Anti-B7-H3 antibody, antibody drug conjugate and medical application thereof

Abstract

The present disclosure relates to anti-B7-H3 antibodies, antibody drug conjugates, and medical uses thereof. In particular, the present disclosure relates to anti-B7-H3 antibodies, antibody drug conjugates based on said anti-B7-H3 antibodies, and their use for the treatment of cancer.

Description

Technical Field

[0001] The present disclosure relates to biological, medical and clinical fields. Specifically, the present disclosure relates to anti-B7-H3 antibodies, antibody-drug conjugates based on the anti-B7-H3 antibodies, and medical uses thereof. Background Art

[0002] B7-H3, also known as CD276, is a membrane protein member of the B7-CD28 family of immunomodulatory proteins. It is a type I membrane protein with a sequence similar to the extracellular domain of programmed death receptor-1 (PD-L1). B7-H3 is a co-stimulatory / co-inhibitory molecule. Early studies have found that B7-H3 can stimulate CD4 + and CD8 + cells, promote the secretion of cytokines such as IFN-γ, and enhance CD8 + B7-H3 plays an inhibitory role in the tumor microenvironment. B7-H3 can affect multiple signaling pathways in tumor cells and inhibit Treg cells, thereby causing immune escape of tumor cells. B7-H3 can also downregulate NK cell cytotoxicity and inhibit NK cell-mediated bacteriolysis by transmitting inhibitory signals. B7-H3 also has non-immunogenic functions and plays an important role in promoting tumor growth, invasion and migration, and abnormal angiogenesis. Several independent studies have shown that higher expression levels of B7-H3 are associated with poorer prognosis in cancer patients. Prostate cancer patients with high expression of B7-H3 are more likely to have disease spread after surgery and have an increased risk of cancer recurrence and cancer-specific death. B7-H3 has also been found in ovarian tumor blood vessels and is associated with poor clinical outcomes.

[0003] B7-H3 is abnormally expressed in a variety of solid cancers, including lung cancer, prostate cancer, pancreatic cancer, colorectal cancer, liver cancer, ovarian cancer and breast cancer, as well as hematological malignancies such as acute leukemia and multiple myeloma. It is expressed at low and limited levels in normal tissues, making it an ideal target for antibody-drug conjugate (ADC) therapy and is likely to become a prognostic and detection indicator for certain cancers.

[0004] ADC is a technology that uses the ability of antibodies to target specific antigens on the surface of tumor cells, accurately delivers anti-tumor drugs (such as cytotoxic agents, cytostatics, small molecule chemotherapeutics, etc.) to tumor target cells, and releases anti-tumor drugs through endocytosis to specifically kill tumors. Antibody-drug conjugates generally consist of three parts: antibodies or antibody-like ligands, small molecule drugs, and linkers (joints) that couple antibodies or antibody-like ligands to drugs. Due to their appropriate molecular weight, high stability, strong targeting, and low toxic side effects, antibody-drug conjugates have been considered to be the most promising anti-tumor drugs. Summary of the invention

[0005] Anti-B7-H3 Antibody

[0006] In one aspect, the present disclosure provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH and the VL are selected from any one of the following groups:

[0007] 1) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 1; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 2;

[0008] 2) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 10;

[0009] 3) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 27;

[0010] 4) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 28;

[0011] 5) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 29;

[0012] 6) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 30;

[0013] 7) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 31;

[0014] 8) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 32;

[0015] 9) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 33;

[0016] 10) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 34;

[0017] 11) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 35;

[0018] 12) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 36;

[0019] 13) the VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 37;

[0020] 14) The VH comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, or HCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 9; and the VL comprises LCDR1 or a variant thereof, LCDR2 or a variant thereof, or LCDR3 or a variant thereof in the sequence shown in SEQ ID NO: 38.

[0021] In some embodiments, the HCDR and the LCDR are identified according to the Kabat, Chothia or IMTG numbering systems.

[0022] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region VH and a light chain variable region VL, wherein:

[0023] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and

[0024] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 25; wherein:

[0025] The sequence of SEQ ID NO: 25 is QNTYGIX1X2X3GAV, and the sequence of SEQ ID NO: 26 is QASQNIX4X5WLS, wherein X1 is selected from I or P; X2 is selected from G, K or R; X3 is selected from Y or R, X4 is selected from N, Q, H, E or R, and X5 is selected from S, A, T, K or R.

[0026] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region VH and a light chain variable region VL, wherein:

[0027] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and

[0028] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8;

[0029] The HCDRs and the LCDRs are identified according to the Kabat numbering system.

[0030] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region VH and a light chain variable region VL, wherein:

[0031] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and

[0032] The light chain variable region comprises LCDR1 shown in any one of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23 and 24, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8;

[0033] The HCDRs and the LCDRs are identified according to the Kabat numbering system.

[0034] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region VH and a light chain variable region VL, wherein:

[0035] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and

[0036] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in any one of SEQ ID NOs: 13, 14, 15 and 16;

[0037] The HCDRs and the LCDRs are identified according to the Kabat numbering system.

[0038] In some specific embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof as described in any of the preceding items comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH and the VL are selected from any one of the following groups:

[0039] 1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1, and

[0040] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2;

[0041] 2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and

[0042] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:10;

[0043] 3) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0044] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27, or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:27;

[0045] 4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0046] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:28, or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:28;

[0047] 5) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0048] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:29, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:29;

[0049] 6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0050] the light chain variable region comprises the amino acid sequence of SEQ ID NO:30, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:30;

[0051] 7) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0052] the light chain variable region comprises the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:31;

[0053] 8) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and

[0054] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:32, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:32;

[0055] 9) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0056] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:33, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:33;

[0057] 10) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and

[0058] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:34, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:34;

[0059] 11) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0060] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:35, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:35;

[0061] 12) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0062] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:36, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:36;

[0063] 13) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0064] the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:37, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:37;

[0065] 14) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

[0066] The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:38 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:38.

[0067] In this context, "at least 85%" means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of the foregoing values, which can be an integer or a decimal.

[0068] In some specific embodiments, the anti-B7-H3 antibody or its antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 1; the VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 2; and the HCDR and the LCDR are determined according to the Kabat numbering system.

[0069] In some specific embodiments, the anti-B7-H3 antibody or its antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 9; the VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 10; and the HCDR and the LCDR are determined according to the Kabat numbering system.

[0070] In some specific embodiments, the anti-B7-H3 antibody or its antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8; the HCDR and the LCDR are determined according to the Kabat numbering system.

[0071] In some specific embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:2.

[0072] In some specific embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:9 and a light chain variable region as shown in SEQ ID NO:10.

[0073] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof as described in any of the preceding items is a rabbit antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, or a chimeric antibody or antigen-binding fragment thereof.

[0074] In some embodiments, in the anti-B7-H3 antibody or antigen-binding fragment thereof as described in any of the preceding items, the heavy chain variable region VH and / or the light chain variable region VL are humanized.

[0075] In some embodiments, the antigen-binding fragment of any of the aforementioned anti-B7-H3 antibodies is selected from any one of the following: Fab, scFv, Fv, Fab', F(ab')2, single domain antibody, scFab, linear antibody, and multispecific antibody.

[0076] In some embodiments, any of the aforementioned anti-B7-H3 antibodies or antigen-binding fragments thereof further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3 and human IgG4, and the light chain constant region is selected from λ and κ light chain constant regions. In some embodiments, the heavy chain constant region is the heavy chain constant region of human IgG1, and the light chain constant region is a κ light chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 11, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 12.

[0077] In some embodiments, any of the aforementioned anti-B7-H3 antibodies or antigen-binding fragments thereof further comprises an Fc.

[0078] In some specific embodiments, any of the aforementioned anti-B7-H3 antibodies comprises Fc of human IgG1, Fc of human IgG2, Fc of human IgG3, or Fc of human IgG4; preferably Fc of human IgG1.

[0079] In some specific embodiments, the anti-B7-H3 antibody comprises a heavy chain set forth in SEQ ID NO: 39, or a sequence at least 85% identical thereto, and a light chain set forth in SEQ ID NO: 40, or a sequence at least 85% identical thereto.

[0080] In other specific embodiments, the anti-B7-H3 antibody comprises the heavy chain set forth in SEQ ID NO:39, and the light chain set forth in SEQ ID NO:40.

[0081] In some specific embodiments, the anti-B7-H3 antibodies or antigen-binding fragments thereof of the present disclosure bind to B7-H3 with a KD value of no greater than 8.5E-08, 8E-08, 7E-08, 6E-08, 5E-08, 4E-08, 3E-08, 2E-08, 1E-08, 9E-09, 8E-09, 7E-09, 6E-09, 5E-09, 4E-09, 3.5E-09, 3E-09, 2.5E-09, 2E-09, 1.5E-09M, 1E-09, 9E-10, 8E-10, 7.9E-10, 7.8E-10, 7.7E-10, 7.6E-10, 7.5E-10, 7.4E-10, or 7.3E-10 (M).

[0082] In other specific embodiments, the anti-B7-H3 antibodies or antigen-binding fragments thereof disclosed herein have an EC of no greater than 0.1, 0.098, 0.095, 0.08, 0.075, 0.074, 0.07, 0.06, 0.05, 0.04, 0.039, 0.036, 0.035, 0.03, 0.029, 0.025, 0.021, 0.02, or 0.016 (μg / mL). 50 , binds to B7-H3.

[0083] In other specific embodiments, the anti-B7-H3 antibodies or antigen-binding fragments thereof disclosed herein have an EC of no greater than 0.4, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34, 0.33, 0.32, 0.31, 0.30, 0.29, 0.28, 0.27 or 0.26 (nM). 50 , binds to cells expressing B7-H3.

[0084] In other specific embodiments, the efficiency of endocytosis of the anti-B7-H3 antibodies or antigen-binding fragments thereof of the present disclosure by target cells is not less than 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.3%.

[0085] Antibody Drug Conjugates

[0086] In another aspect, the present disclosure provides an antibody-drug conjugate comprising any one of the above-mentioned antibody-drug conjugates or anti-B7-H3 antibodies or antigen-binding fragments thereof.

[0087] In some embodiments, the antibody drug conjugates of the present disclosure have the following structure:

[0088]

[0089] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0090] L is a linker;

[0091] D is the drug;

[0092] n is an integer from 1 to 10 or a decimal.

[0093] In some embodiments, L has -L a -L b -L c -L d -Shown structure, L a Connected to Ab, L d Connected to D.

[0094] In some embodiments, L a Selected from -(succinimidyl-3-yl-N)-(CH2)mC(=O)-, -(succinimidyl-3-yl-N)-(CH2CH2O)pC(=O)- and (CH2)qC(=O)-; wherein, Indicates the connection site with Ab, * indicates the connection site with L b m is an integer from 0 to 8, preferably, m is 5; p is an integer from 1 to 8, preferably, p is 2; q is an integer from 0 to 4, preferably, q is 0 or 1.

[0095] In some embodiments, L bSelected from -(CH2)rC(=O)-, -NH-(CH2CH2O)s-(CH2)tC(=O)-, -NH-(CH2)wC(=O)- and a single bond, wherein r is an integer from 1 to 6, preferably, r is 3 or 4; s is an integer from 1 to 6, preferably, s is 4; t is an integer from 0 to 4, preferably, t is 2; w is an integer from 0 to 4, preferably, w is 2.

[0096] In some embodiments, L c is a peptide residue consisting of 1 to 7 amino acids, or is a single bond; wherein the amino acids are selected from valine, citrulline, glycine, phenylalanine, alanine, proline, isoleucine, lysine, serine, glutamic acid and aspartic acid, and the amino acids are unsubstituted or each independently substituted by one or more substituents, wherein each substituent is independently selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 Cycloalkyl and Where # indicates the connection site.

[0097] In some embodiments, L d Selected from NH-CH2-O-CH2-C(=O)-, and single bond; where a * Indicates that L c The attachment site, b * Indicates the attachment site to D.

[0098] In some specific factual scenarios, L has -L a -L b -L c -L d - the structure shown, wherein:

[0099] L a for Indicates the connection site with Ab, * indicates the connection site with L b The connection site,

[0100] L b is a single bond or -NH-(CH2)wC(=O)-, w is an integer from 0 to 4, preferably, w is 2;

[0101] L c is a -valyl-citrullyl-, -glycyl-glycyl-phenylalanyl-glycyl-, -glycyl- or -yl bond, and

[0102] L d for -NH-CH2-O-CH2-C(=O)- or single

[0103] key; a * Indicates that L c The attachment site, b * Indicates the attachment site to D.

[0104] In some specific factual scenarios, L has -L a -L b -L c -L d - the structure shown, wherein:

[0105] L a -(succinimidyl-3-yl-N)-(CH2)5-C(=O)-, or -(succinimidyl-3-yl-N)-(CH2CH2O)2-C(=O)-;

[0106] L b is a single bond,

[0107] L c is -valyl-citrullyl-, or -glycyl-glycyl-phenylalanyl-glycyl-;

[0108] L d for or NH-CH2-O-CH2-C(=O), a * Indicates that L c The attachment site, b * Indicates the attachment site to D.

[0109] In some specific factual scenarios, L has -L a -L b -L c -L d - the structure shown, wherein:

[0110] L a is -CH2-C(=O)-;

[0111] L b is -NH-(CH2CH2O)4-(CH2)2-C(=O)-, -NH-(CH2)2-C(=O)- or a single bond;

[0112] L c -Valyl-citrullyl-, or single bond, e* indicates b f* indicates the connection site with L d The attachment site of

[0113] L d for a * Indicates that L c The attachment site, b * Indicates the attachment site to D.

