM His-HAc, pH 5.5 buffer containing 10% DMSO, then ultrafiltered six times with 30 mM His-HAc, pH 5.5, without DMSO. Finally, the sample was sterilized by filtration using a 0.2 μm PES membrane to obtain 15 mg of the antibody-drug conjugate WBP301088-X2 (DAR4) at a concentration of 7.162 mg / mL, with a yield of 75%.

[0475] The drug loading rate (DAR) of the antibody-drug conjugate WBP301088-X2 (DAR4) was 4.02, and the SEC purity was 99.20%.

[0476] 2) The antibody-drug conjugate WBP301088-X2 (DAR6) was prepared as follows:

[0477] 20 mg of 11.3 mg / ml WBP301088 mAb was placed in a 50 ml centrifuge tube. 30 mM His-HAc, pH 5.5 buffer was added to dilute the antibody concentration to 5 mg / ml. 100 mM EDTA aqueous solution was added to 5% of the total reaction volume. After vortexing, the antibody was reduced by adding 2 mg / ml TCEP aqueous solution (the molar ratio of TCEP to antibody was 8:1). After vortexing, the reaction was incubated on a refrigerated thermomixer at 37°C for 2 hours. The aforementioned linker-payload DMA solution was added to a final drug-to-antibody molar ratio of 12:1. DMA was added to 10% of the total reaction volume. After vortexing, the reaction was incubated on a refrigerated thermomixer at 4°C for 1 hour. The sample storage buffer was replaced using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). The sample was first ultrafiltered three times with 30 mM His-HAc, pH 5.5 buffer containing 10% DMSO, then ultrafiltered six times with 30 mM His-HAc, pH 5.5, without DMSO. Finally, the sample was sterilized by filtration using a 0.2 μm PES membrane to obtain 10.8 mg of the antibody-drug conjugate WBP301088-X2 (DAR6) at a concentration of 4.933 mg / mL, with a yield of 54%.

[0478] The drug loading rate (DAR) of the antibody-drug conjugate WBP301088-X2 (DAR6) was 5.68, and the SEC purity was 99.57%.

[0479] 3) The antibody-drug conjugate WBP301088-X2 (DAR8) was prepared as follows:

[0480] 20 mg of 11.3 mg / ml WBP301088 mAb was placed in a 50 ml centrifuge tube. 30 mM His-HAc, pH 5.5 buffer was added to dilute the antibody concentration to 5 mg / ml. 100 mM EDTA aqueous solution was added to 5% of the total reaction volume. After vortexing, the antibody was reduced by adding 2 mg / ml TCEP aqueous solution at a TCEP to antibody molar ratio of 15:1. After vortexing, the reaction was incubated on a refrigerated thermomixer at 37°C for 2 hours. The aforementioned linker-payload DMA solution was added to a final drug-to-antibody molar ratio of 18:1. DMA was added to 10% of the total reaction volume. After vortexing, the reaction was incubated on a refrigerated thermomixer at 4°C for 1 hour. The sample storage buffer was replaced using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). The sample was first ultrafiltered three times with 30 mM His-HAc, pH 5.5 buffer containing 10% DMSO, then ultrafiltered six times with 30 mM His-HAc, pH 5.5, without DMSO. Finally, the sample was sterilized by filtration using a 0.2 μm PES membrane to obtain 10.2 mg of the antibody-drug conjugate WBP301088-X2 (DAR8) at a concentration of 4.278 mg / mL, with a yield of 51.0%.

[0481] The drug loading rate (DAR) of the antibody-drug conjugate WBP301088-X2 (DAR8) was 7.45, and the SEC purity was 98.73%.

[0482] 2.3. Preparation of Cytotoxin (Payload)

[0483] The preparation scheme of cytotoxin P-III-30 is as follows:

[0484] first step

[0485] Under nitrogen, DIEA (60.6 mg, 0.47 mmol) was added dropwise 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). The mixture was reacted at 0°C for 2 hours. LCMS indicated complete reaction. The reaction solution was added dropwise to 20 mL of water with stirring. The precipitated solid was filtered to afford 60.2 mg of P-III-30 as a gray solid, yield: 61%. MS-ESI: m / z 522.2 [M+H]+.

[0486] 1H NMR (400MHz, DMSO-d6) δ8.42 (d, J=8.7Hz, 1H), 7.79 (d, J=11.0Hz, 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=4.6Hz, 1H), 4.09-3.99 (m, 1H), 3.22-3.11 (m, 2H), 2.40 (s, 3H), 2.28 (dd, J=13.7, 7.2Hz, 1H), 2 .22-2.08(m, 3H), 1.94-1.78(m, 2H), 1.08(d, J=6.1Hz, 3H), 0.87(t, J=7.3Hz, 3H).

