Anti-b7-h3 antibodies, antibody drug conjugates, and medical uses 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 the anti-B7-H3 antibodies, and uses thereof for treating cancer.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the fields of biology, medicine, and clinic. In particular, the present disclosure relates to anti-B7-H3 antibodies, antibody drug conjugates based on the anti-B7-H3 antibodies, and medical uses thereof. BACKGROUND

[0002] B7-H3, also known as CD276, is a member of the membrane protein family of immune regulatory proteins B7-CD28, and is a type I membrane protein similar to the sequence of the extracellular domain of programmed death receptor-1 (PD-L1). B7-H3 is a costimulatory / inhibitory molecule. Early studies found that B7-H3 can stimulate CD4 + and CD8 + cells, promote the secretion of cytokines such as IFN-γ, and enhance the cytotoxicity of CD8 + T cells. However, most literatures believe that B7-H3 plays an inhibitory role in the tumor microenvironment. B7-H3 can affect multiple signaling pathways of tumor cells, inhibit Treg cells, and thus cause immune escape of tumor cells. B7-H3 can also down-regulate the cytotoxicity of NK cells by transmitting inhibitory signals, and inhibit NK cell-mediated lysis. 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 poor 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 various 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, and is expressed at low levels in normal tissues, thus becoming 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 to precisely deliver anti-tumor drugs (such as cytotoxic agents, cytostatic agents, small molecule chemotherapeutics, etc.) to tumor target cells, and then release the anti-tumor drugs through endocytosis to specifically kill tumors. Antibody drug conjugates generally consist of three parts: an antibody or antibody-like ligand, a small molecule drug, and a linker that couples the antibody or antibody-like ligand to the drug. Due to the appropriate molecular weight, high stability, strong targeting ability, and small side effects, antibody drug conjugates have been considered as the most promising anti-tumor drugs. SUMMARY

[0005] Anti-b7-h3 antibodies

[0006] In one aspect, the present disclosure provides an anti-B7-H3 antibody or antigen binding fragment thereof comprising 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 a HCDR1 or a variant thereof, a HCDR2 or a variant thereof, a HCDR3 or a variant thereof in the sequence set forth in SEQ ID NO: 1; the VL comprises a LCDR1 or a variant thereof, a LCDR2 or a variant thereof, a LCDR3 or a variant thereof in the sequence set forth in SEQ ID NO: 2;

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

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

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

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

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

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

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

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

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

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

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

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

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

[0021] In some embodiments, the HCDRs and the LCDRs are determined according to the Kabat, Chothia, or IMTG numbering system.

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

[0023] the heavy chain variable region comprises a HCDR1 set forth in SEQ ID NO: 3, a HCDR2 set forth in SEQ ID NO: 4, a HCDR3 set forth in SEQ ID NO: 5; and

[0024] the light chain variable region comprises a LCDR1 set forth in SEQ ID NO: 6, a LCDR2 set forth in SEQ ID NO: 7, a LCDR3 set forth in SEQ ID NO: 8;

[0025] the sequence of SEQ ID NO: 25 is QNTYGIX1X2X3GAV, the sequence of SEQ ID NO: 26 is QASQNIX4X5WLS, wherein XI 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.

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

[0027] the heavy chain variable region comprises a HCDR1 set forth in SEQ ID NO: 3, a HCDR2 set forth in SEQ ID NO: 4, a HCDR3 set forth in SEQ ID NO: 5; and

[0028] the light chain variable region comprises a LCDR1 set forth in SEQ ID NO: 6, a LCDR2 set forth in SEQ ID NO: 7, a LCDR3 set forth in SEQ ID NO: 8;

[0029] the HCDRs and the LCDRs are determined 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 a HCDR1 set forth in SEQ ID NO: 3, a HCDR2 set forth in SEQ ID NO: 4, a HCDR3 set forth in SEQ ID NO: 5; and