[0114] In some specific factual scenarios, L has -L a -L b -L c -L d - the structure shown, wherein:

[0115] L a is -C(=O)-;

[0116] L b is -(CH2)3-C(=O)- or -(CH2)4-C(=O)-;

[0117] L c -Valyl-Citrulyl- or e* represents the b f* indicates the connection site with L d The attachment site of

[0118] L d for a * Indicates that L c The attachment site, b * Indicates the attachment site to D.

[0119] In some embodiments, according to the antibody drug conjugates of the present disclosure, the drug is selected from: cytotoxic agents, radioisotopes, immunomodulators, cytokines, lymphokines, chemokines, growth factors, tumor necrosis factors, hormones, hormone antagonists, enzymes, oligonucleotides, DNA, RNA, siRNA, RNAi, microRNA, photosensitive therapeutic agents, anti-angiogenic agents, pro-apoptotic agents, peptides, lipids, carbohydrates, chelating agents or combinations thereof. In some specific embodiments, according to the antibody drug conjugates of the present disclosure, the drug is a cytotoxic agent, or a pharmaceutically acceptable salt thereof.

[0120] In some specific embodiments, in the antibody drug conjugate according to the present disclosure, the drug is monomethyl auristatin E (MMAE), or a pharmaceutically acceptable salt thereof.

[0121] In some embodiments, according to the antibody drug conjugate of the present disclosure, the drug is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), eribulin, exotecan, maytansine, SN-38, pyrrolobenzodiazepine and duocarmycin.

[0122] In some specific embodiments, in the antibody drug conjugate according to the present disclosure, the drug is monomethyl auristatin F (MMAF), or a pharmaceutically acceptable salt thereof.

[0123] In some specific embodiments, in the antibody-drug conjugate according to the present disclosure, the drug is eribulin, or a pharmaceutically acceptable salt thereof.

[0124] In some specific embodiments, in the antibody-drug conjugate according to the present disclosure, the drug is exotecan, or a pharmaceutically acceptable salt thereof.

[0125] In some specific embodiments, in the antibody-drug conjugate according to the present disclosure, the drug is maytansine, or a pharmaceutically acceptable salt thereof.

[0126] In some specific embodiments, in the antibody-drug conjugates according to the present disclosure, the drug is SN-38, or a pharmaceutically acceptable salt thereof.

[0127] In some specific embodiments, in the antibody drug conjugate according to the present disclosure, the drug is a pyrrolobenzodiazepine, or a pharmaceutically acceptable salt thereof.

[0128] In some specific embodiments, in the antibody-drug conjugate according to the present disclosure, the drug is duocarmycin, or a pharmaceutically acceptable salt thereof.

[0129] In some specific embodiments, the antibody drug conjugates of the present disclosure have the following structure:

[0130]

[0131] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0132] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0133] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0134]

[0135] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0136] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0137] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0138]

[0139] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0140] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0141] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0142]

[0143] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0144] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0145] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0146]

[0147] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0148] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0149] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0150]

[0151] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0152] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0153] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0154]

[0155] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0156] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0157] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0158]

[0159] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0160] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0161] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0162]

[0163] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0164] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0165] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0166]

[0167] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0168] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0169] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0170]

[0171] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0172] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0173] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0174]

[0175] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0176] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0177] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0178]

[0179] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0180] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0181] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0182]

[0183] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0184] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0185] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0186]

[0187] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0188] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0189] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0190]

[0191] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0192] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0193] In other specific embodiments, the antibody drug conjugates disclosed herein have the following structure:

[0194]

[0195] wherein Ab is an anti-B7-H3 antibody or antigen-binding fragment thereof according to the present disclosure;

[0196] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0197] In a more preferred embodiment, the antibody drug conjugate of the present disclosure has the structure shown in Formula 11.

[0198] In a more preferred embodiment, the antibody drug conjugate of the present disclosure has a structure as shown in Formula 12.

[0199] In a more preferred embodiment, the antibody drug conjugate of the present disclosure has the structure shown in Formula 13.

[0200] In a more preferred embodiment, the antibody drug conjugate of the present disclosure has a structure as shown in Formula 14.

[0201] In a more preferred embodiment, the antibody drug conjugate of the present disclosure has the structure shown in Formula 15.

[0202] In some specific embodiments, Ab comprises a heavy chain variable region and a light chain variable region, wherein:

[0203] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and

[0204] The light chain variable region comprises LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:8.

[0205] In some embodiments, Ab comprises a heavy chain variable region and a light chain variable region, wherein:

[0206] The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:10.

[0207] In some embodiments, Ab comprises a heavy chain and a light chain, wherein:

[0208] The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 39 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 39, and

[0209] The light chain comprises the amino acid sequence as shown in SEQ ID NO:40 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:40.

[0210] The present disclosure also provides a method for preparing ADC, which may further include: combining the anti-B7-H3 antibody or antigen-binding fragment thereof of the present disclosure with a drug-linker compound (or drug-linker compound (linker-drug, LD), such as LD-1 to LD-18 shown in the present invention) under conditions sufficient to form an antibody conjugate.

[0211] In one embodiment of the present disclosure, the drug is coupled to the antibody or its antigen-binding fragment via a linker unit (e.g., a linker). The loading of the ligand-drug conjugate can be controlled by the following non-limiting methods, including but not limited to:

[0212] (1) controlling the molar ratio of the drug linker fragment and the antibody (or its antigen-binding fragment),

[0213] (2) Control the reaction time and temperature,

[0214] (3) Select different reaction reagents.

[0215] In some specific embodiments, the drug loading n is 3.7-4.3, or n is 3.8-4.3 (eg, 4).

[0216] In some other specific embodiments, n is 1.8-2.2 (such as 2). In some other specific embodiments, n is 2.5-2.8 (such as 2.7).

[0217] Pharmaceutical composition

[0218] In another aspect, the present disclosure provides a pharmaceutical composition comprising:

[0219] A preventive or therapeutically effective amount of any of the aforementioned antibody-drug conjugates or anti-B7-H3 antibodies or antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

[0220] Isolated nucleic acids

[0221] In another aspect, the present disclosure also provides an isolated nucleic acid encoding the anti-B7-H3 antibody or antigen-binding fragment thereof described in any one of the aforementioned items.

[0222] It should be understood that even though the present application provides specific nucleotide sequence examples, the nucleotide sequences capable of "encoding the anti-B7-H3 antibodies or antigen-binding fragments thereof of the present disclosure" are not limited to these specific sequences, because equivalent nucleotide sequences can be used to encode the same amino acid sequence according to the codon preference and codon degeneracy of the expression host.

[0223] In another aspect, the present disclosure further provides a host cell, which comprises the aforementioned nucleic acid or the aforementioned vector or is used to express the anti-B7-H3 antibody or antigen-binding fragment thereof of the present disclosure.

[0224] Prevention or treatment methods

[0225] In another aspect, the present disclosure also provides a method for treating cancer, comprising administering to a subject a preventively or therapeutically effective amount of any of the aforementioned antibody-drug conjugates, anti-B7-H3 antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions.

[0226] In another aspect, the present disclosure also provides a method for inhibiting the growth or proliferation of cancer cells, the method comprising administering to a subject a preventively or therapeutically effective amount of any of the aforementioned antibody-drug conjugates, anti-B7-H3 antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions.

[0227] In some embodiments, the cancer is a solid tumor.

[0228] In some embodiments, the cancer is a hematological neoplasm.

[0229] In some embodiments, the cancer is selected from the group consisting of prostate cancer (e.g., castration-resistant prostate cancer), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), pancreatic cancer (e.g., pancreatic ductal carcinoma), colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, kidney cancer, urothelial carcinoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, triple-negative breast cancer, glioblastoma, pancreatic ductal carcinoma, colon cancer, osteosarcoma, soft tissue sarcoma, thyroid cancer, uterine cancer (e.g., endometrial cancer), multiple myeloma, rhabdomyosarcoma, squamous cell carcinoma, hematological tumors, mesothelioma, endometrial cancer, acute myeloid leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0230] In some specific embodiments, the antibody drug conjugate, anti-B7-H3 antibody or antigen-binding fragment thereof, nucleic acid or pharmaceutical composition of the present disclosure is used to prevent or treat prostate cancer, melanoma, breast cancer, ovarian cancer, lung cancer, sarcoma or intrauterine cancer.

[0231] In some embodiments, the cancer is associated with aberrant expression of B7-H3.

[0232] In some embodiments, cancer cells of the cancer express B7-H3 protein.

[0233] use

[0234] In another aspect, the present disclosure also provides use of any of the aforementioned antibody-drug conjugates, anti-B7-H3 antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions in the preparation of drugs for treating or preventing cancer.

[0235] The present disclosure also provides the use of a combination of an anti-B7-H3 antibody or an antigen-binding fragment thereof and a cytotoxic agent as described in any of the above items in the preparation of a drug for treating or preventing cancer, wherein the cytotoxic agent is selected from any of the following: monomethyl auristatin E (MMAE) or a pharmaceutically acceptable salt thereof, monomethyl auristatin F (MMAF) or a pharmaceutically acceptable salt thereof, eribulin or a pharmaceutically acceptable salt thereof, exitecan or a pharmaceutically acceptable salt thereof, maytansine or a pharmaceutically acceptable salt thereof, SN-38 or a pharmaceutically acceptable salt thereof, pyrrolobenzodiazepine or a pharmaceutically acceptable salt thereof, duocarmycin or a pharmaceutically acceptable salt thereof. Preferably, the pharmaceutically acceptable salt is a mesylate (as an example, eribulin mesylate, exitecan mesylate).

[0236] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof and the cytotoxic agent are in the same or different containers.

[0237] In some embodiments, the aforementioned anti-B7-H3 antibody or antigen-binding fragment thereof is provided for use in treating cancer, wherein the anti-B7-H3 antibody or antigen-binding fragment thereof and a cytotoxic agent are administered in combination.

[0238] In some embodiments, the cancer is a solid tumor.

[0239] In some embodiments, the cancer is a hematological neoplasm.

[0240] In some embodiments, the cancer is selected from the group consisting of prostate cancer (e.g., castration-resistant prostate cancer), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), pancreatic cancer (e.g., pancreatic ductal carcinoma), colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, kidney cancer, urothelial carcinoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, triple-negative breast cancer, glioblastoma, pancreatic ductal carcinoma, colon cancer, osteosarcoma, soft tissue sarcoma, thyroid cancer, uterine cancer (e.g., endometrial cancer), multiple myeloma, rhabdomyosarcoma, squamous cell carcinoma, hematological tumors, mesothelioma, endometrial cancer, acute myeloid leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0241] In some specific embodiments, the cancer is selected from the group consisting of prostate cancer, melanoma, breast cancer, ovarian cancer, lung cancer, sarcoma, and intrauterine cancer.

[0242] In some embodiments, the cancer is associated with aberrant expression of B7-H3 protein.

[0243] In some embodiments, cancer cells of the cancer express B7-H3 protein.

[0244] In another aspect, the present disclosure also provides any of the above-mentioned antibody-drug conjugates, anti-B7-H3 antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions for use as a drug. In some embodiments, the present disclosure also provides any of the above-mentioned antibody-drug conjugates, anti-B7-H3 antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions for use as a drug for treating cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological tumor. In some embodiments, the cancer is selected from the group consisting of prostate cancer (e.g., castration-resistant prostate cancer), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), pancreatic cancer (e.g., pancreatic ductal carcinoma), colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, kidney cancer, urothelial carcinoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, triple-negative breast cancer, glioblastoma, pancreatic ductal carcinoma, colon cancer, osteosarcoma, soft tissue sarcoma, thyroid cancer, uterine cancer (e.g., endometrial cancer), multiple myeloma, rhabdomyosarcoma, squamous cell carcinoma, hematological tumors, mesothelioma, endometrial cancer, acute myeloid leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0245] In some specific embodiments, the cancer is selected from the group consisting of prostate cancer, melanoma, breast cancer, ovarian cancer, lung cancer, sarcoma, and intrauterine cancer.

[0246] On the other hand, the anti-B7-H3 antibody or antigen-binding fragment thereof disclosed in the present invention is used for immunoassay. The immunoassay includes contacting the biological sample with the anti-B7-H3 antibody or antigen-binding fragment thereof disclosed in the present invention to detect cancer cells in the biological sample from the patient, or the expression level of B7-H3. Typically, the anti-B7-H3 antibody or antigen-binding fragment thereof disclosed in the present invention can be coupled with a fluorescent label or other label.

[0247] Reagents / Kits

[0248] In another aspect, the present disclosure further provides a drug kit comprising at least one container, wherein each container independently comprises: any one of the aforementioned antibody-drug conjugates, anti-B7-H3 antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions of the present disclosure.

[0249] In diagnostic or research applications, the kit comprises one or more of the following components: assay reagents, buffers, and an antibody or antigen-binding fragment thereof of the present disclosure.

[0250] Additionally, these reagents / kits may include instructions. BRIEF DESCRIPTION OF THE DRAWINGS

[0251] Figure 1 : Binding ability of 112-59 mutant antibody to B7-H3-his.

[0252] Figures 2 to 3 : EC50 of the binding activity of the mutated antibody to Calu-6 cells.

[0253] Figure 4 :The inhibitory results of different ADCs on HCC1954 breast cancer xenografts in NSG female mice.

[0254] Figure 5 :Tumor inhibitory activity of ADC in the HCC1806 breast cancer model of athymic nude female mice.

[0255] Figure 6 : Tumor inhibitory activity of ADC in the Calu-6 lung cancer model of CD1 Nude female mice.

[0256] Figure 7 : Tumor inhibitory activity of ADC in the OVCAR3 ovarian cancer model of NSG female mice.

[0257] Figure 8 :The inhibition rate of ADC molecules on PC-3 prostate cancer transplanted tumors in Athymic Nude female mice. DETAILED DESCRIPTION

[0258] the term

[0259] The terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0260] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising", "having", "including", etc. should be understood as meaning "including but not limited to". Unless otherwise specified, "comprising" includes "consisting of", for example, for VH comprising HCDR1 as shown in SEQ ID NO: 3, it clearly covers the amino acid sequence of HCDR1 as shown in SEQ ID NO: 3.