[0487] 2.4 ELISA-based detection of the binding activity of antibody-drug conjugates and their unconjugated antibodies to human B7H3 antigen

[0488] In this example, ELISA was used to detect whether there was a change in the specific binding of the antibody to the B7H3 antigen protein before and after conjugation with the drug. The information of the antigen and detection antibody used is as follows.

[0489] 2.4.1 ELISA-based binding activity detection

[0490] A 96-well ELISA plate was coated with recombinant human B7H3 antigen (see table above) at 2 μg / mL, 30 μL / well, at 4°C overnight. The next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing three times with PBST, serial dilutions of antibody WBP301088, antibody-drug conjugate WBP301088-X2 (DAR6), or isotype control antibody hIgG1 were added and incubated for 1 hour. After washing three times with PBST, 100 μL / well of a 1:5000 dilution of HRP-labeled anti-human secondary antibody was added and incubated for 1 hour. After incubation, the plate was washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190). The results are shown in Figure 13 , which show that there was no significant change in the binding activity of the antibody to the human B7H3 antigen protein before and after conjugation with the drug.

[0491] 2.4.2 ELISA-based detection of binding activity to B7 family proteins

[0492] Human PD-L1 protein (see the table above), 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 immobilized on 96-well plates by incubation overnight at 4°C. The 96-well plates were then blocked by incubation with 1% BSA in PBS at 37°C for 1 hour. After blocking, the 96-well plates were washed three times with PBST (PBS containing 0.05% Tween 20). Serial dilutions of antibody WBP301088, antibody-drug conjugate WBP301088-X2 (DAR6), or isotype control antibody hIgG1 isotype were prepared in binding buffer (PBS containing 0.05% Tween 20 and 0.5% BSA) and incubated with each B7 family protein immobilized on a 96-well plate for 1 hour at 37°C. Following incubation, the 96-well plate was washed three times with PBST, incubated with the secondary antibody (see table above) in binding buffer at 37°C for 1 hour, washed again, developed with TMB, and stopped using 1M H2SO4. The results are shown in Figure 14.

[0493] As can be seen from Figure 14, there is no significant change in the binding activity of the antibody to the human B7H3 antigen protein before and after conjugation with the drug, and the antibody only specifically binds to human B7-H3 and does not specifically bind to other B7 family proteins.

[0494] Example 3. Testing the inhibitory effect of antibody-drug conjugates on tumor cell proliferation in vitro

[0495] use The chemiluminescent cell viability assay (CTG method) was used to evaluate the inhibitory effects of the anti-B7H3 ADC WBP301088-X2 (DAR6) and the reference molecule ADC-1 on cell proliferation after incubation with B7H3-positive human tumor cells for 7 days.

[0496] Cells in the logarithmic growth phase were collected and plated at a density of 1000-3000 cells / well. The cell plates were placed in a 37°C, 5% CO2 incubator for overnight culture. On the second day of the experiment, WBP301088-X2 (DAR6) was diluted 3-fold with complete culture medium to obtain 9 concentration gradients (starting with the highest concentration of 1000nM). After the drug was added, 50μL / well was added to the cell culture plate. Complete culture medium was used as a blank control, and 3 replicates were set up; the plates were continued to be incubated in a 37°C, 5% CO2 incubator for 5 days. After the incubation was completed, the cell culture plate was removed and equilibrated to room temperature. 50μL of CTG detection reagent was added to each well. After shaking and mixing, the plate was placed in the dark for 10 minutes, and the signal value was read using an enzyme-linked microplate reader. GraphPad Prism software was used to draw an S-type dose-response curve using a nonlinear regression model and calculate the IC 50 Cell viability calculation formula = (Lum 待测药 -Lum 空白对照 ) / (Lum 溶剂空白对照 -Lum 空 白对照 )×100%.

[0497] The experimental results are shown in the table below, where CAL-120 is from DSMZ and other cell lines are from ATCC.

[0498] Table 17: Antibody-drug conjugates' inhibitory activity on human tumor cell proliferation

[0499] According to the results in Table 17, the antibody-drug conjugate WBP301088-X2 (DAR6) of the present application has proliferation inhibitory activity against a variety of B7H3-positive tumor cells, and its activity is better than that of the reference ADC-1.

[0500] Example 4. Evaluation of the efficacy of antibody-drug conjugates in mice bearing human lung cancer cells Calu-6

[0501] To investigate the inhibitory effect of WBP301088-X2 (DAR6) on tumor formation in vivo, B7H3-positive human lung cancer Calu-6 cells were used to form xenografts in mice, and the in vivo anti-tumor effect of WBP301088-X2 (DAR6) was evaluated.