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

[0033] the HCDRs and the LCDRs are determined 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 a HCDR1 set forth in SEQ ID NO: 3, a HCDR2 set forth in SEQ ID NO: 4, a HCDR3 set forth in SEQ ID NO: 5; and

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

[0037] the HCDRs and the LCDRs are determined 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 one of the above, 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 an amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO: 1, and

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

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

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

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

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

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

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

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

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

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

[0050] the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 30 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 30;

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

[0052] the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9, and

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

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

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

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

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

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

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

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

[0061] 12) the heavy chain variable region comprises an amino acid sequence as set forth in 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 an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 36;

[0063] 13) the heavy chain variable region comprises an amino acid sequence as set forth in 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 an amino acid sequence as set forth in SEQ ID NO: 37 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 37;

[0065] 14) the heavy chain variable region comprises an amino acid sequence as set forth in 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 an amino acid sequence as set forth in SEQ ID NO: 38 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 38.

[0067] In the present 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, and can be an integer or a fraction.

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

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

[0070] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 4, and HCDR3 of SEQ ID NO: 5; and a light chain variable region comprising LCDR1 of SEQ ID NO: 6, LCDR2 of SEQ ID NO: 7, and LCDR3 of SEQ ID NO: 8.

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

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

[0073] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof of any of the preceding embodiments is a rabbit-derived 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, the anti-B7-H3 antibody or antigen-binding fragment thereof of any of the preceding embodiments, the heavy chain variable region VH and / or the light chain variable region VL is humanized.

[0075] In some embodiments, the antigen-binding fragment of any of the preceding 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 multi-specific antibody.

[0076] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof of any of the preceding embodiments further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is selected from the group consisting of human IgGl, human IgG2, human IgG3, and human IgG4, and the light chain constant region is selected from the group consisting of lambda and kappa light chain constant regions. In some embodiments, the heavy chain constant region is a heavy chain constant region of human IgGl and the light chain constant region is a kappa 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 foregoing anti-B7-H3 antibodies or antigen binding fragments thereof further comprises an Fc.

[0078] In some particular embodiments, any of the foregoing anti-B7-H3 antibodies comprises an Fc of human IgGl, an Fc of human IgG2, an Fc of human IgG3, or an Fc of human IgG4; preferably an Fc of human IgGl.

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

[0080] In some particular embodiments, the anti-B7-H3 antibody comprises a heavy chain as set forth in SEQ ID NO: 39 or a sequence at least 85% identical thereto, and a light chain as set forth in SEQ ID NO: 40 or a sequence at least 85% identical thereto.

[0081] In some particular embodiments, the anti-B7-H3 antibody or antigen binding fragment thereof of the present disclosure binds B7-H3 with a KD value no higher 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-09 M, 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 some particular embodiments, the anti-B7-H3 antibody or antigen binding fragment thereof of the present disclosure binds B7-H3 with an EC 50 no higher 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 (pg / mL).

[0083] In some particular embodiments, the anti-B7-H3 antibody or antigen binding fragment thereof of the present disclosure binds B7-H3-expressing cells with an EC 50 no higher 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).

[0084] In other specific embodiments, the anti-B7-H3 antibody or antigen binding fragment thereof of the present disclosure is endocytosed by the target cell with an efficiency of no 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 of the antibody drug conjugates or anti-B7-H3 antibodies or antigen binding fragments thereof.

[0087] In some embodiments, the antibody drug conjugate of the present disclosure has 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 a drug;

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

[0093] In some embodiments, L has the structure of -L a -L b -L c -L d -CH2CH2O)-, -CH2CH2S)-, -CH2CH2NR1-, -CH2C(=O)-, -CH2C(=S)-, -CH2P(=O)2-, -CH2NR1C(=O)-, -CH2NR1C(=S)-, -CH2NR1P(=O)2-, -CH2OC(=O)-, -CH2OC(=S)-, -CH2OP(=O)2-, -CH2SC(=O)-, -CH2SP(=O)2-, -CH2NR1C(=NCN)- a attached to Ab, L d is attached to D.