[0261] It should be understood that the ordinal numbers "first", "second", "Formula 1", "Formula 2", "1)", "2)", etc. in the present disclosure are only used to distinguish different technical features, elements, components, and steps, and are not intended to limit the level, sequence, or quantity.

[0262] The target refers to the object to which the therapeutic active ingredient (antibody drug conjugate, anti-B7-H3 antibody or its antigen-binding fragment) of the present disclosure is directed. The target can be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, mature protein, isoform, modified form, free, surface expressed). In the present disclosure, the target refers in particular to a protein. As an example, the antibody drug conjugate, anti-B7-H3 antibody or its antigen-binding fragment of the present disclosure targets B7-H3.

[0263] The nucleotide or amino acid information of B7-H3 as a target is well known in the art, and can be obtained, for example but not limited to, from literature or databases.

[0264] In the context of the present disclosure, B7-H3 should be understood broadly, including various forms of B7-H3 molecules at various stages, such as, but not limited to, molecules generated during amplification, replication, transcription, splicing, processing, translation, and modification of the B7-H3 gene, such as cDNA, mRNA, precursor protein, mature protein, and fragments thereof. In specific embodiments, B7-H3 refers to human B7-H3. In some specific embodiments, human B7-H3 refers to mature B7-H3 protein, especially B7-H3 expressed on the cell surface.

[0265] In the field of antibodies, targets usually exist in the form of antigens. "Antigen" refers to a molecule or part thereof that can be selectively recognized or bound by an antigen binding molecule (e.g., an antibody drug conjugate, an anti-B7-H3 antibody, or an antigen binding fragment thereof of the present disclosure). An antigen may have one or more epitopes.

[0266] "Epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. An epitope can be formed by continuous amino acids (linear epitope); or comprise non-continuous amino acids (conformational epitope). An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids. Routine methods in the art can be used to screen for antibodies or their antigen-binding fragments that bind to a specific epitope, such as, but not limited to, alanine scanning, peptide cleavage analysis, epitope extraction, and chemical modification of the antigen.

[0267] "Antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and full-length antibodies, as long as they exhibit the desired antigen-binding activity. Typically, natural IgG antibodies are heterotetrameric proteins consisting of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH) and three constant domains (CH1, CH2, and CH3). From N to C-terminus, each light chain has a variable region (VL) and a constant light domain (CL). Depending on the context, a technician can determine the specific meaning of "antibody".

[0268] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. Antibodies are divided into five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0269] "Specific binding" means that the anti-B7-H3 antibody or antigen-binding fragment thereof of the present disclosure binds to the targeted antigen or epitope with higher affinity than other antigens or epitopes thereof. -7An equilibrium dissociation constant (KD) of M or less is considered to be "specific" binding. KD can be measured using known methods, such as FACS or surface plasmon resonance. "Specific binding" does not exclude cross-reactivity to homologous antigens of other species (such as cynomolgus monkeys (Macacafascicularis), chimpanzees (Pan troglodytes) or marmosets (Callithrix jacchus). In this regard, "anti-B7-H3...", "specifically binds to B7-H3..." means that the anti-B7-H3 antibody or its antigen-binding fragment can specifically recognize or bind to B7-H3 (or its epitope).

[0270] "Affinity" is used to describe the strength of the non-covalent interaction between an anti-B7-H3 antibody or antigen-binding fragment thereof and its antigen or epitope. Affinity is usually represented by the equilibrium dissociation constant (KD). "Kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, and "kdis" or "kd" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. "KD" is obtained from the ratio of kd to ka (i.e., kd / ka), expressed as a molar concentration.

[0271] "Fully human antibodies" include antibodies having variable and constant regions of human germline immunoglobulin sequences.

[0272] A "humanized antibody" refers to an antibody produced by grafting non-human CDR sequences into the framework of a human antibody variable region.

[0273] A "chimeric antibody" is an antibody formed by fusing the variable region of an antibody from a first species (such as mouse or rabbit) with the constant region of an antibody from another species (such as human).

[0274] "Antibody fragments" or "antigen-binding fragments" are molecules other than intact antibodies that contain a portion of an intact antibody that retains the antigen-binding ability of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0275] "Fab" consists of a light chain and the CH1 and variable region of a heavy chain.

[0276] "Fab' fragment", which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0277] "F(ab')2 fragment" is a bivalent fragment formed by two Fab' fragments linked by a disulfide bridge at the hinge region.

[0278] "Fab'-SH" refers to Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group.

[0279] "Single-chain Fab fragment" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following orders from N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein the linker is a peptide linker. In some embodiments, the peptide linker is a polypeptide of at least 30 amino acids, preferably a polypeptide of 32-50 amino acids.

[0280] "Diabodies" have two antigen-binding sites and include a heavy chain variable domain (VH) and a light chain variable domain (VL) connected to each other in the same polypeptide chain.

[0281] A "linear antibody" comprises a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding regions.

[0282] A "Fv fragment" has the VL and VH domains of one arm of an antibody.

[0283] A "single domain antibody" is an antibody fragment that comprises all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.

[0284] A single-chain antibody fragment (scFv) is an antibody composed of a heavy chain variable region and a light chain variable region connected by a linker.

[0285] "Fc" contains two heavy chain fragments including CH2 and CH3 domains. The two heavy chain fragments are held together by disulfide bonds and by the hydrophobic interaction of the CH3 domains.

[0286] "Variable region" refers to the domain of an antibody or antigen-binding fragment that binds antigen.

[0287] "Complementarity determining region" or "CDR" refers to the region within the variable region that primarily contributes to antigen binding. VH and VL each contain four framework regions (FR) and three complementarity determining regions (CDR). VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL has the following sequence from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0288] "Framework" or "FR" refers to the variable domain residues outside the hypervariable region (CDR) residues. The FR of the variable domain is usually composed of four FR domains: FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences usually appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0289] The heavy chain variable region of the antibody that specifically binds to B7-H3 is represented as VH, and the light chain variable region is represented as VL. The three CDR regions in VH are represented as HCDR1, HCDR2, and HCDR3, respectively; the three CDR regions in VL are represented as LCDR1, LCDR2, and LCDR3, respectively.

[0290] The amino acid sequence boundaries of a CDR can be determined by various well-known schemes, for example: the Kabat numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), Chothia numbering system, and ImMunoGenTics (IMGT) numbering system (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278). Exemplarily, in the table below, the amino acid sequences of the "alternative" CDR1, CDR2 and CDR3 of the heavy chain variable region and light chain variable region of the humanized antibody 112-59 are compared with the CDRs defined according to Kabat.

[0291] Table 1-1. Relationship between CDR numbering systems

[0292]

[0293] The correspondence between various numbering systems is well known to those skilled in the art. In other words, when a CDR sequence under one numbering system and its position in an antibody are provided, the technician is capable of determining the corresponding CDR sequence under another numbering system and its position in an antibody. Technical solutions corresponding to different numbering systems will be regarded as equivalent technical solutions.

[0294] "Antibody drug conjugate" (ADC) refers to a molecule formed by connecting an antibody (or its antigen-binding fragment) to a drug (such as a cytotoxic agent) via a linker.

[0295] "Cytotoxic agents" refer to chemical molecules that have the ability to destroy growth in target cells. In principle, cytotoxic agents can kill cells at sufficient concentrations, but due to lack of specificity, when killing tumor cells, they can also cause apoptosis or death of normal cells, leading to side effects. Cytotoxic agents include toxins (such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals), radioactive isotopes, chemotherapeutic drugs, antibiotics, and nucleolytic enzymes.

[0296] The term "linker unit" or "linker fragment" or "linker" refers to a chemical structure fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end, and may also be connected to other linkers before being connected to the drug. The linker may be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including linkers containing peptidyl that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as linkers containing glucuronides that can be degraded by glucuronidase. Peptide linkers may include, for example, dipeptides, such as valyl-citrullyl, phenylalanyl-lysyl or valyl-alanyl; or tripeptides, such as glycyl-phenylalanyl-glycyl; or tetrapeptides, such as glycyl-glycyl-phenylalanyl-glycyl. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0297] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved through the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (e.g., iodinated, brominated or chlorinated), benzyl halides (e.g., iodinated, brominated or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

[0298] As an example, the linker disclosed herein has -L a -L b -L c -L d -structure, in which:

[0299] L a Selected from -(succinimidyl-3-yl-N)-(CH2)mC(=O)-, -(succinimidyl-3-yl-N)-(CH2CH2O)pC(=O)- and (CH2)qC(=O)-, wherein Indicates the connection site with Ab, * indicates the connection site with L b m is an integer between 0 and 8, preferably, m is 5; p is an integer between 1 and 8, preferably, p is 2; q is an integer between 0 and 4, preferably, q is 0 or 1;

[0300] L b Selected from -(CH2)rC(=O)-, -NH-(CH2CH2O)s-(CH2)tC(=O)-, -NH-(CH2)wC(=O)- and a single bond, wherein r is an integer from 1 to 6, preferably, r is 3 or 4; s is an integer from 1 to 6, preferably, s is 4; t is an integer from 0 to 4, preferably, t is 2; w is an integer from 0 to 4, preferably, w is 2;

[0301] L c is a peptide residue consisting of 1 to 7 amino acids or a single bond; wherein the amino acids are selected from valine, citrulline, glycine, phenylalanine, alanine, proline, isoleucine, lysine, serine, glutamic acid and aspartic acid, and the amino acids are unsubstituted or each independently substituted by one or more substituents, wherein each substituent is independently selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, C3-7 Cycloalkyl and Where # indicates the substitution site;

[0302] L d Selected from NH-CH2-O-CH2-C(=O)-, and single

[0303] key; where a * Indicates that L c The attachment site, b * Indicates the attachment site to D.

[0304] The term "peptide residue" in this context refers to a short peptide formed by linearly connecting amino acid residues through amide bonds. The term "amino acid" refers to an organic compound that contains an amino group and a carboxyl group in its molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure. When amino acids are combined with each other, a molecule of water is lost because some groups participate in the formation of amide bonds, so the amino acid unit in the peptide is called an "amino acid residue".

[0305] The term "single bond" refers to a covalent bond between two atoms in a compound molecule formed by sharing a pair of electrons.

[0306] The term "alkyl" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., C 1-20 The alkyl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment. The substituents are preferably selected from one or more of D atoms, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0307] The term "C 1-6 "Alkyl" is a lower alkyl group containing 1 to 6 carbon atoms. 1-6 Non-limiting examples of "alkyl" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group can be substituted or unsubstituted.

[0308] The term "halogenated C 1-6 "Alkyl" refers to C 1-6 The hydrogen on the alkyl group is replaced by one or more halogens, where C 1-6 Alkyl is as defined above.

[0309] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl. Polycyclic cycloalkyls include cycloalkyls of spirocyclic, condensed or bridged rings.

[0310] The term "C 3-7 "Cycloalkyl" refers to a cycloalkyl group containing 3 to 7 ring carbon atoms, and cycloalkyl is as defined above.

[0311] The term "alkoxy" refers to -O-(alkyl), wherein the definition of alkyl is as described above. Non-limiting examples include: methoxy, ethoxy, propoxy and butoxy, etc. Alkoxy can be substituted or unsubstituted, and when substituted, it can be substituted at any usable point of attachment. Substituents are preferably selected from one or more of D atoms, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0312] The term "C 1-6 "Alkoxy" means an alkoxy group containing 1 to 6 carbon atoms, and alkoxy is as defined above.

[0313] The term "aryl" refers to a monocyclic all-carbon aromatic ring (i.e., a monocyclic aromatic group) or a polycyclic aromatic ring system (i.e., a polycyclic aromatic group) having a conjugated π electron system, which has 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., a 6- to 14-membered aromatic group). The aryl group is preferably an aromatic group having 6 to 10 ring atoms (i.e., a 6- to 10-membered aromatic group). The monocyclic aromatic group is, for example, phenyl. Non-limiting examples of the polycyclic aromatic group include: naphthyl, anthracenyl, phenanthryl, etc. The polycyclic aromatic group also includes a phenyl group fused with one or more heterocyclic groups or cycloalkyl groups, or a naphthyl group fused with one or more heterocyclic groups or cycloalkyl groups, wherein the connection point is on the phenyl group or the naphthyl group, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic aromatic ring system, and non-limiting examples include:

[0314]

[0315] The aryl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of D atoms, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, oxo, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0316] The term "heteroaryl" refers to a monocyclic heteroaromatic ring (i.e., a monocyclic heteroaryl) or a polycyclic heteroaromatic ring system (i.e., a polycyclic heteroaryl) having a conjugated π electron system, which contains at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur in the ring (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but does not include -OO-, -OS-, or -SS-), and has 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 5- to 14-membered heteroaryl). The heteroaryl is preferably a heteroaryl having 5 to 10 ring atoms (i.e., a 5- to 10-membered heteroaryl), and more preferably a heteroaryl having 5 or 6 ring atoms (i.e., a 5- or 6-membered heteroaryl).

[0317] The term "cycloalkyloxy" refers to a cycloalkyl-O- group in which cycloalkyl is as defined above.

[0318] The term "heterocyclyloxy" refers to heterocyclyl-O-, wherein heterocyclyl is as defined above.

[0319] The term "aryloxy" refers to an aryl-O- group in which aryl is as defined above.

[0320] The term "heteroaryloxy" refers to heteroaryl-O-, wherein heteroaryl is as defined above.

[0321] The term "alkylthio" refers to an alkyl-S- group in which alkyl is as defined above.

[0322] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0323] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0324] The term "haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.

[0325] The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups, wherein alkyl is as defined above.

[0326] The term "hydroxy" refers to an -OH group.