[0502] Female BALB / c Nude mice aged 6-8 weeks (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were used as experimental animals. 6 Calu-6 lung cancer cells were inoculated subcutaneously on the right back of 6-8 week old female BALB / c Nude mice. The mice were observed for tumor growth. When the tumor volume reached about 139 mm 3Tumor-bearing mice were randomly divided into blank control or treatment groups (n=6 mice per group). WBP301088-X2 or reference ADC-1 were injected intravenously (IV) once on the day of grouping (Day 0) at a dose of 1 mg / kg or 3 mg / kg, respectively. The experiment was terminated on Day 28. Grouping and dosing were as follows. Tumor volume and mouse body weight were measured twice weekly and recorded.

[0503] Blank control (negative control group): normal saline

[0504] WBP301088-X2 (treatment group): 1 mg / kg

[0505] WBP301088-X2 (treatment group): 3 mg / kg

[0506] Reference ADC-1 (treatment group, positive control group): 1 mg / kg

[0507] Reference ADC-1 (treatment group, positive control group): 3 mg / kg

[0508] All samples were diluted with physiological saline.

[0509] At the end of the experiment, the mice were euthanized and the tumor inhibition rate (TGI) was calculated as follows:

[0510] TGI (%) = [1-(T i -T0) / (V i -V0)]×100

[0511] T i : Mean tumor volume of the treatment group and the positive control group on the i-th day of administration;

[0512] T0: mean tumor volume of the treatment group and the positive control group on day 0 of administration;

[0513] V i : Mean tumor volume of the negative control group on the i-th day of administration;

[0514] V0: mean tumor volume of the negative control group on day 0 of administration.

[0515] The experimental results are shown in Figure 15, Table 18 and Table 19.

[0516] Table 18. Tumor volume of each group at different time points (mm 3 )

[0517] Note: Tumor volume is expressed as mean ± standard error; days are days after the start of drug administration.

[0518] Table 19. Evaluation of the anti-tumor efficacy of the tested antibody-drug conjugates in a Calu-6 cell subcutaneous xenograft tumor mouse model (calculated based on the tumor volume on day 24 after administration)

[0519] Note: Tumor volume is expressed as mean ± standard error; tumor growth inhibition is expressed as T / C (T / C (%) = T i / V i × 100) and TGI (TGI (%) = [1-(T i -T0) / (V i -V0)]×100); p value was calculated based on tumor volume (p>0.05 represents no statistical difference, p<0.01 represents a significant difference).

[0520] The results in Figure 15, Table 18 and Table 19 show that the antibody-drug conjugate WBP301088-X2 of the present application exhibited significant dose-dependent tumor inhibition activity after a single administration and the tumor inhibition effects at both doses of 1 mg / kg and 3 mg / kg were significantly better than those of the reference ADC-1.

[0521] Example 5. Evaluation of the efficacy of antibody-drug conjugates on human melanoma cell A375 tumor-bearing mice

[0522] To investigate the inhibitory effect of WBP301088-X2 (DAR6) on tumor formation in vivo, B7H3-positive human melanoma A375 cells were used to form xenografts in mice, and the in vivo anti-tumor effect of WBP301088-X2 (DAR6) was evaluated.

[0523] Female NOD / SCID mice aged 6-8 weeks (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used as experimental animals. 6 A375 human melanoma cells were inoculated subcutaneously on the right back of 6-8 week old female NOD / SCID mice and the tumor growth was observed. When the tumor volume reached about 89 mm 3 Tumor-bearing mice were randomly divided into blank control or treatment groups (n=6 mice per group). WBP301088-X2 or reference ADC-1 were injected intravenously (iv) once on the day of grouping (day 0) at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, respectively. The experiment was terminated on day 20. Tumor volume and mouse body weight were measured twice weekly and recorded.

[0524] Blank control (negative control group): normal saline

[0525] WBP301088-X2 (treatment group): 1 mg / kg

[0526] WBP301088-X2 (treatment group): 3 mg / kg

[0527] WBP301088-X2 (treatment group): 10 mg / kg

[0528] Reference ADC-1 (treatment group): 3 mg / kg

[0529] Reference ADC-1 (treatment group): 10 mg / kg

[0530] All samples were diluted with physiological saline.

[0531] At the end of the experiment, the mice were euthanized and TGI was calculated. TV (Relative tumor inhibition rate). TGI TV The calculation formula for (relative tumor inhibition rate) is:

[0532] Among them, mean RTV treat : Mean RTV value of the drug-treated group;

[0533] mean RTV vehicle : Average RTV value of the Vehicle group (vehicle group, in this example, the group only administered with normal saline);

[0534] The calculation formula of the RTV is:

[0535] Among them, V nt : Tumor volume of mouse numbered n on day t;

[0536] V n0 : Tumor volume of mice numbered n on day 0;

[0537] RTV n : The relative tumor volume of the mouse numbered n on day t.

[0538] The experimental results are shown in Figure 16, Table 20 and Table 21.

[0539] Table 20 Changes in tumor volume in different groups

[0540] Note: Data are expressed as mean ± standard error.