[0094] In some embodiments, L a is selected from -(succinimid-3-yl-N)-(CH2)m-C(=O)-, -(succinimid-3-yl-N)-(CH2CH2O)p-C(=O)- and (CH2)q-C(=O)-; wherein, represents the attachment site to Ab, * represents the attachment site to 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 bis selected from -(CH2)r-C(=0)-, -NH-(CH2CH2O)s-(CH2)t-C(=0)-, -NH- (CH2)w-C(=0)- 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 the group consisting of valine, citrulline, glycine, phenylalanine, alanine, proline, isoleucine, lysine, serine, glutamic acid and aspartic acid, which are unsubstituted or each independently substituted by one or more substituents, wherein each substituent is independently selected from the group consisting of halogen, hydroxy, cyano, amino, C 1-6 alkyl, halo-C 1-6 alkyl, C 1-6 alkoxy, C 3-7 cycloalkyl and wherein # indicates the point of attachment.

[0097] In some embodiments, L d is selected from NH-CH2-O-CH2-C(=0)-, and a single bond; wherein a * indicates the point of attachment to L c , b * indicates the point of attachment to D.

[0098] In some specific embodiments, L has the structure of -L a -L b -L c -L d - as indicated, wherein:

[0099] L a is indicates the point of attachment to Ab, * indicates the point of attachment to L b , b

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

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

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

[0103] Key; a * Indicates with L c The connection site, b * This indicates the connection site with D.

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

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

[0106] L b It is a single key.

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

[0108] L d for Or NH-CH2-O-CH2-C(=O), a * Indicates with L c The connection site, b * This indicates the connection site with D.

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

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

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

[0112] L c -Valyl-citrullyl-, Or a single bond, e* indicates a relationship with L b The connection site, f* represents the connection point with L. d Connection sites;

[0113] L d for a * represents the point of attachment to L c b * represents the point of attachment to D.

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

[0115] L a is -C(=0)-;

[0116] L b is -(CH2)3-C(=0)- or -(CH2)4-C(=0)-;

[0117] L c is -valyl-arginyl- or e*represents the point of attachment to L b f*represents the point of attachment to L d ;

[0118] L d is a * represents the point of attachment to L c b * represents the point of attachment to D.

[0119] In some embodiments, the antibody drug conjugate according to the present disclosure, wherein the drug is selected from the group consisting of: a cytotoxic agent, a radioisotope, an immunomodulatory agent, 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 photosensitizing therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelator, or a combination thereof. In some specific embodiments, the antibody drug conjugate according to the present disclosure, wherein the drug is a cytotoxic agent, or a pharmaceutically acceptable salt thereof.

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

[0121] In some embodiments, the antibody drug conjugate according to the present disclosure, wherein the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), eribulin, exatecan, 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 exetecan, 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 conjugate 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 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 conjugate of the present disclosure has the structure as shown below:

[0130]

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

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

[0133] In some specific embodiments, the antibody drug conjugate of the present disclosure has the structure as shown below:

[0134]

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

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

[0137] In some specific embodiments, the antibody drug conjugate of the present disclosure has the structure as shown below:

[0138]

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

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

[0141] In other specific embodiments, the antibody drug conjugate of the present disclosure has the structure shown below:

[0142]

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

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

[0145] In other specific embodiments, the antibody drug conjugate of the present disclosure has the structure shown below:

[0146]

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

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

[0149] In other specific embodiments, the antibody drug conjugate of the present disclosure has the structure shown below:

[0150]

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

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

[0153] In other specific embodiments, the antibody drug conjugate of the present disclosure has the structure shown below:

[0154]

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

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

[0157] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0158]

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

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

[0161] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0162]

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

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

[0165] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0166]

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

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

[0169] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0170]

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

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

[0173] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0174]

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

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

[0177] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0178]

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

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

[0181] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0182]

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

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

[0185] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0186]

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

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

[0189] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0190]