[0327] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0328] The term "amino" refers to -NH2.

[0329] The term "cyano" refers to -CN.

[0330] N-ethyldiisopropylamine is abbreviated as DIEA.

[0331] N,N-Dimethylformamide is referred to as DMF.

[0332] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphorus is abbreviated as HATU.

[0333] 1-Hydroxybenzotriazole is abbreviated as HOBt.

[0334] 2-Bromoacetic anhydride is abbreviated as BrAc2O.

[0335] "Substituted" refers to one or more hydrogen atoms in a group, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms, which are independently replaced by a corresponding number of substituents. Those skilled in the art can determine possible or impossible substitutions (by experiment or theory) 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.

[0336] The term "drug loading" (Drug-to-Antibody Ratio, DAR) refers to the average number of cytotoxic agents conjugated to each antibody in an antibody drug conjugate population, and can also be expressed as the ratio of the amount of cytotoxic agents to the amount of antibody. The scope of drug loading can be 1-20, preferably 1-10 cytotoxic agents (D) connected to each antibody (Ab). In the embodiments of the present disclosure, drug loading is expressed as n, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or the mean (which can be an integer or decimal) of any two numerical values. n is preferably 1-10, more preferably 1-5, or an integer or decimal of 2-5. Conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA tests, monoclonal antibody size variant assays (CE-SDS) and HPLC feature determination drug loading can be used. Although for a specific conjugate molecule, the cytotoxic agent to antibody ratio has an exact value, it should be understood that when used to describe a sample containing many molecules, the value will often be an average value. In some embodiments, the DAR is between about 1 and about 6, and typically is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0. In some embodiments, a DAR of 'about x' means that the measured value of the DAR is within 20% of x.

[0337] In the expression "...having at least 85% sequence identity...", identity refers to the degree (percentage) to which the amino acids / nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned. During the alignment process, gaps are introduced if necessary to obtain maximum sequence identity. Sequence identity is determined by techniques known in the art, such as computer software (BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign).

[0338] As an example, "the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1" means that the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5, and amino acid mutations are allowed to be introduced in regions outside the CDRs so as to have at least 85% sequence identity with SEQ ID NO: 1.

[0339] "Amino acid mutation" includes amino acid substitution, deletion, insertion, modification or any combination thereof. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, as long as the final construct has the desired characteristics, such as amino acid substitutions that reduce homodimerization in the Fc region. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity. Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and / or carboxyl terminus of the polypeptide chain. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, that is, an amino acid is replaced with another amino acid having different structural and / or chemical properties. Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc.

[0340] In the present disclosure, various methods can be used to indicate the same amino acid mutation. For example, the amino acid residue at a specific site can be indicated by position + amino acid residue, for example, 366S, 368A means that the amino acid residue at site 366 is S and the amino acid residue at site 368 is A.

[0341] As a supplement, in the anti-B7-H3 antibody or antigen-binding fragment thereof of the present application, although the C-terminus of the Fc region is a complete C-terminus ending with PGK, it can still be a truncated C-terminus. Exemplarily, one or two C-terminal residues are removed from the truncated C-terminus (e.g., a truncated C-terminus ending with PG). The composition comprising the antibody may include: an antibody with a complete C-terminus and an antibody with a truncated C-terminus.

[0342] Exemplary Anti-B7-H3 Antibodies or Antigen-Binding Fragments thereof

[0343] In a specific embodiment, the anti-B7-H3 antibody or antigen-binding fragment thereof of the present disclosure comprises a VH and a VL selected from any one of the following groups:

[0344] 1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, and

[0345] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2;

[0346] 2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and

[0347] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10;

[0348] 3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and

[0349] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27;

[0350] 4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and

[0351] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28;

[0352] 5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and

[0353] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29;

[0354] 6) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and

[0355] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30;

[0356] 7) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and

[0357] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31;

[0358] 8) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and

[0359] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32;

[0360] 9) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33;

[0361] 10) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34;

[0362] 11) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35;

[0363] 12) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 36;

[0364] 13) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37;

[0365] 14) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38;

[0366] Among them, CDR is determined according to the Kabat numbering system.

[0367] Exemplary Antibody Drug Conjugates

[0368] In a specific embodiment, the antibody drug conjugate of the present disclosure has the following structure:

[0369] in:

[0370] Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof according to the present disclosure;

[0371] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0372] In a specific embodiment, the antibody drug conjugate of the present disclosure has the following structure:

[0373] in:

[0374] Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof according to the present disclosure;

[0375] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0376] In a specific embodiment, the antibody drug conjugate of the present disclosure has the following structure:

[0377]

[0378] in:

[0379] Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof according to the present disclosure;

[0380] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0381] In a specific embodiment, the antibody drug conjugate of the present disclosure has the following structure:

[0382]

[0383] in:

[0384] Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof according to the present disclosure;

[0385] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0386] In a specific embodiment, the antibody drug conjugate of the present disclosure has the following structure:

[0387]

[0388] in:

[0389] Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof according to the present disclosure;

[0390] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0391] In a specific embodiment, the antibody drug conjugate of the present disclosure has the following structure:

[0392]

[0393] in:

[0394] Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof according to the present disclosure;

[0395] n is a DAR value, and n is an integer or decimal between 2 and 5, and preferably 4.

[0396] The skilled person will note that in the preferred antibody drug conjugates disclosed herein, the anti-B7-H3 antibody or antigen-binding fragment thereof and the L of the linker are a The components form a connection of two single bonds.

[0397] "Nucleic acid" is used interchangeably with "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The nucleic acids are synthetic, naturally occurring, and non-naturally occurring. Unless otherwise indicated, a nucleic acid sequence also encompasses conservative variants (e.g., degenerate substitutions) and complementary sequences thereof as well as explicitly specified sequences. The term "nucleic acid molecule" includes a plurality of nucleic acid molecules.

[0398] "Vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is attached. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which an exogenous DNA segment is attached. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV), in which an exogenous DNA segment is attached to the viral genome. Some vectors are capable of autonomous replication in host cells, while others can be integrated into the host's genome, thereby replicating along with the host genome.

[0399] "Host cell" and "cell line" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced and their progeny, regardless of the number of generations of the progeny. The progeny are not allowed to be completely identical to the parent cell in terms of nucleic acid, but may contain mutations, as long as the progeny with the mutation have the same desired function or activity as the original cell.

[0400] The host cell cannot develop into a complete animal or plant individual.

[0401] Host cells include prokaryotic and eukaryotic host cells. Eukaryotic host cells include, but are not limited to, mammalian cells, insect cells, plant cells, and fungal cells. Mammalian cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Exemplary host cells include, but are not limited to, CHO, NSO, COS, SP2 cells, HeLa cells, BHK cells, human hepatocellular carcinoma cells, A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast, such as, but not limited to, Pichia, Saccharomyces, Hansenula polymorpha, and Kluyveromyces.

[0402] The anti-B7-H3 antibodies or antigen-binding fragments thereof of the present disclosure can be produced using recombinant methods.

[0403] In a specific embodiment, a host cell comprising the expression vector is cultured under conditions suitable for expression of the antibody, and the antibody is recovered (optionally purified) from the host cell (or its culture).

[0404] A "pharmaceutical composition" refers to a mixture containing one or more antibody-drug conjugates, anti-B7-H3 antibodies or antigen-binding fragments thereof of the present disclosure and other chemical components, such as pharmaceutically acceptable carriers, diluents, buffers or excipients.

[0405] "Pharmaceutically acceptable carrier, diluent, buffer or excipient" refers to an ingredient in a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers, diluents, buffers or excipients include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0406] "Subject" or "individual" refers to an animal, preferably a mammal. According to a specific embodiment, the subject is a mammal, including, for example, camels, horses, cows, pigs, sheep, cats, dogs, rabbits, rats, guinea pigs, mice, primates (e.g., humans). In a specific embodiment, the subject is a human.

[0407] In specific embodiments, the subject is an individual susceptible to, suspected of having, or already having a disease.

[0408] "Treatment" means providing an antibody drug conjugate, anti-B7-H3 antibody or antigen binding fragment thereof, or a pharmaceutical composition thereof, of the present disclosure to a subject. The subject has one or more symptoms of a disease. Typically, the antibody drug conjugate, anti-B7-H3 antibody or antigen binding fragment thereof is administered in an amount effective to alleviate the symptoms of the disease in the treated subject or subject population.

[0409] In specific embodiments, the selection of an effective amount can be determined by those skilled in the art based on consideration of a variety of factors (e.g., through clinical trials), including the disease to be treated, the symptoms involved, the route of administration, the severity of the disease, the subject's weight, the subject's immune status, and other factors known to those skilled in the art.

[0410] The effective amount in the specific embodiment can be obtained from the dose-response curve derived from the animal model test system, and it is allowed to be determined according to the doctor's judgment and the situation of each subject. As an example, the amount of the drug required for the subject's one-time administration can be conveniently obtained by calculating the product of the subject's body weight and the unit weight dose required for the subject's one-time medication. For example, in the process of preparing the drug, it is generally believed that the adult body weight is 50-70kg, and the dosage can be initially determined by the equivalent dose conversion relationship between the unit weight dose of the experimental animal and the human. For example, it can be determined according to the guidance opinions proposed by drug administration agencies such as SFDA and FDA. In some embodiments, the dosage of humans and mice can be converted using the body surface area conversion coefficient of 0.0026 for humans and mice.

[0411] The active ingredient (e.g., antibody drug conjugate, anti-B7-H3 antibody or antigen-binding fragment thereof) herein can be administered once, or can be divided into many smaller unit doses and administered at certain time intervals. It should be understood that the dosage, duration, and interval of treatment are functions of the disease being treated, and can be determined by inference using animal or clinical trial data. The administration may include a single administration, or two or more administrations separated by appropriate time intervals. Two adjacent administrations may be spaced 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months apart.

[0412] It should be understood that when a numerical range is disclosed, for example, "A to B" is a concise way of writing that, although not every point value in the range is given, it is deemed that the integers and decimals within the range have been explicitly disclosed.

[0413] “And / or”, for example, “A and / or B” should be understood to mean “A and B” or “A or B”.

[0414] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur. As used in the specification and claims, the singular forms "a", "an" include the corresponding plural referents unless the content clearly dictates otherwise.

[0415] Example

[0416] The following examples are provided to provide a better understanding of the subject matter of the present invention. These examples should not be considered to limit the subject matter described. Rather, it should be understood that the embodiments and embodiments described in the present invention are only for illustrative purposes, and various modifications or changes thereof will be apparent to those skilled in the art and are included within the scope of the present invention.

[0417] Example 1. Screening of B7-H3 Antibodies

[0418] 1. Screening of rabbit anti-human B7-H3 antibodies

[0419] First, the extracellular region of human B7-H3 protein (UniProt number: Q5ZPR3) was expressed as an immunogen, and the C-terminus of the B7-H3 protein amino acid sequence (Leu29-Pro245) was fused with the antibody Fc sequence and then cloned into an expression vector. The expression vector was transiently transfected into 293 cells, and the cell culture fluid was collected and purified after 7 days.

[0420] To prepare rabbit anti-human B7-H3 monoclonal antibody, human B7-H3 extracellular region protein was used to immunize New Zealand rabbits (purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd.) for a total of 5 immunizations. B cells from immunized New Zealand rabbits were isolated and cultured monoclonally. The titer of monoclonal B cell supernatant was detected by ELISA. The immunogen B7-H3 protein was coated on the microplate at 1 μg / mL at 4°C overnight, and then blocked with blocking solution at 37°C for 1 hour. After washing the plate, the B cell supernatant was added to the plate and incubated at 37°C for 1 hour. After washing the plate, HRP-labeled goat anti-rabbit IgG (manufacturer: Jakson) was added and reacted at 37°C for 1 hour. After washing the plate, TMB solution was added and reacted at room temperature in the dark for 5 minutes, and then 2M H2SO4 was added to terminate the reaction. The absorbance was detected at a wavelength of 450nm on an ELISA reader. Based on the binding activity with B7-H3, a monoclonal clone 36F3 with high binding activity was screened (OD value was 2.3 measured at a wavelength of 450 nm). The variable region sequences of 36F3 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0421] 36F3VH: SEQ ID NO: 1

[0422]

[0423] 36F3VL: SEQ ID NO: 2

[0424]

[0425] Table 1-2. CDR regions of 36F3 antibody

[0426] CDR sequence Serial number HCDR1 SYAMS SEQ ID NO: 3 HCDR2 FIGSGGSASYATWAKG SEQ ID NO: 4 HCDR3 GSSSSPTNL SEQ ID NO: 5 LCDR1 QASQNINSWLS SEQ ID NO: 6 LCDR2 QASKLAS SEQ ID NO: 7 LCDR3 QNTYGIIGYGAV SEQ ID NO: 8

[0427] 2. Humanization of Rabbit Anti-Human B7-H3 Antibody

[0428] The heavy chain variable region and light chain variable region of the 36F3 antibody were compared with the human IgG gene sequence database to identify the best matching human germline IgG gene sequence. Then the CDR regions of the heavy chain variable region and light chain variable region of the rabbit antibody were transplanted to the framework sequences of the matching heavy chain variable region gene and light chain variable region gene, respectively. Specifically, the human germline IgG heavy chain and light chain selected by 36F3 were IGHV3-23*04 and IGKV1-21*01, respectively, and the corresponding humanized antibody was 112-59. The VH and VL sequences of 112-59 were connected to the heavy chain constant region and light chain constant region of the human antibody to obtain the full-length sequence of the 112-59 antibody. The sequence of the 112-59 antibody is as follows:

[0429] 112-59-VH: SEQ ID NO: 9

[0430]

[0431] 112-59-VL: SEQ ID NO: 10

[0432]

[0433] Heavy chain constant region: SEQ ID NO: 11

[0434]

[0435] Light chain constant region: SEQ ID NO: 12

[0436]

[0437] 3.112-59 Antibody Mutation

[0438] The LCDR1 or LCDR3 of the light chain of 112-59 was mutated to obtain a new light chain variable region, and the new light chain variable region was recombined with the light chain constant region and the heavy chain of 112-59 to obtain a new antibody that binds to B7-H3. The specific mutations are as follows:

[0439] Table 2. Sequences of LCDR3 of 112-59 mutant antibodies

[0440]

[0441] Table 3. LCDR1 sequences of 112-59 mutant antibodies

[0442]

[0443] LCDR3 general formula: QNTYGIX1X2X3GAV (SEQ ID NO: 25), wherein X1 is selected from I or P; X2 is selected from G, K or R; X3 is selected from Y or R.