[0541] Table 21 Relative tumor inhibition rate (TGI) of different groups TV )

[0542] The results in Figure 16, Table 20 and Table 21 indicate that the antibody-drug conjugate WBP301088-X2 of the present application exhibited significant dose-dependent tumor inhibition activity after a single administration.

[0543] Example 6. Evaluation of the efficacy of antibody-drug conjugates on human brain cancer cell U87 tumor-bearing mice

[0544] To investigate the inhibitory effect of WBP301088-X2 (DAR6) on tumor formation in vivo, the anti-tumor effect of WBP301088-X2 (DAR6) was evaluated in vivo after xenografts were formed in mice using B7H3-positive human brain cancer cell U87.

[0545] Female NCG mice aged 6-8 weeks (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used as experimental animals. 6 U87 human brain cancer cells were inoculated subcutaneously on the right side of the neck of 6-8 week old female NCG mice. The growth of the tumor was observed. When the tumor volume reached about 187 mm 3 Tumor-bearing mice were randomly divided into blank control or treatment groups (6 mice per group). On the day of grouping (day 0), WBP301088-X2 or reference ADC-1 were injected intravenously (iv) once at doses of 1 mg / kg and 3 mg / kg, respectively. The experiment was terminated on day 20. Grouping and dosing were as follows. Tumor volume and mouse body weight were measured twice weekly and recorded.

[0546] Blank control (negative control group): normal saline

[0547] WBP301088-X2 (treatment group): 1 mg / kg

[0548] WBP301088-X2 (treatment group): 3 mg / kg

[0549] Reference ADC-1 (treatment group, positive control group): 1 mg / kg

[0550] Reference ADC-1 (treatment group, positive control group): 3 mg / kg

[0551] All samples were diluted with physiological saline.

[0552] At the end of the experiment, the mice were euthanized and the tumor inhibition rate (TGI) was calculated as follows:

[0553] TGI (%) = [1-(T i -T0) / (V i -V0)]×100

[0554] T i: Mean tumor volume of the treatment group and the positive control group on the i-th day of administration;

[0555] T0: mean tumor volume of the treatment group and the positive control group on day 0 of administration;

[0556] V i : Mean tumor volume of the negative control group on the i-th day of administration;

[0557] V0: mean tumor volume of the negative control group on day 0 of administration.

[0558] The experimental results are shown in Figure 17, Table 22 and Table 23.

[0559] Table 22. Tumor volume of each group at different time points (mm 3 )

[0560] Note: Tumor volume is expressed as mean ± standard error; days are days after the start of drug administration.

[0561] Table 23. Evaluation of the anti-tumor efficacy of the tested antibody-drug conjugates in a subcutaneous U87MG cell xenograft tumor mouse model (calculated based on the tumor volume on day 20 after administration)

[0562] Note: Tumor volume is expressed as mean ± standard error; tumor growth inhibition is expressed as T / C (T / C (%) = T i / V i × 100) and TGI (TGI (%) = [1-(T i -T0) / (V i -V0)]×100); p value was calculated based on tumor volume (p>0.05 represents no statistical difference, p<0.01 represents a significant difference).

[0563] The results in Figure 17, Table 22 and Table 23 show that the antibody-drug conjugate WBP301088-X2 of the present application exhibited significant dose-dependent tumor inhibition activity after a single administration, and the tumor inhibition effect of the antibody-drug conjugate WBP301088-X2 at a dose of 3 mg / kg was significantly better than that of the reference ADC-1.

[0564] Example 7. Evaluation of the efficacy of antibody-drug conjugates on human prostate cancer cell PC-3 tumor-bearing mice

[0565] To investigate the inhibitory effect of WBP301088-X2 (DAR6) on tumor formation in vivo, B7H3-positive human prostate cancer cell PC-3 xenografts were established in mice, and the in vivo anti-tumor effect of WBP301088-X2 (DAR6) was evaluated.

[0566] Female BALB / c Nude mice aged 6-8 weeks (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were used as experimental animals. 6 Human prostate cancer cells PC-3 were inoculated subcutaneously on the right side of the neck of 6-8 week old female BALB / c nude mice and the tumor growth was observed. When the tumor volume reached about 155 mm 3 Tumor-bearing mice were randomly divided into blank control or treatment groups (6 mice per group). On the day of grouping (day 0), WBP301088-X2 or reference ADC-1 were injected intravenously (iv) once at a dose of 2 mg / kg or 6 mg / kg, respectively. The experiment was terminated on day 21. Grouping and dosing were as follows. Tumor volume and mouse body weight were measured twice weekly and recorded.

[0567] Blank control (negative control group): normal saline

[0568] WBP301088-X2 (treatment group): 2 mg / kg

[0569] WBP301088-X2 (treatment group): 6 mg / kg

[0570] Reference ADC-1 (treatment group, positive control group): 2 mg / kg

[0571] Reference ADC-1 (treatment group, positive control group): 6 mg / kg

[0572] All samples were diluted with physiological saline.