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

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

[0193] In other specific embodiments, the antibody drug conjugates of the present disclosure have the structure shown below:

[0194]

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

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

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

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

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

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

[0201] In more preferred embodiments, 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 set forth in SEQ ID NO: 3, HCDR2 set forth in SEQ ID NO: 4, HCDR3 set forth in SEQ ID NO: 5; and

[0204] the light chain variable region comprises LCDR1 set forth in SEQ ID NO: 6, LCDR2 set forth in SEQ ID NO: 7, LCDR3 set forth 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 an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO: 9, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence comprising 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 an amino acid sequence set forth in SEQ ID NO: 39 or an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO: 39, and

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

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

[0211] In one embodiment of the present disclosure, the drug is conjugated to the antibody or antigen-binding fragment thereof via a linking unit (exemplarily, 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 antigen-binding fragment thereof),

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

[0214] (3) selecting different reaction reagents.

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

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

[0217] Pharmaceutical compositions

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

[0219] a prophylactically or therapeutically effective amount of the antibody drug conjugate or anti-B7-H3 antibody or antigen-binding fragment thereof of any one of the preceding items, 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 of any one of the preceding items.

[0222] It is understood that even though specific nucleotide sequence examples are provided herein, the nucleotide sequences that can "encode an anti-B7-H3 antibody or antigen binding fragment thereof of the present disclosure" are not limited to these specific sequences, as equivalent nucleotide sequences can be employed to encode the same amino acid sequence, depending on the codon preference of the expression host, codon degeneracy.

[0223] In another aspect, the present disclosure also provides a host cell comprising the aforementioned nucleic acid or the aforementioned vector or for expressing an anti-B7-H3 antibody or antigen binding fragment thereof of the present disclosure.

[0224] Methods of prevention or treatment

[0225] In another aspect, the present disclosure also provides a method of treating cancer, comprising administering to a subject a prophylactically or therapeutically effective amount of the aforementioned antibody drug conjugate, anti-B7-H3 antibody or antigen binding fragment thereof, nucleic acid or pharmaceutical composition.

[0226] In another aspect, the present disclosure also provides a method for inhibiting the growth or proliferation of cancer cells, comprising administering to a subject a prophylactically or therapeutically effective amount of the aforementioned antibody drug conjugate, anti-B7-H3 antibody or antigen binding fragment thereof, nucleic acid or pharmaceutical composition.

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

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

[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, renal cancer, urothelial cancer, 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 tumor, mesothelioma, endometrial cancer, acute myeloid leukemia, non-Hodgkin 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 for preventing or treating prostate cancer, melanoma, breast cancer, ovarian cancer, lung cancer, sarcoma, or endometrial cancer.

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

[0232] In some embodiments, the cancer is associated with aberrant expression of B7-H3.

[0233] Uses

[0234] In another aspect, the present disclosure also provides use of the antibody drug conjugate, the anti-B7-H3 antibody or antigen binding fragment thereof, the nucleic acid, or the pharmaceutical composition of any of the foregoing in the manufacture of a medicament for treating or preventing cancer.

[0235] The present disclosure also provides use of the combination of the anti-B7-H3 antibody or antigen binding fragment thereof and the cytotoxic agent of any of the foregoing in the manufacture of a medicament 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, exetecan 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 mesylate salt (as an example, eribulin mesylate, exetecan 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 foregoing anti-B7-H3 antibody or antigen binding fragment thereof is provided for use in treating cancer, which is administered in combination with a cytotoxic agent.

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

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

[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, renal cancer, urothelial cancer, 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 neoplasm, mesothelioma, endometrial cancer, acute myeloid leukemia, non-Hodgkin 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, endometrial cancer.

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

[0243] In some embodiments, the cancer is associated with aberrant expression of B7-H3 protein.

[0244] In some embodiments, the cancer is associated with aberrant expression of B7-H3 protein.