[0444] LCDR1 general formula: QASQNIX4X5WLS (SEQ ID NO: 26), wherein X4 is selected from N, Q, H, E or R, and X5 is selected from S, A, T, K or R.

[0445] The sequence of the VL of the 112-59 mutant antibody is as follows:

[0446] 112-59-5VL: SEQ ID NO: 27

[0447]

[0448] 112-59-6VL: SEQ ID NO: 28

[0449]

[0450]

[0451] 112-59-7VL:SEQ ID NO:29

[0452]

[0453] 112-59-9VL:SEQ ID NO:30

[0454]

[0455] 112-59QS-VL:SEQ ID NO:31

[0456]

[0457] 112-59NA-VL:SEQ ID NO:32

[0458]

[0459] 112-59NT-VL:SEQ ID NO:33

[0460]

[0461] 112-59HS-VL:SEQ ID NO:34

[0462]

[0463] 112-59ES-VL:SEQ ID NO:35

[0464]

[0465] 112-59RS-VL:SEQ ID NO:36

[0466]

[0467] 112-59NK-VL:SEQ ID NO:37

[0468]

[0469] 112-59NR-VL:SEQ ID NO:38

[0470]

[0471]

[0472] The above variable region sequence is connected to the human heavy chain constant region and light chain constant region to obtain the full-length sequence of the antibody. The sequence of an exemplary antibody is as follows:

[0473] 112-59 heavy chain: SEQ ID NO: 39

[0474]

[0475] 112-59 light chain: SEQ ID NO: 40

[0476]

[0477] Example 2. Synthesis of linkers and toxins

[0478] The toxin (Drug) used to prepare ADC is pre-coupled with a linker to prepare linker-drug compounds LD-1 to LD-18; exemplary toxins are MMAE (purchased from MedChemExpress, product number HY-100374), Eribulin mesylate (Eribulin, MedChemExpress, product number HY-13442) and Exatecan mesylate (Exatecan purchased from Advance ChemBlocks, product number 10484).

[0479] 1. Preparation of LD-1

[0480]

[0481] The corresponding ADC structure is shown in Equation 1.

[0482]

[0483] Maleimidocaproic acid (compound 18, 12 mg) was added to a solution of anhydrous DMF (2 mL) containing compound 6 (MMAE purchased from Medchem Express, HY-15162) (62 mg, TFA salt), followed by DIEA (0.02 mL), and HATU (20 mg). The reaction mixture was stirred at room temperature (22 ° C). After 15 minutes, the crude reaction mixture was directly purified by RP-HPLC to obtain a white solid compound 19 (62 mg, TFA salt) after lyophilization, i.e., LD-1.

[0484] LCMS: m / z 1316.6 [M+H + ].

[0485] 2. Preparation of LD-2:

[0486]

[0487]

[0488] The corresponding ADC structure is shown in Equation 2.

[0489]

[0490] MC-VC-PABC-PNP (15 mg, purchased from MedChemExpress, Cat. No. HY-20336) and Eribulin mesylate (17 mg, MedChemExpress, HY-13442) were dissolved in anhydrous DMF (1 mL). DIEA (0.01 mL) was added and the mixture was stirred at room temperature for 6 hours. The crude product was directly purified by RP-HPLC to give LD-2 (19 mg) as a white solid.

[0491] LCMS: m / z 1328.8 [M+H + ].

[0492] 3. Preparation of LD-3:

[0493]

[0494] The corresponding ADC structure is shown in Equation 3.

[0495]

[0496] To a solution of MC-GGFG-OH (purchased from Innopep San Diego, 16 mg,) and exotecan mesylate (purchased from Advanced ChemBlocks Product No. 10484, 16 mg) in anhydrous DMF (1 mL), PyAOP ((7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 16 mg) was added, followed by DIEA (0.015 mL). The reaction mixture was stirred at room temperature for 15 minutes and directly purified by RP-HPLC to give LD-3 (22 mg) as a yellow solid.

[0497] LCMS: m / z 947.4 [M+H + ].

[0498] 4. Preparation of LD-4:

[0499]

[0500] The corresponding ADC structure is shown in Equation 4.

[0501] LD-4 was prepared according to Example 43 in Patent No. US9808537B2.

[0502] 5. Preparation of LD-5:

[0503] LD-5 was purchased from MedchemExpress, Cat.#HY-128975, and its structure is shown below:

[0504]

[0505] The corresponding ADC structure is shown in Equation 5.

[0506] 6. Preparation of LD-6

[0507]

[0508] The corresponding ADC structure is shown in Equation 6.

[0509]

[0510] Saturated NaHCO (0.2 mL) and bromoacetic anhydride (9 mg) were added to a solution of acetonitrile / water (6 / 4, v / v, 2 mL) containing compound 6 (20 mg). After the reaction mixture was stirred at room temperature (22 ° C) for 10 minutes, the reaction mixture was directly purified by RP-HPLC to obtain white solid compound 7 (17 mg), i.e. LD-6, after freeze-drying.

[0511] LCMS: m / z 1243.6 [M+H + ].

[0512] 7. Preparation of LD-7:

[0513]

[0514] The corresponding ADC structure is shown in Equation 7.

[0515]

[0516] To a solution of compound 3 (purchased from MedChemExpress, HY-13442) (12 mg, TFA salt) in acetonitrile / water (6 / 4, 1 mL), saturated NaHCO3 aqueous solution (0.1 mL) was added, followed by 2-bromoacetic anhydride (6 mg, Toronto Research Chemicals). The mixture was stirred at room temperature. After 10 minutes, the mixture was purified by RP-HPLC to obtain LD-7 (11 mg) as a white solid.

[0517] LCMS: m / z 1255.6 [M+H + ].

[0518] 8. Preparation of LD-8

[0519]

[0520] The corresponding ADC structure is shown in Equation 8.

[0521]

[0522] To a solution of anhydrous DMF (2 mL) containing compound 12 (65 mg, prepared with reference to WO2022026915) and MMAE (72 mg), DIEA (0.002 mL) was added, followed by HOBt (3 mg). The reaction mixture was stirred at room temperature (22 ° C) for 18 hours and diluted with water (20 mL). The reaction mixture was extracted with ether (40 mL), the organic phase was dried with NaSO4, and concentrated to dryness under reduced pressure to obtain crude compound 13, which was finally dissolved in methanol (2 mL). Zinc powder (200 mg) was added to a methanol solution containing compound 13, and formic acid (0.2 mL) was added. The reaction was stirred at room temperature for 30 minutes. The solid was removed by filtration, and the filtrate was directly purified by RP-HPLC to obtain white solid compound 15 (72 mg) after lyophilization.

[0523] LCMS: m / z 1471.6 [M+H + ].

[0524] Compound 15 (70 mg) was dissolved in acetonitrile / water (6 / 4, v / v, 3 mL) solution, NaOH (1 M, 0.3 mL) was added, and the reaction mixture was stirred at room temperature (22° C.) to obtain compound 16. After 8 hours, hydrochloric acid (1 M, 0.12 mL) was added to the crude product of compound 16, and then bromoacetic anhydride (14 mg) was added. The crude reaction mixture was directly purified by RP-HPLC to obtain white solid compound 17 (46 mg) after freeze-drying, i.e., LD-8.

[0525] LCMS: m / z 1426.7 [M+H + ].

[0526] 9. Preparation of LD-9

[0527]

[0528] The corresponding ADC structure is shown in Equation 9.

[0529]

[0530] Under argon atmosphere, Ag2O (15 mmol) was added to a stirred solution of ethyl bromide-α-D-glucose 28 (4.6 g) and compound 28 (Fmoc-Tyr-OtBu, 4.1 g) in anhydrous acetonitrile (100 mL) at 0°C, and the solution was stirred at room temperature for 4 hours. The mixture was filtered and the filtrate was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 30 (5.3 g) as a white solid.

[0531] Compound 30 (4g) was redissolved in TFA / dichloromethane (1 / 1, v / v, 80mL). After 30 minutes, the mixture was diluted with dichloromethane (200mL) and washed with water (50mL×4). The organic layer was evaporated to dryness to obtain crude acid 31, which was dissolved in DMF (50mL). p-Aminobenzyl alcohol (0.65g) was added to the solution, followed by DIEA (1.8mL) and HATU (1.9g). The reaction mixture was stirred at room temperature for 20 minutes and then diluted with ethyl acetate (200mL). The mixture was washed with 0.5M hydrochloric acid (100mL) and water (150mL). The organic layer was dried (over Na2SO4) and evaporated to dryness under reduced pressure. The residue was ground with hexane / ether (1 / 1, 100mL) to obtain crude compound 32 as a tan solid, which was dissolved in DMF (30mL). Diisopropylamine (30 mL) was added, and the mixture was stirred at room temperature for 2 hr.

[0532] The reactant was then concentrated under reduced pressure to about 20mL and diluted with DMF (30mL). Fmoc-Gly-Gly-OH (1.8g) and DIEA (1.8mL) were added, followed by HATU (1.9g), and the mixture was stirred at room temperature. After 20 minutes, the reaction mixture was diluted with ethyl acetate (150mL). The mixture was washed with 0.5M hydrochloric acid (150mL) and water (200mL). The organic layer was dried (over Na2SO4) and evaporated under reduced pressure to dryness. The crude product was purified by column chromatography to obtain compound 33 as a light yellow solid (3.2g).

[0533] Compound 33 (2.4 g) was dissolved in DMF (20 mL). DIEA (0.5 mL) and bis-PNP carbonate (1.5 g) were added to the solution, and the mixture was stirred at room temperature for 16 hours. The reaction was then diluted with ethyl acetate (200 mL) and washed with water (3 × 100 mL). The organic layer was dried (over Na2SO4) and evaporated to dryness under reduced pressure. The residue was purified by column chromatography to obtain compound 34 (2.3 g) as a white solid.

[0534] To the solution of compound 34 (0.12g) and MMAE (0.072g) in DMF (2mL), DIEA (0.018mL) is added and the reaction is stirred at room temperature for 24 hours. Then the mixture is diluted with ethyl acetate (30mL) and washed with hydrochloric acid (0.5M, 30mL). The organic layer is washed with water (20mL), dried (Na2SO4) and evaporated under reduced pressure to dryness. The residue is redissolved in MeOH (3mL) and MeONa (4.4M in MeOH, 0.1mL) is added. The reaction mixture is stirred at room temperature for 2 hours, then neutralized with 1N hydrochloric acid (0.5mL). The mixture is directly purified by RP-HPLC to obtain compound 35, which is a white solid (TFA salt, 72mg).

[0535] Compound 35 (14 mg, TFA salt) was dissolved in acetonitrile / water (6 / 4, 1 mL), saturated NaHCO3 aqueous solution (0.1 mL) was added, and then 2-bromoacetic anhydride (6 mg, Toronto Research Chemicals) was added. The mixture was stirred at room temperature. After 10 minutes, the mixture was purified by RP-HPLC to obtain LD-9 (12 mg) as a white solid.

[0536] LCMS: m / z 1426.5 [M+H + ].

[0537] 10. Preparation of LD-10

[0538]

[0539] The corresponding ADC structure is shown in Equation 10.

[0540]

[0541] Compound 24 (10 mg, synthesized according to the literature: Bioconjugate Chem. 2006, 17, 831-840) was dissolved in a solution of DMF (1 mL), eribulin mesylate (8 mg) was added, and DIEA (0.005 mL) was then added. The mixture was stirred at room temperature for 16 hours, then diluted with ethyl acetate (20 mL) and washed with water (20 mL) and brine (20 mL) in sequence. The organic layer was concentrated under reduced pressure to obtain a residue, which was suspended in acetonitrile / water (6 / 4, 2 mL). LiOH (1N, 0.07 mL) was added, and the reaction mixture was stirred at room temperature. After 6 hours. The crude mixture was purified by RP-HPLC to obtain compound 25, which was a white powder, which was dissolved in acetonitrile / water (6 / 4, 1 mL), saturated NaHCO3 aqueous solution (0.06 mL) was added, and 2-bromoacetic anhydride (4 mg, Toronto Research Chemicals) was then added. The mixture was stirred at room temperature. After 10 min, the mixture was purified by RP-HPLC to give LD-10 (8 mg) as a white solid.

[0542] LCMS: m / z 1278.6 [M+H + ].

[0543] 11. Preparation of LD-11

[0544]

[0545] The corresponding ADC structure is shown in Equation 11.

[0546]

[0547] To 2-tert-butylhydrazine-1,2-dicarboxylate (76.8mg, 0.33mmol) and 3-bromo-2-(bromomethyl)propionic acid tert-butyl ester (200mg, 0.66mmol) in 3mL anhydrous tetrahydrofuran, add NaH (60%, 80mg, 2.0mmol). The mixture was stirred at room temperature for 15 minutes, then the reaction was quenched in 1mL water with 60 μL AcOH. The mixture was then purified by preparative HPLC. The pure components were freeze-dried to give 204mg of white solid A1.

[0548] LCMS: m / z 373.6 [M+H + ].