[0573] At the end of the experiment, the mice were euthanized and the tumor inhibition rate (TGI) was calculated as follows:

[0574] TGI (%) = [1-(T i -T0) / (V i -V0)]×100)

[0575] T i : Mean tumor volume of the treatment group and the positive control group on the i-th day of administration;

[0576] T0: mean tumor volume of the treatment group and the positive control group on day 0 of administration;

[0577] V i : Mean tumor volume of the negative control group on the i-th day of administration;

[0578] V0: mean tumor volume of the negative control group on day 0 of administration.

[0579] The experimental results are shown in Figure 18, Table 24 and Table 25.

[0580] Table 24. Tumor volume of each group at different time points (mm 3 )

[0581] Note: Tumor volume is expressed as mean ± standard error; days are days after the start of drug administration.

[0582] Table 25. Evaluation of the anti-tumor efficacy of the tested antibody-drug conjugates in a PC-3 cell subcutaneous xenograft tumor mouse model (calculated based on the tumor volume on day 21 after administration)

[0583] Note: Tumor volume is expressed as mean ± standard error; tumor growth inhibition is expressed as T / C (T / C (%) = T i / V i × 100) and TGI (TGI (%) = [1-(T i -T0) / (V i -V0)]×100); p value was calculated based on tumor volume (p>0.05 represents no statistical difference, p<0.01 represents a significant difference).

[0584] The results in Figure 18, Table 24 and Table 25 show that the antibody-drug conjugate WBP301088-X2 of the present application exhibited significant dose-dependent tumor inhibition activity after a single administration, and the tumor inhibition effect was significantly better than that of the reference ADC-1 at doses of 2 mg / kg and 6 mg / kg.

[0585] Example 8. Evaluation of the efficacy of antibody-drug conjugates in a prostate cancer human xenograft (PDX) model

[0586] To investigate the efficacy of WBP301088-X2 (DAR6) in a human prostate cancer xenograft model, human prostate cancer PR9586 and PR9587 tumor tissues (obtained from Crown Biotech (Zhongshan) Co., Ltd.) were subcutaneously xenografted into NPG and NOG male mice to evaluate the anti-tumor effect of WBP301088-X2 (DAR6).

[0587] 1. Test drugs and materials

[0588] Blank control (control group): normal saline

[0589] WBP301088-X2 (treatment group): 10 mg / kg

[0590] 2. Preparation of test drugs: All samples were diluted with normal saline.

[0591] 3. Experimental animals: NPG mice were purchased from Beijing Weitongda Biotechnology Co., Ltd., and NOG mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0592] 4. Experimental methods:

[0593] Human prostate cancer PR9586 and PR9587 tumor tissues were inoculated subcutaneously into NPG or NOG mice, and the tumors were grown to 500-800 mm. 3 At about 14 days, tumor tissues were collected and mice were euthanized. The collected tumor tissues were cut into tumor masses with a diameter of 2-3 mm and inoculated subcutaneously at the right anterior scapula of NPG or NOG mice. When the average tumor volume of the NPG or NOG mice inoculated with the tumor mass reached about 100-200 mm 3 The mice were randomly divided into blank control group or treatment group, with 6 mice in each group.

[0594] WBP301088-X2 was administered intravenously (iv) on the day of grouping (day 0) and once every two weeks for a total of 2 injections, with each dose of 10 mg / kg. The experiment was terminated on day 25 (for mice vaccinated with PR9586) or day 48 (for mice vaccinated with PR9587). After the start of dosing, 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 diameter and b represents the minor diameter).

[0595] At the end of the experiment, the mice were euthanized and the tumor inhibition rate TGI was calculated:

[0596] TGI (%) = [1-(T i -T0) / (V i -V0)]×100

[0597] in

[0598] T i : Mean tumor volume of the treatment group on the i-th day of administration;

[0599] T0: mean tumor volume of the treatment group on day 0 of administration;

[0600] V i : Mean tumor volume of the negative control group on the i-th day of administration;

[0601] V0: mean tumor volume of the negative control group on day 0 of administration.

[0602] 5. Experimental results:

[0603] The experimental results are shown in Figure 19A, Figure 19B, Table 26 and Table 27.

[0604] Table 26. Evaluation of the anti-tumor efficacy of the tested antibody-drug conjugates in the PR9586 human tumor xenograft model (calculated based on the tumor volume on day 25 after the first dose)

[0605] Note: p value was calculated based on tumor volume (p>0.05 means no statistical difference, p<0.01 means significant difference).

[0606] Table 27. Evaluation of the anti-tumor efficacy of the tested antibody-drug conjugates in the PR9587 human tumor xenograft model (calculated based on the tumor volume on day 48 after the first dose)

[0607] Note: p value was calculated based on tumor volume (p>0.05 means no statistical difference, p<0.01 means significant difference).