[0549]

[0550] 3,4-dibromofuran-2,5-dione (140 mg, 0.55 mmol) was added to 8.0 mL of acetic acid (AcOH) solution containing tert-butyl 2-[[tert-butoxycarbonyl-(tert-butoxycarbonylamino)amino]methyl]prop-2-enoate (A1) (204 mg, 0.55 mmol). The mixture was stirred at reflux for 11 days under an argon atmosphere, then concentrated to 3 mL and purified by preparative HPLC to give 43 mg of white solid A2 (22%).

[0551] LCMS: m / z 354.8 [M + H + ].

[0552]

[0553] To a stirred solution of 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (350 mg) in anhydrous dichloromethane (10 mL) was added N-hydroxysuccinimide (230 mg) followed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (400 mg). The mixture was stirred at room temperature for 30 minutes and the reaction was concentrated to dryness under reduced pressure. The residue was directly purified by RP-HPLC to give compound 4 as a white solid (337 mg) after freeze drying.

[0554] LCMS: m / z 452.0 [M+H + ].

[0555]

[0556] Compound 4 (12 mg) was added to a DMF (1 mL) solution containing compound 6 (31 mg, purchased from MedChemExpress, catalog number: HY-100374), followed by DIEA (0.01 mL). The reaction mixture was stirred at room temperature (22°C). After 3 hours, the crude reaction mixture was directly purified by RP-HPLC to obtain a white solid compound 8 (17 mg), i.e., LD-11, after freeze-drying.

[0557] LCMS: m / z 1243.6 [M+H + ].

[0558] 12. Preparation of LD-12

[0559]

[0560] The corresponding ADC structure is shown in Equation 12.

[0561]

[0562] To a solution of compound 1 (77 mg, MedChemExpress, HY-41189) in anhydrous N,N-dimethylformamide (2 mL) was added eribulin (compound 2, mesylate, 82 mg, MedChemExpress, HY-13442) followed by N-ethyldiisopropylamine (0.035 mL). The reaction mixture was stirred at room temperature (22°C). After 6 hours, piperidine (0.1 mL) was added and the reaction was allowed to stir at room temperature for 15 minutes. The reaction mixture was directly purified by RP-HPLC to give compound 3 (110 mg, trifluoroacetate) as a white solid after lyophilization.

[0563] Compound 4 (20 mg, synthesized according to the method reported in PCT / US2022 / 078563) was added to a solution of N,N-dimethylformamide (2 mL) containing compound 3 (50 mg), and then N-ethyldiisopropylamine (0.01 mL) was added. The reaction mixture was stirred at room temperature. After 3 hours, the crude mixture was purified by RP-HPLC to obtain a white solid compound 5 (43 mg) after lyophilization, i.e., LD-12.

[0564] LCMS: m / z 1471.6 [M+H + ].

[0565] 13. Preparation of LD-13

[0566]

[0567]

[0568] The corresponding ADC structure is shown in Equation 13;

[0569]

[0570] Compound 20 (60 mg, purchased from InnoPep) and isotecan mesylate (compound 21, 53 mg, purchased from Advance ChemBlocks, item number 10484) were dissolved in anhydrous N,N-dimethylformamide (2 mL) solution, N-ethyldiisopropylamine (0.05 mL) was added, and then O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphorus (38 mg) was added. The mixture was stirred at room temperature for 15 minutes. Piperidine (0.1 mL) was added. After 30 minutes, the crude mixture was purified by RP-HPLC and lyophilized to obtain compound 22 (67 mg, trifluoroacetate) as a yellow powder.

[0571] To a solution of compound 22 (44 mg) in N,N-dimethylformamide (2 mL) was added 4 (25 mg, synthesized according to the method reported in PCT / US2022 / 078563), followed by N-ethyldiisopropylamine (0.02 mL). The reaction mixture was stirred at room temperature. After 1 hour, the crude mixture was purified by RP-HPLC and lyophilized to give compound 23, LD-13, as a yellow solid (42 mg).

[0572] LCMS: m / z 1090.2 [M+H + ].

[0573] 14. Preparation of LD-14

[0574]

[0575] The corresponding ADC structure is Equation 14;

[0576]

[0577] Compound 4 (same structure as described above) (6 mg) was added to a DMF (1 mL) solution of compound 26 (TFA salt, 10 mg, synthesized according to patent number: US 9808537B2), followed by DIEA (0.007 mL). The mixture was stirred at room temperature for 2 hours and then purified by RP-HPLC to obtain compound LD-14 (10 mg) as a yellow powder.

[0578] LCMS: m / z 1177.3 [M+H + ].

[0579] 15. Preparation of LD-15

[0580]

[0581]

[0582] The corresponding ADC structure is shown in Equation 15.

[0583]

[0584] To a solution of compound 25 (TFA salt, 13 mg) in DMF (1 mL) was added compound 4 (5 mg) followed by DIEA (0.007 mL). The mixture was stirred at room temperature for 2 hours and then purified by RP-HPLC to give compound LD-15 (11 mg) as a white powder.

[0585] LCMS: m / z 1495.5 [M+H + ].

[0586] 16. Preparation of LD-16

[0587]

[0588]

[0589] The corresponding ADC structure is Equation 16;

[0590]

[0591] Compound 4 (described above, 6 mg) was added to a DMF (1 mL) solution of compound 27 (TFA salt, 10 mg, synthesized according to patent number: WO 2022 / 048883A1), followed by DIEA (0.007 mL). The mixture was stirred at room temperature for 2 hours and then purified by RP-HPLC to obtain compound LD-16 (10 mg) as a yellow powder.

[0592] LCMS: m / z 1170.1 [M+H + ].

[0593] 17. Preparation of LD-17

[0594]

[0595] The corresponding ADC structure is Equation 17;

[0596]

[0597] Compound 6 (0.18 mmol) was dissolved in 1 mL of anhydrous DMF, and DIEA (0.44 mmol) and glutaric anhydride (0.21 mmol) were added respectively. After the reaction solution was stirred at room temperature for ten minutes, dichloromethane (1 mL), pentafluorophenol (0.89 mmol) and EDC (0.89 mmol) were added. After the reaction solution was stirred at room temperature for thirty minutes, the solvent was dried under reduced pressure, and compound LD-17 (white powder) was obtained after purification by HPLC.

[0598] LCMS: m / z 1403.9 [M+H + ].

[0599] 18. Preparation of LD-18

[0600]

[0601] The corresponding ADC structure is shown in Equation 18.

[0602]

[0603] Compound 36 (synthesized from adipic acid, 20 mg) was added to a solution of compound 35 (14 mg, TFA salt) in DMF (6 / 4, 1 mL), followed by DIEA (0.005 mL). The mixture was stirred at room temperature. After reacting for 30 minutes, the mixture was purified by RP-HPLC to obtain LD-18 (13 mg) as a white solid.

[0604] LCMS: m / z 1600.8 [M+H + ].

[0605] Example 3. Synthesis of ADC

[0606] The antibody disclosed herein is reacted with a drug linker (LD) to prepare an ADC drug, and the DAR value of the prepared ADC is determined.

[0607] 1. Treat the antibody with a reducing agent (such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT)) to reduce part or all of the cysteine ​​disulfide residues to form highly nucleophilic cysteine ​​thiol groups (-CH2SH). The partially or completely reduced antibody is then reacted with a drug linker or a linker reagent and an electrophilic functional group (such as maleimide or bromoacetyl) to finally prepare an ADC.

[0608] The structural formulas of the prepared ADC-1 to ADC-27 are respectively shown in the corresponding relationship in Table 4 and the structures shown in Example 2.

[0609] 2. ADC DAR value determination method:

[0610] The DAR value of the ADC of the present invention was analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). ADC was separated in a chromatographic column using a MabPac HIC-Butyl analytical column (4.6x100mm, 5μm, Cat. No. 088558, ThermoFisher, USA). 25mM sodium phosphate buffer (pH 6.8) containing 1.5M ammonium sulfate was used as buffer solution A, and 25mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile was used as buffer B, with 85% A and 15% B being stable as initial conditions, using 85% A and 15% B to 5% A and 95% B linear gradient elution for 30 minutes, and using 5% A and 95% B for additional elution for 5 minutes. The flow rate and temperature were set to 0.5mL / min and 25°C. ADC drug distribution was detected at 214 and 280nm for calculating the DAR value, and the formula is as follows:

[0611]

[0612] 3. Preparation of MC linker ADC:

[0613] The antibody was dissolved in PBS at pH 7.2, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride: antibody was 2.3 to 3.3:1). After incubation at 37°C for about 120 minutes, the drug linker using the MC linker was added to the reduced antibody (the molar ratio of the drug linker: antibody was 5:1), and 0 to 10% DMSO was added, reacted at room temperature for 1 hour, eluted and purified with G25 resin, desalted, filtered with a 0.2 μm filter under sterile conditions, ADC was obtained, and stored frozen. The average DAR value of the ADC was determined to be between 3.7 and 4.3 using the above-mentioned hydrophobic interaction chromatography-high performance liquid chromatography analysis.

[0614] 4. Preparation of Br-linker ADC

[0615] The antibody was dissolved in 100 mM, pH 8.0 phosphate buffer, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride: antibody was 2.3 to 3.3:1). After incubation at 37°C for about 120 minutes, the drug linker using a Br linker was added to the reduced antibody (the molar ratio of drug linker: antibody was 5:1), and 0 to 10% DMSO was added, reacted at room temperature for 1 hour, eluted and purified with G25 resin, and desalted, filtered with a 0.2 μm filter under sterile conditions to obtain ADC, which was frozen for storage. The above-mentioned hydrophobic interaction chromatography-high performance liquid chromatography analysis determined that the average DAR value of ADC was between 3.7 and 4.3.

[0616] 5. Preparation of DiBr linker ADC

[0617] The antibody was dissolved in 100 mM, pH 8.0 phosphate buffer, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride: antibody was 10:1). After incubation at 37°C for about 120 minutes, the drug linker using a DiBr linker was added to the reduced antibody (the molar ratio of the drug linker: antibody was 5:1), and 0 to 10% DMSO was added, reacted at room temperature for 1 hour, eluted and purified with G25 resin, and desalted, filtered with a 0.2 μm filter under sterile conditions to obtain ADC, which was frozen for storage. The above-mentioned hydrophobic interaction chromatography-high performance liquid chromatography analysis determined that the average DAR value of the ADC was between 4.0 and 4.3.

[0618] 6. Preparation of Pfp-linker ADC

[0619] The antibody was dissolved in PBS at pH 7.2, and the drug linker using the Pfp linker was added to the reduced antibody (the molar ratio of drug linker: antibody was 6 to 9:1), and 0 to 10% DMSO was added, reacted at room temperature for 6 to 16 hours, eluted and purified with G25 resin, desalted, filtered with a 0.2 μm filter under sterile conditions, and ADC was obtained and stored frozen. The above-mentioned hydrophobic interaction chromatography-high performance liquid chromatography analysis determined that the average DAR value of the ADC was between 1.5 and 1.9.

[0620] 7. Exemplary prepared ADCs are summarized in Table 4 below:

[0621] Table 4

[0622]

[0623] 8. Preparation of control antibodies:

[0624] The MGA017 antibody was prepared with reference to Mol Cancer Ther (2020) 19(11): 2235–2244, and its sequence is as follows:

[0625] MGA017 heavy chain: SEQ ID NO: 41

[0626]

[0627] MGA017 light chain: SEQ ID NO: 42

[0628]

[0629] 112cDa (i.e. Ab2) was prepared with reference to sequences No. 51 and No. 53 in EP2703486A1, and its sequence is as follows:

[0630] 112 cDa heavy chain: SEQ ID NO: 43

[0631]

[0632] 112 cDa light chain: SEQ ID NO: 44

[0633]

[0634] Test Example 1. Antibody Affinity Detection

[0635] The affinity kinetics of the 112-59 mutant molecule was analyzed by OCTET R8 (Sartorius). The IgG antibody was captured by AHC biosensor (Cat.#2202005011, Sartorius), and a series of concentration gradients of human B7-H3-His (Cat.#B73-H52E2, ACRO) antigen diluted in PBST (pH=7.4) (PBS+0.05% Tween20) buffer were used as analytes. The ligand immobilization amount was 1.5nm, and the antigen-antibody binding kinetics were monitored in real time for 60s, and the dissociation kinetics were tracked for 120s. The reaction signal was detected in real time by OCTET R8 to obtain the binding and dissociation curves. After each experimental cycle of dissociation was completed, the biosensor chip was washed and regenerated with 10mM Gly-HCl pH 1.5 (Cat.#BR-1003-54, Cytiva). The data were analyzed using Sartorius's Octet Analysis Studio 12.2 (CFR 11) software with a 1:1 (Kinetic Analysis) binding model. The ka (kon), kd (koff) and KD values ​​determined by this method are shown in Tables 5 and 6 below.

[0636] Table 5. Antibody affinity

[0637] Antibody KD(M) ka(1 / Ms) kdis(1 / s) 112-59-5 3.108E-09 2.603E-05 8.089E-04 112-59-6 4.072E-09 3.406E-05 1.387E-03 112-59-7 8.197E-08 3.698E-05 3.031E-02 112-59-9 3.900E-08 2.394E-05 9.335E-03 112-59 1.113E-09 4.956E-05 5.517E-04

[0638] Table 6. Affinity of antibodies

[0639] Antibody KD(M) ka(1 / Ms) kdis(1 / s) 112-59RS 1.135E-09 5.383E05 6.108E-04 112-59NT 7.636E-10 5.223E05 3.988E-04 112-59QS 1.886E-09 4.940E05 9.318E-04 112-59NR 7.892E-10 5.613E05 4.430E-04 112-59NK 7.226E-10 5.271E05 3.809E-04 112-59NA 7.986E-10 4.936E05 3.942E-04 112-59 9.503E-10 4.974E05 4.727E-04

[0640] Test Example 2. ELISA method to detect the binding of antibody and antigen

[0641] The binding activity of the 112-59 point mutation antibody to the antigen was detected by ELISA. The B7-H3-His solution (manufacturer: ACRO; item number: B73-H52E2) was diluted to a concentration of 2 μg / mL with PBS buffer and added to the wells of the ELISA plate at 100 μL / well for 16 hours at 4°C. Then wash the plate 3 times with PBST and pat dry the residual liquid. Add 3% BSA blocking solution to the wells of the ELISA plate at 200 μL / well and incubate at 37°C for 2 hours. Take out the blocked ELISA plate and wash it 3 times with PBST. Add the primary antibody, i.e. the antibody to be tested, to the corresponding wells of the coated plate at 100 μL / well, 15 μg / mL in the first well, 5-fold gradient dilution, and incubate at 37°C for 1.5 hours. Wash the plate 5 times, add the secondary antibody Anti-Human IgG (Fc-specific), and dilute it 3000 times. Add 100 μL of secondary antibody to each well of the ELISA plate and incubate at 37°C for 1 hour. Wash the plate 5 times and pat dry any remaining droplets. Add 100 μL of TMB colorimetric solution to each well and react at 37°C in the dark for 20 minutes. Add 100 μL / well of stop solution (2MHCL) to the ELISA plate to stop the reaction and read the absorbance value of OD450. The experimental results are shown in the following table and Figure 1 shown.

[0642] The results showed that the binding activity of the antibody after point mutation was not significantly different from that of the molecule before mutation.

[0643] Table 7. Binding ability of 112-59 mutant antibody to B7-H3 protein

[0644] Antibody 112-59HS 112-59NT 112-59RS 112-59QS 112-59ES EC50(μg / mL) 0.0159 0.0349 0.0204 0.0976 0.0282 Antibody 112-59NR 112-59NK 112-59NA 112-59 - EC50(μg / mL) 0.0357 0.0738 0.0390 0.0381 -

[0645] Test Example 3. Binding of Antibodies to Cells Expressing B7-H3

[0646] Flow cytometry was used to analyze the binding activity of antibodies to cells. Target cells Calu-6 (Shanghai Cell Bank) were treated, the viability was measured and counted, and 2×10 cells were dispensed according to the number of samples. 5 pcs / tube, prepare staining buffer (1%-2% FBS-PBS). Wash the cells by centrifugation and remove the culture medium. Add the primary antibody, i.e., the B7-H3 antibody in this application, with an initial concentration of 50 μg / mL, dilute 5-fold into 8 gradients, and set up a blank control PBS with a volume of 100-200 μL, incubate at 4°C for 30 min. Add the secondary antibody (Alexa Fluor 488 Sheep Anti-Human IgG (H+L) Manufacturer: Thermo Catalog No.: A11013): Wash 3 times by centrifugation at 1200rpm for 5min, dilute the secondary antibody 750 times, the volume is 100-200μL, the blank control is PBS, and incubate at 4℃ for 30min. Wash 3 times by centrifugation at 1200rpm for 5min; On the machine: 150μL staining buffer is diluted to a round-bottom 96-well plate, and the fluorescence intensity of living cells is analyzed, and the Green fluorescence channel is selected.

[0647] The results are shown in Tables 8-1, 8-2 and Figure 2-Figure 3 As shown, the EC50 value of the binding activity of the mutated molecule to Calu-6 cells is comparable to that of 112-59.

[0648] Table 8-1. Binding of 112-59 mutant antibodies to Calu-6 cells

[0649] Antibody 112-59HS 112-59NT 112-59RS 112-59QS 112-59 EC50(nM) 0.2558 0.3677 0.3228 0.3703 0.3921

[0650] Table 8-2. Binding of 112-59 mutant antibodies to Calu-6 cells

[0651]

[0652] Test Example 4. Binding experiment of antibody and cells expressing B7-H3

[0653] The cell lines selected in this experiment are: A375 (ATCC CRL-1619 human malignant melanoma cells), HCC-1806 (ATCC CRL-2335 human breast squamous carcinoma cells), HCC-1954 (ATCC CRL-2338 human breast ductal carcinoma cells), OVCAR3 (ATCC HTB-161 human ovarian adenocarcinoma cells), Calu-6 (ATCC HTB-56 human lung cancer cells), RH41 (DSMZACC 592 human alveolar rhabdomyosarcoma cells), MFE-280 (DSMZ ACC 410 human endometrial adenocarcinoma cells).

[0654] A375 cells were cultured in DMEM medium containing 10% fetal bovine serum, HCC-1806, HCC-1954, and RH41 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, Calu-6 cells were cultured in EMEM medium containing 10% fetal bovine serum, OVCAR3 cells were cultured in RPMI-1640 medium containing 20% ​​fetal bovine serum and 0.01 mg / mL bovine insulin, and MFE-280 cells were cultured in 45% EMEM, 45% RPMI-1640 mixed medium containing 10% fetal bovine serum, 2 mM L-glutamine, 1× insulin-transferrin-selenium medium supplement at 37°C in a 5% CO2 incubator.

[0655] The experimental process is as follows:

[0656] The cells in the logarithmic growth phase were dissociated with 0.25% trypsin, resuspended in staining buffer (Biolengend), washed, counted, and adjusted to 4.5×10 5 Cells / 100μL staining buffer were added to a 96-well plate, 45μL per well. 5μL human TruStain (BioLegend) receptor blocker was added to each well to prevent nonspecific binding of antibodies to human tumor cells. The antibody to be tested was diluted to a starting concentration of 200nM, diluted in staining buffer, and then diluted 3 times to obtain a total of 11 concentration points including 200nM and zero point. 50μL of the diluted antibody to be tested was added to each well of the 96-well plate, and the highest final concentration was 100nM. Mix well and then react at 4°C for 15 minutes. After the reaction, the cells were washed in staining buffer, and then PE-labeled constant region (Fc)-specific antibody (rabbit anti-human IgG PE conjugate, BioLegend, 410707) was suspended in 5μL / 2×10 5 Cells / 100 μL staining buffer were added and reacted at 4°C for 15 minutes. After the cell reaction, the cells were washed in staining buffer and resuspended in 100 μL staining buffer. The single cell readings in the PE channel were analyzed using a Novocyte 3000 (Agilent) device. The Sigmoidal, 4PL four-parameter equation was used to draw a dose-response curve with the antibody protein concentration as the horizontal axis and the corresponding PE channel reading as the vertical axis. EC was generated after analysis. 50 Value. The equation is:

[0657] Y=Bottom+(X^Hillslope)*(Top-Bottom) / (X^HillSlope+EC50^HillSlope),

[0658] MFI multiple = the highest PE reading of the experimental group treated with antibodies / PE reading of the group without antibody addition; The FACS binding activity results of the anti-hB7-H3 antibody test substances to human B7-H3 are shown in Table 9.

[0659] Table 9. Binding ability of anti-B7-H3 antibodies to cell lines

[0660]

[0661] - indicates not tested. The results show that the anti-B7-H3 antibody 112-59 in the present application can specifically bind to cells expressing B7-H3 with high binding ability. In addition, on the same cell line, the total amount of antigen that can be recognized and labeled by the anti-B7-H3 antibody 112-59 in the present application is higher than that of the control antibodies MGA017 and 112cDa, which is specifically manifested by a significant increase in the MFI multiple ratio.

[0662] Test Example 5. Antibody Internalization Experiment of Antibodies and Cells Expressing B7-H3

[0663] The cell line and culture method selected in this experiment are consistent with those in Test Example 4. The experimental process is as follows:

[0664] Cells in the logarithmic growth phase were dissociated with 0.25% trypsin, resuspended in cell culture medium, and centrifuged at 200g for 5 minutes. The precipitated cells were resuspended in cell culture medium and counted. A cell suspension was prepared with a concentration of 5000 cells / 50μL culture medium, and 50μL of the prepared cell suspension was added to each well of the 96-well plate. The 96-well plate containing cells was cultured overnight in a 37°C incubator to allow the cells to adhere to the wall and grow. The test antibody was fluorescently labeled using Incucyte Fabfluor-PH Orange Antibody Labeling Reagent (Sartorius, 4812). This pH-sensitive fluorescent reagent binds to the Fab region of the antibody and emits orange fluorescence in an environment of pH 4.5-5.5 when the antibody is endocytosed into the lysosome. The specific method for fluorescently labeling antibodies is as follows: the antibody labeling reagent and the antibody to be tested are mixed in a molecular weight ratio of 1 to 3 in the cell culture medium, mixed evenly, and placed at 37°C for 15 minutes. 50μl of the labeled antibody was added to each well of the 96-well plate cultured overnight, with a final concentration of 3μg / mL. Put into Incucyte machine Select the adherent cell mode and use a 20x objective lens to take pictures in two channels, orange fluorescence and visible light, every 1-2 hours for 24 hours, and take pictures at three different positions in each well.

[0665] After the experiment, use The integrated image analysis tool uses the Cell-by-cell method to analyze images, cut and measure cell background signals and fluorescence signals, and minimize the impact of background fluorescence. After the Cell-by-cell analysis is completed, the software gives the average cell fluorescence intensity cumulative value reflecting the enhancement of the fluorescence signal: OCUxμm2. The Classification function is then used to analyze the ratio of endocytosis-positive cells representing an increase in fluorescence area: cells with positive fluorescence signals / all cells%. The threshold for background fluorescence positivity is selected as OCU=0.05 during the analysis. The average cell fluorescence intensity and endocytosis-positive cell ratio values ​​are exported from the software and plotted using Prism software. The results of the endocytosis activity of the anti-hB7-H3 antibody test substance on different cell lines are shown in Table 10.

[0666] Table 10. Internalization efficiency of anti-B7-H3 antibodies in different cell lines

[0667]

[0668] - indicates not tested.

[0669] The results showed that the anti-B7-H3 antibody 112-59 in the present application can efficiently and specifically bind to a variety of solid tumor cells expressing B7-H3, and is efficiently internalized by the cells. In addition, on multiple cell lines, the internalization rate of the 112-59 antibody in the present application and the total amount of internalized antibody within 24 hours were higher than those of the control antibodies MGA017 and 112cDa, specifically manifested in a higher ratio of positive cells within 24 hours and a higher average total fluorescence intensity per cell.

[0670] Test Example 6. Cytotoxicity test of ADC molecules

[0671] This experiment uses CellTiterGlo2 (Promega) reagent to evaluate the anti-proliferative effect of the drug. The cell line and culture method selected in the experiment are consistent with those in Test Example 4. The experimental process is as follows:

[0672] The cells were 2×10 3 -5×10 3 The cells were inoculated into 96-well plates at a density of 50 μL / well. After culturing for 24 h, ADC molecules diluted with culture medium at different concentrations were added at 100 μL / well. Duplicate wells were set for each concentration, and solvent controls and cell-free culture medium wells of corresponding concentrations were set. After culturing in a 37°C, 5% CO2 incubator for 96 h (Calu-6) or 144 h (HCC-1954, OVCAR3, MFE-280), 100 μL CellTiterGlo2 was added to each well, and the plates were placed on an orbital oscillator at room temperature for 15 minutes to mix. The luminescence values ​​were measured and the IC values ​​of the ADC molecules for killing various cells were calculated. 50Value (nM). The results are shown in the table below.

[0673] Table 11-1. IC values ​​of different antibody conjugates ADC on inhibition of Calu-6 cell proliferation 50 Value (nM)

[0674] ADC ADC-6 ADC-7 ADC-16 ADC-17 ADC-18 ADC-19 IC50(nM) 561.75 3.04 0.17 0.0076 0.029 0.0083

[0675] Table 11-2. IC values ​​of different antibody conjugates ADC on HCC-1954 cell proliferation inhibition 50 Value (nM)

[0676] ADC ADC-1 ADC-2 ADC-3 ADC-6 ADC-7 IC50(nM) 0.078 0.11 0.069 667 2.39 ADC ADC-10 ADC-12 ADC-16 ADC-17 ADC-19 IC50(nM) 0.13 0.0295 0.14 0.074 0.022

[0677] Table 11-3. IC values ​​of different antibody conjugates ADC on inhibition of OVCAR3 cell proliferation 50 Value (nM)

[0678]

[0679] Table 11-4. IC of ADC on the inhibition of proliferation of different cells 50 Value (nM)

[0680] cell OVCAR3 MFE-280 ADC-24 0.8 11.1

[0681] As can be seen from Table 11-1 to Table 11-4, the ADC molecules (ADC-3, -10, -12, -17, -19, -24) in the present application have obvious killing effects on tumor cells with different B7-H3 expression levels. It shows that the ADC molecules described in the present application can specifically kill positive cells through B7-H3-mediated endocytosis. And they have obvious inhibitory effects on cancer cell lines from breast cancer, ovarian cancer, lung cancer, endometrial cancer and melanoma, respectively, indicating that the obtained ADC has a good inhibitory effect on various solid tumor types.

[0682] Test Example 7. In vivo efficacy test of ADC molecules

[0683] 1. ADC efficacy test in breast cancer cell line HCC1954 transplanted mouse model

[0684] 2×10 6 HCC1954, a breast cancer cell line expressing human B7-H3, was transplanted subcutaneously into female severely immunodeficient NSG mice (purchased from Jackson Laboratories). After transplantation, when the tumor size reached an average of 150 mm 3 (120mm 3 -180mm 3At 1.0 mg / kg, 3.0 mg / kg, or 10.0 mg / kg of ADC-6, ADC-7, or ADC-17 prepared in Example 3 were intravenously injected into the mice once. In the control group, 4 mL / kg of PBS was intravenously injected into the mice. 28 days thereafter, the tumor size of the mice and the body weight of the mice were measured.

[0685] TGI(%)=1-[(T d -T0) / (Cd-C0)]×100%, where:

[0686] T d and C d To measure the average tumor volume of the treated and control animals on the same day, T0 and C0 are the average tumor volumes of the treated and control animals on the first day of administration. Figure 4 And Table 12.