[0608] The results showed that the antibody-drug conjugate WBP301088-X2 of the present application exhibited significant tumor-suppressing activity after administration.

[0609] Example 9. Evaluation of the efficacy of antibody-drug conjugates in a prostate cancer microtumor xenograft model

[0610] To investigate the efficacy of WBP301088-X2 (DAR6) in a humanized prostate cancer xenograft model, the anti-tumor effect of WBP301088-X2 (DAR6) was evaluated in a humanized prostate cancer microtumor xenograft mouse model.

[0611] 1. Test drugs and materials

[0612] Blank control (control group): normal saline

[0613] WBP301088-X2 (treatment group): 10 mg / kg

[0614] Collagenase digestion solution:

[0615] Prepare collagenase digestion solution by mixing the components in the table according to the manufacturer's instructions.

[0616] 2. Preparation of test drugs: All samples were diluted with normal saline.

[0617] 3. Experimental Animals: CB17 SCID male mice, 6-8 weeks old, weighing approximately 18-22 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0618] 4. Experimental methods:

[0619] The tumor tissues of human prostate cancer transplant models LD1-2027-410644 and LD1-2034-362055 (obtained from Shanghai Lidi Biotechnology Co., Ltd.) were inoculated subcutaneously into mice. 3 Tumor tissue was surgically removed from mice aseptically, and non-tumor and necrotic tissue were removed in a biosafety cabinet to ensure the purity of the inoculated tumor tissue. The mice were then euthanized. The treated tumor tissue was cut into 1-3 cubic millimeter pieces and digested with collagenase digestion solution at 37°C for 1-2 hours. The pieces were centrifuged at 1200 rpm for 3 minutes, the supernatant removed, and the cells resuspended in 10 ml of PBS containing 1% FBS and counted on a hemocytometer. Murine cells were removed, the supernatant removed by centrifugation at 1200 rpm for 3 minutes, and the cells resuspended in RPMI1640 cell culture medium. The cells were counted on a hemocytometer to adjust the cell density.

[0620] The cell suspension was filled into capsules. CB17 SCID mice were randomly divided into blank control or treatment groups (three mice per group). Each mouse received one capsule subcutaneously on each flank, for a total of six capsules per group. Each capsule contained 5,000 tumor cells. The day of inoculation was designated Day 0, and animals were dosed with a single dose via tail vein injection for 10 days.

[0621] After the experiment, the mice were euthanized, the capsules were removed, and cell viability was detected using CellTiter-Glo. The capsules were cut into pieces and an equal volume of PBS and CellTiter-Glo were added. TM Reagents, measure luminescence values. In the light signal and system, the luminescence value is proportional to the amount of ATP, and ATP is positively correlated with the number of living cells. Therefore, cell viability can be obtained by detecting the ATP content.

[0622] 5. Experimental results:

[0623] The experimental results are shown in Figure 20A, Figure 20B and Table 28.

[0624] Table 28. Experimental endpoint cell viability test results

[0625] The results shown in FIG. 20A , FIG. 20B , and Table 28 indicate that WBP301088-X2 has a significant anti-tumor effect.

[0626] Example 10. Pharmacokinetic study of single-dose antibody-drug conjugates

[0627] Purpose of the test

[0628] Following a single intravenous infusion of the antibody-drug conjugate WBP301088-X2(DAR6) in cynomolgus monkeys, the serum concentrations of WBP301088-X2(DAR6), WBP301088-total antibody, and cytotoxin P-III-30 were measured at different time points. The pharmacokinetic parameters of WBP301088-X2(DAR6), WBP301088-total antibody, and cytotoxin P-III-30 were calculated to provide a basis for preclinical and clinical studies.

[0629] Experimental methods

[0630] Eighteen cynomolgus monkeys, half male and half female, were weighed the day before administration and randomly divided into three groups (3 females and 3 males / group) based on body weight, namely a low-dose group (1 mg / kg), a medium-dose group (3 mg / kg), and a high-dose group (10 mg / kg). Each animal was given WBP301088-X2 solution by intravenous infusion.

[0631] Blood was collected from the forelimb vein of cynomolgus monkeys in each group. The processed blood samples collected at 0 h before administration and 0.5, 1, 4, 8, 24, 48, 72, 96, 168, 240, 336, 504, and 672 h after administration were used as PK samples. After blood collection at each time point, serum was obtained by centrifugation for sample analysis.

[0632] ELISA was used to quantify the concentrations of total WBP301088 antibodies and WBP301088-X2 in cynomolgus macaque serum samples. LC-MS / MS was used to quantify the concentration of P-III-30 in cynomolgus macaque serum samples. The experimental results are shown in Table 29.

[0633] Table 29. Exposure parameters of different doses of WBP301088-X2 (DAR6) in cynomolgus monkeys after a single intravenous infusion

[0634] Experimental Conclusion

[0635] Under these experimental conditions, after single intravenous administration of 1, 3, and 10 mg / kg of WBP301088-X2 to cynomolgus monkeys, no significant pharmacokinetic differences were observed between the antibody-drug conjugate and the total antibody (WBP301088), with both exhibiting dose-dependent increases in pharmacokinetic activity. This indicates that WBP301088-X2 exhibits good plasma stability in vivo. Furthermore, low plasma concentrations of P-III-30 were detected, demonstrating that the ADC exhibits slow release in monkeys, a stable conjugation mechanism, low systemic exposure to P-III-30, and a high safety profile for WBP301088-X2.

[0636] Example 11: Pharmacokinetic and toxicity studies of antibody-drug conjugates after multiple administration

[0637] Purpose of the test

[0638] WBP301088-X2(DAR6) was administered intravenously to cynomolgus monkeys once every three weeks for a total of two doses. The purpose was to observe the nature, extent, dose-effect, and time-effect relationships of potential toxic reactions induced by the antibody-drug conjugate and to identify target organs or tissues of toxicity to provide a reference for subsequent studies.

[0639] Experimental methods

[0640] Six cynomolgus monkeys, half male and half female, weighing 2.3-3.4 kg, were divided into a low-dose group (30 mg / kg) and a high-dose group (80 mg / kg). Dosing was performed once every three weeks for a total of two doses, with a dosing volume of 5 mL / kg. Animal tolerance and drug-related toxicity were assessed through clinical observation, body weight and food intake, hematology, blood biochemistry, urine, and gross anatomy.

[0641] Experimental Conclusion

[0642] Cynomolgus monkeys were intravenously administered 30 mg / kg and 80 mg / kg of WBP301088-X2 (DAR6) once every three weeks for a total of two doses. The monkeys were autopsied four days after the second dose (i.e., 25 days after the start of the drug administration).

[0643] The results showed that all animals tolerated the antibody-drug conjugate well during the trial, with no abnormalities in body weight, food consumption, electrocardiogram (ECG) parameters, coagulation parameters, or gross anatomy, indicating the safety of the antibody-drug conjugate.

[0644] Although some representative embodiments and details have been shown for the purpose of illustrating the present invention, it will be apparent to those skilled in the art that various changes and modifications can be made thereto without departing from the scope of the subject invention. In this regard, the scope of the present invention is limited only by the claims.

Claims

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

6.

2. The antibody-drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (I) a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 8 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 9; or a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 10 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 11; or (II) a heavy chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, and a light chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9; or a heavy chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10, and a light chain variable region that is at least 95%, 96%, 97%, 98% or 99% identical 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 its 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 its framework region compared to SEQ ID NO: 9 or 11.

3. The antibody-drug conjugate of claim 1 or 2, wherein the antibody is selected from the group consisting of: a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

4. The antibody-drug conjugate of claim 1 or 2, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fv, ScFv, Fab'-SH, sdAb, VHH, diabody and linear antibody.

5. The antibody-drug conjugate of any one of claims 1 to 3, wherein 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.

6. The antibody-drug conjugate of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises: (I) a heavy chain with an amino acid sequence as set forth in SEQ ID NO: 12 and a light chain with an amino acid sequence as set forth in SEQ ID NO: 13; or a heavy chain with an amino acid sequence as set forth in SEQ ID NO: 14 and a light chain with an amino acid sequence as set forth in SEQ ID NO: 15; or (II) a heavy chain at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12, and a light chain at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; or a heavy chain at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14, and a light chain at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

15.

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein the cytotoxic drug comprises a structure represented by formula (A-1), a tautomer, an enantiomer, a diastereomer, or a mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof, in, M is -L 2 -L 1 -C(O)-; L 2 is -O- or -S-; L 1 -(C(R 1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkyl or 3-6 membered saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a replace; m is selected from 1, 2, 3 or 4; the heteroatoms in the 3-6 membered saturated heterocyclic group are selected from N, O and S, and the number of heteroatoms is 1-3; R 1a 、R 1b and R 2a are each independently selected from hydrogen, halogen, hydroxy, amino, and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more R; Each R is independently hydrogen or halogen.

8. The antibody-drug conjugate of claim 7, wherein the L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a Selected from: hydrogen, halogen and C1-C6 alkyl; R 1b Selected from: hydrogen, halogen and C1-C6 alkyl.

9. The antibody-drug conjugate of claim 7 or 8, wherein L 1 -(C(R 1a )(R 1b )) m -CH2-; m is 1 or 2.

10. The antibody-drug conjugate of claim 9, wherein L 1 Selected from:

11. The antibody-drug conjugate of claim 7, wherein L 1 is a C3-C6 saturated cycloalkyl or a 3-6 membered saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and the 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a Replacement, R 2a Each is independently selected from the group consisting of: hydrogen, halogen and C1-C6 alkyl.