[0687] Table 12. Inhibition rate of ADC molecules on HCC1954 breast cancer transplanted tumors in NSG female mice (Day 28)

[0688]

[0689] The results showed that ADC-17 of the present invention can significantly inhibit the growth of HCC1954 transplanted tumors in mice.

[0690] 2. ADC efficacy test in breast cancer cell line HCC1806 transplanted mouse model

[0691] 5×10 6 HCC1806, a breast cancer cell line expressing human B7-H3, was transplanted subcutaneously into female immunodeficient Athymic Nude mice (purchased from Charles River Laboratories). After transplantation, when the tumor size reached an average of 150 mm 3 (120mm 3 -180mm 3 At 1.0, 3.0, or 10.0 mg / kg ADC-6, ADC-7, or ADC-17 prepared in Example 3 was intravenously injected into the mice once. In the control group, 4 mL / kg PBS was intravenously injected into the mice. Thereafter, 14 days later, the tumor size of the mice and the body weight of the mice were measured.

[0692] TGI(%)=1-[(T d -T0) / (Cd-C0)]×100%, where:

[0693] T d and Cd To measure the average tumor volume of the treated and control animals on the same day, T0 and C0 are the average tumor volumes of the treated and control animals on the first day of administration. Figure 5 And Table 13.

[0694] Table 13. Inhibition rate of ADC molecules on HCC1806 breast cancer transplanted tumors in Athymic Nude female mice (Day 14)

[0695]

[0696] The results showed that ADC-17 of the present invention can significantly inhibit the growth of HCC1806 transplanted tumors in mice.

[0697] 3. ADC efficacy test in lung cancer cell line Calu-6 transplanted mouse model

[0698] 1×10 7 Calu-6, a lung cancer cell line expressing human B7-H3, was transplanted subcutaneously into female immunodeficient CD1Nude mice (purchased from Charles River Laboratories). After transplantation, when the tumor size reached an average of 150 mm 3 (120mm 3 -180mm 3 At 1.0, 3.0, or 10.0 mg / kg ADC-6, ADC-7, or ADC-17 prepared in Example 3 was intravenously injected into the mice once. In the control group, 4 mL / kg PBS was intravenously injected into the mice. 24 days thereafter, the tumor size of the mice and the body weight of the mice were measured.

[0699] TGI(%)=1-[(T d -T0) / (Cd-C0)]×100%, where:

[0700] T d and C d To measure the average tumor volume of the treated and control animals on the same day, T0 and C0 are the average tumor volumes of the treated and control animals on the first day of administration. Figure 6 And Table 14.

[0701] Table 14. Inhibitory effect of ADC molecules on Calu-6 lung cancer transplanted tumors in CD1 Nude female mice (Day 24)

[0702]

[0703] The results showed that ADC-17 of the present invention can significantly inhibit the growth of Calu-6 transplanted tumors in mice.

[0704] 4. ADC efficacy testing in ovarian cancer cell line OVCAR3 transplanted mouse model

[0705] 5×10 6 Ovarian cancer cell line OVCAR3 expressing human B7-H3 was transplanted subcutaneously into female severely immunodeficient NSG mice (purchased from Charles River Laboratories). After transplantation, when the tumor size reached an average of 150 mm 3 (120mm 3 -180mm 3 At 1:10 pm (day 0), the mice were divided into groups, and 0.1, 0.3, 1.0, 3.0, 6.0, 10.0 or 20.0 mg / kg ADC-6, ADC-7, or ADC-17 prepared in Example 3 was intravenously injected into the mice once. In the control group, 4 mL / kg PBS was intravenously injected into the mice. 21 days thereafter, the tumor size of the mice and the body weight of the mice were measured.

[0706] TGI(%)=1-[(T d -T0) / (Cd-C0)]×100%, where:

[0707] T d and C d To measure the average tumor volume of the treated and control animals on the same day, T0 and C0 are the average tumor volumes of the treated and control animals on the first day of administration. Figure 7 And Table 15.

[0708] Table 15. Inhibitory effect of ADC molecules on OVCAR3 ovarian cancer transplanted tumors in NSG female mice (Day 21)

[0709]

[0710] The results showed that ADC-17 of the present invention can significantly inhibit the growth of OVCAR3 transplanted tumors in mice.

[0711] 5. ADC efficacy testing in PC-3 prostate cancer cell line transplanted mouse model

[0712] 2×10 6 PC-3 prostate cancer cell line expressing human B7-H3 was transplanted subcutaneously into female immunodeficient Athymic Nude mice (purchased from Charles River Laboratories). After transplantation, when the tumor size reached an average of 150 mm 3 (120mm 3 -180mm3 At 1:10 pm (day 0), the mice were divided into groups, and 3.0 mg / kg ADC-6, ADC-7, and ADC-17 prepared in Example 3 were intravenously injected into the mice once. In the control group, 4 mL / kg PBS was intravenously injected into the mice. 24 days thereafter, the size of the tumors transplanted into the mice and the body weight of the mice were measured.

[0713] TGI(%)=1-[(T d -T0) / (Cd-C0)]×100%, where:

[0714] T d and C d To measure the average tumor volume of the animals in the treatment group and the control group on the same day, T0 and C0 are the average tumor volumes of the animals in the treatment group and the control group on the first day of administration.

[0715] Results Figure 8 And Table 16.

[0716] Table 16. Inhibition rate of ADC molecules on PC-3 prostate cancer transplanted tumors in Athymic Nude female mice (Day 24)

[0717]

[0718] The results showed that ADC-17 of the present invention can significantly inhibit the growth of PC-3 transplanted tumors in mice.

Claims

1. An anti-B7-H3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 1; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 2; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 9; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 10, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38; Preferably, the amino acid sequences of the HCDR and the LCDR are determined according to the Kabat, Chothia or IMTG numbering systems; More preferably, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 1; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 2, and the amino acid sequences of the HCDR and the LCDR are determined according to the Kabat numbering system.

2. An anti-B7-H3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and The light chain variable region comprises LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 25, wherein: The sequence of SEQ ID NO: 25 is QNTYGIX1X2X3GAV, The sequence of SEQ ID NO: 26 is QASQNIX4X5WLS, in: X1 is selected from I or P; X2 is selected from G, K or R; X3 is selected from Y or R, X4 is selected from N, Q, H, E or R, X5 is selected from S, A, T, K or R; Preferably, the heavy chain variable region and the light chain variable region are selected from any one of the following groups: 1) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and The light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8; 2) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and The light chain variable region comprises LCDR1 shown in any one of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23 and 24, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8; and 3) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and The light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in any one of SEQ ID NOs: 13, 14, 15 and 16.

3. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-B7-H3 antibody or antigen-binding fragment thereof is a rabbit antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, or a chimeric antibody or antigen-binding fragment thereof.

4. The anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the heavy chain variable region and the light chain variable region are selected from any one of the following groups: 1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:10; 2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2; 3) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27, or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:27; 4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:28, or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:28; 5) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:29, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:29; 6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:30, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:30; 7) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:31; 8) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:32, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:32; 9) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:33, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:33; 10) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:34, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:34; 11) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:35, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:35; 12) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:36, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:36; 13) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:37, or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:37; and 14) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:38 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

38.

5. The anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding fragment is selected from any one of the following: Fab, scFv, Fv, Fab', F(ab')2, single domain antibody, scFab, linear antibody and multispecific antibody.

6. The anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, further comprising a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3 and human IgG4, and the light chain constant region is selected from λ and κ light chain constant regions; More preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:11, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:

12.

7. The anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein: The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 39 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 39, and The light chain comprises the amino acid sequence as shown in SEQ ID NO:40 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

40. 8 . An antibody-drug conjugate comprising the anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 .

9. The antibody-drug conjugate according to claim 8, which has the following structure: in: Ab is the anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; L is a linker; D is the drug; n is an integer from 1 to 10 or a decimal.

10. The antibody drug conjugate according to claim 9, wherein L has -L a -L b -L c -L d - structure, where L a Connected to Ab, L d Connected to D, where: L a Selected from -(succinimidyl-3-yl-N)-(CH2)mC(=O)-, -(succinimidyl-3-yl-N)-(CH2CH2O)pC(=O)- and (CH2)qC(=O)-, The wavy line Indicates the connection site with Ab, * indicates the connection site with L b m is an integer from 0 to 8, preferably, m is 5; p is an integer from 1 to 8, preferably, p is 2; q is an integer from 0 to 4, preferably, q is 0 or 1; L b is selected from -(CH2)rC(=O)-, -NH-(CH2CH2O)s-(CH2)tC(=O)-, -NH-(CH2)wC(=O)- and a single bond, wherein: r is an integer of 1-6, preferably, r is 3 or 4; s is an integer of 1-6, preferably, s is 4; t is an integer of 0-4, preferably, t is 2; w is an integer of 0-4, preferably, w is 2; L c is a peptide residue consisting of 1 to 7 amino acids, or is a single bond; wherein the amino acids are selected from valine, citrulline, glycine, phenylalanine, alanine, proline, isoleucine, lysine, serine, glutamic acid and aspartic acid, and the amino acids are unsubstituted or each independently substituted by one or more substituents, wherein each substituent is independently selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 Cycloalkyl and Where # indicates the connection site; L d Selected from NH-CH2-O-CH2-C(=O)-, and single bond; where a * Indicates that L c The attachment site, b * Indicates the attachment site to D.

11. The antibody drug conjugate of claim 10, wherein said L has a structure selected from the group consisting of: i)L a for L b is a single bond or -NH-(CH2)2-C(=O)-, L c is a -valyl-citrullyl-, -glycyl-glycyl-phenylalanyl-glycyl-, -glycyl- or -yl bond, and L d for -NH-CH2-O-CH2-C(=O)- or a single bond; ii) L a is -(succinimidyl-3-yl-N)-(CH2)5-C(=O)-, or -(succinimidyl-3-yl-N)-(CH2CH2O)2-C(=O)-, L b is a single bond, L c is -valyl-citrullyl-, or -glycyl-glycyl-phenylalanyl-glycyl-, and L d for or NH—CH2—O—CH2—C(═O); iii) L a is -CH2-C(=O)-, L b is -NH-(CH2CH2O)4-(CH2)2-C(=O)-, -NH-(CH2)2-C(=O)- or a single bond, L c is -valyl-citrullyl-, or single key, L d for and iv) L a is -C(=O)-, L b is -(CH2)3-C(=O)- or -(CH2)4-C(=O)-, L c -Valyl-Citrulyl- or L d for in, Wavy Lines Indicates the connection site with Ab, * indicates the connection site with L b The attachment site, a * Indicates that L c The attachment site, b * represents the connection site with D, and e* represents the connection site with L b f* indicates the connection site with L d connection site.

12. The antibody drug conjugate of any one of claims 14 to 16, wherein the drug is selected from a cytotoxic agent, a radioisotope, an immunomodulator, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, a photosensitive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelating agent or a combination thereof; preferably, the drug is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), eribulin, exotecan, maytansine, SN-38, pyrrolobenzodiazepine and duocarmycin.

13. The antibody drug conjugate according to any one of claims 8 to 12, which has a structure selected from any one of the following: in: Ab is the anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; n is an integer or decimal between 2 and 5, preferably 4; More preferably, the antibody-drug conjugate has a structure shown in Formula 11, Formula 12, Formula 13, Formula 14, Formula 15 or Formula 16.

14. A pharmaceutical composition comprising: 1) a therapeutically effective amount of an anti-B7-H3 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, or A therapeutically effective amount of the antibody drug conjugate of any one of claims 8 to 13, and 2) One or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

15. An isolated nucleic acid encoding: The anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

16. An expression vector, which expresses: The anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

17. A host cell comprising: The isolated nucleic acid of claim 15, or The expression vector of claim 16.

18. A method for treating cancer, comprising administering to a subject a preventively or therapeutically effective amount of any of the following: The anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, The antibody-drug conjugate according to any one of claims 8 to 13, or The pharmaceutical composition according to claim 14; The cancer is a solid tumor or a blood tumor; Preferably, the cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, pancreatic cancer, colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, kidney cancer, urothelial carcinoma, head and neck cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, glioblastoma, pancreatic ductal carcinoma, colon cancer, osteosarcoma, soft tissue sarcoma, thyroid cancer, uterine cancer, multiple myeloma, rhabdomyosarcoma, squamous cell carcinoma, hematological tumors, mesothelioma, endometrial cancer, acute myeloid leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, follicular lymphoma and mantle cell lymphoma; Preferably, the cancer is selected from the group consisting of prostate cancer, melanoma, breast cancer, ovarian cancer, lung cancer, sarcoma and intrauterine cancer; Most preferably, the cancer is associated with abnormal expression of B7-H3 protein.

19. A method for inhibiting the growth or proliferation of cancer cells, the method comprising administering a preventively or therapeutically effective amount of any of the following: The anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, The antibody drug conjugate according to any one of claims 8 to 13, or The pharmaceutical composition according to claim 14; The cancer is a solid tumor or a hematological tumor; Preferably, the cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, pancreatic cancer, colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, kidney cancer, urothelial carcinoma, head and neck cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, glioblastoma, pancreatic ductal carcinoma, colon cancer, osteosarcoma, soft tissue sarcoma, thyroid cancer, uterine cancer, multiple myeloma, rhabdomyosarcoma, squamous cell carcinoma, hematological tumors, mesothelioma, endometrial cancer, acute myeloid leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, follicular lymphoma and mantle cell lymphoma; Preferably, the cancer is selected from the group consisting of prostate cancer, melanoma, breast cancer, ovarian cancer, lung cancer, sarcoma and endometrial adenocarcinoma; Most preferably, the cancer is associated with abnormal expression of B7-H3 protein.

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