12. The antibody-drug conjugate of claim 11, wherein L 1 is optionally replaced by one or more R 2a Substituted C3-C6 saturated cycloalkyl; R 2a Each is independently selected from the group consisting of: hydrogen, halogen and C1-C6 alkyl.

13. The antibody-drug conjugate of claim 12, wherein L 1 1, 2 or 3 R 2a Replaces: R 2a Each is independently selected from the group consisting of: hydrogen, halogen and C1-C6 alkyl.

14. The antibody-drug conjugate of claim 13, wherein L 1 Selected from:

15. The antibody-drug conjugate according to any one of claims 1 to 14, wherein the linker unit L is -L a -L b -L c -, -L a -for -L b -Selected from: Preferably -L c -for 16. The antibody-drug conjugate of claim 15, wherein the linker unit L is Preferably 17. The antibody-drug conjugate according to any one of claims 1 to 16, wherein the structure of the antibody-drug conjugate is as shown in formula (A-2): in, p represents the average number of connections, and p is an integer or decimal selected from 1 to 10; Ab as described in any one of claims 1-6; M as described in any one of claims 7-14; L is as described in claim 15 or 16.

18. The antibody-drug conjugate of any one of claims 1 to 17, wherein p is an integer or decimal of 2-8, such as p is an integer or decimal of 4-8, such as p is an integer or decimal of 4-6 or 6-8.

19. The antibody-drug conjugate according to claim 18, wherein the structure of the antibody-drug conjugate is as shown in Formula (A-2a) or Formula (A-2b): in, p represents the average number of connections, and p is selected from an integer or decimal of 1 to 10, preferably an integer or decimal of 2-8, for example, p is an integer or decimal of 4-8, for example, p is an integer or decimal of 4-6 or 6-8; Ab is an antibody or antigen-binding fragment thereof targeting B7H3, comprising: HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and HCDR3, LCDR1 with an amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 7, LCDR2 with an amino acid sequence as set forth in SEQ ID NO: 5, and LCDR3 with an amino acid sequence as set forth in SEQ ID NO: 6; L 2 is -O- or -S-; preferably -O-; X1 is selected from the group consisting of 1, 2 or 3 R 2a Substituted C3-C6 saturated cycloalkyl; X2 is selected from -(C(R 1a )(R 1b )) m -CH2-; m is selected from 1 or 2; R 1a 、R 1b or R 2a Each is independently selected from hydrogen, halogen or C1-C6 alkyl optionally substituted by 1, 2 or 3 R; Each R is independently hydrogen or halogen.

20. The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate is selected from the following structural formula: in, p represents the average number of connections, and p is selected from an integer or decimal of 1 to 10, preferably an integer or decimal of 2-8, for example, p is an integer or decimal of 4-8, for example, p is an integer or decimal of 4-6 or 6-8; Ab is as described in any one of claims 1-6.

21. The antibody-drug conjugate of claim 20, wherein the antibody-drug conjugate is selected from: or a pharmaceutically acceptable salt thereof; wherein, p represents the average number of connections, and p is an integer or decimal selected from 1 to 10, preferably an integer or decimal of 2-8, for example, p is an integer or decimal of 4-8, for example, p is an integer or decimal of 4-6 or 6-8; Ab is an anti-B7H3 antibody, which comprises a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 2, a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 3, a LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, a LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and a LCDR3 with an amino acid sequence as shown in SEQ ID NO: 6; for example, it comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 10 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 11; for example, it comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 12 and a light chain with an amino acid sequence as shown in SEQ ID NO:

13.

22. The antibody-drug conjugate of claim 21, wherein the antibody-drug conjugate is selected from: in, p represents the average number of connections, and p is selected from an integer or decimal of 1 to 10, preferably an integer or decimal of 2-8; for example, p is an integer or decimal of 4-8, for example, p is an integer or decimal of 4-6 or 6-8, for example, p is 7.45, 5.68 or 4.02; WBP301088 is an anti-B7H3 antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 12, and the light chain amino acid sequence of which is shown in SEQ ID NO:

13.

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

24. Use of the antibody-drug conjugate according to any one of claims 1 to 22, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 23 in the preparation of a medicament for treating and / or preventing a disease or condition mediated by B7H3, preferably, the disease or condition is cancer, for example, the disease or condition is a disease or condition in which B7H3 is positively expressed.

25. The method of claim 24, wherein the cancer is selected from 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 carcinoma, lymphoma (such as non-Hodgkin's lymphoma), chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma.

26. A method for treating and / or preventing a disease or condition mediated by B7H3, comprising administering to a subject in need thereof an antibody-drug conjugate according to any one of claims 1 to 22, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 23, wherein the disease or condition is cancer, for example, a disease or condition in which B7H3 is positively expressed.

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