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

[0246] In another aspect, the anti-B7-H3 antibody or antigen-binding fragment thereof of the present disclosure is used for immunoassay. The immunoassay comprises detecting cancer cells in a biological sample from a patient, or the expression amount of B7-H3, by contacting the biological sample with the anti-B7-H3 antibody or antigen-binding fragment thereof of the present disclosure. Typically, the anti-B7-H3 antibody or antigen-binding fragment thereof of the present disclosure can be coupled with a fluorescent label or other label.

[0247] Reagent / kit

[0248] In another aspect, the present disclosure also provides a kit comprising at least one container, each of which independently comprises: the antibody drug conjugate, the anti-B7-H3 antibody or antigen-binding fragment thereof, the nucleic acid, or the pharmaceutical composition of any one of the foregoing of the present disclosure.

[0249] In diagnostic or research use, the kit comprises one or more of the following components: an analytical reagent, a buffer, and the antibody or antigen-binding fragment thereof of the present disclosure.

[0250] In addition, the reagent / kit can comprise an instruction. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

[0254] Figure 5 : Tumor inhibition activity of ADC in Athymic Nude female mice HCC1806 breast cancer model.

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

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

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

[0258] The terminology

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

[0260] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", "include", "including", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". "Comprise" includes "consist of", for example, for VH comprising HCDR1 as set forth in SEQ ID NO: 3, it explicitly covers HCDR1 of the amino acid sequence as set forth in SEQ ID NO: 3.

[0261] It should be understood that the ordinal terms "first", "second", "Formula 1", "Formula 2", "1)", "2)", etc. in the present disclosure are merely used to distinguish different technical features, elements, components, steps, and are not intended to imply a level, order, number, etc.

[0262] Target refers to the object that the therapeutically active ingredient (antibody drug conjugate, anti-B7-H3 antibody or antigen binding fragment thereof) of the present disclosure is directed to. 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 to a protein, in particular. As an example, the antibody drug conjugate, anti-B7-H3 antibody or antigen binding fragment thereof 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, for example, but not limited to, can be obtained from literature or databases.

[0264] In the context of the present disclosure, B7-H3 should be interpreted broadly, including various forms of molecules of B7-H3 at various stages, for example, but not limited to, molecules produced during amplification, replication, transcription, splicing, processing, translation, modification of 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, in particular, cell surface expressed B7-H3.

[0265] In the context of antibodies, targets are often in the form of antigens. An "antigen" refers to a molecule or portion thereof that is selectively recognized by an antigen binding molecule, e.g., an antibody drug conjugate of the disclosure, an anti-B7-H3 antibody, or antigen binding fragment thereof. An antigen can have one or more epitopes.

[0266] An "epitope" refers to a region on an antigen to which an antibody or antigen binding fragment thereof specifically binds. An epitope can be formed by contiguous amino acids (linear epitope); or comprise non-contiguous 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. Antibodies or antigen binding fragments thereof that bind to a particular epitope can be screened using routine methods in the art, such as, but not limited to, alanine scanning, peptide cleavage analysis, epitope extraction, chemical modification of antigens.

[0267] An "antibody" is used in the broadest sense, and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, full-length antibodies, so long as they exhibit the desired antigen-binding activity. Typically, a natural IgG antibody is a heterotetrameric protein composed of two light chains and two heavy chains that are linked via disulfide bonds. From N- to C-terminus, each heavy chain has one variable region (VH), three constant domains (CH1, CH2, and CH3). From N- to C-terminus, each light chain has one variable region (VL), one constant light domain (CL). Depending on the context, one skilled in the art 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 classified into five major classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0269] "Specifically binds" refers to an anti-B7-H3 antibody or antigen binding fragment thereof of the disclosure binds to a targeted antigen or epitope thereof with a higher affinity than to other antigens or epitopes thereof. Typically, an anti-B7-H3 antibody or antigen binding fragment thereof of the disclosure binds to a targeted antigen or epitope thereof with an affinity of about 1 x 10 -7A binding equilibrium dissociation constant (KD) of M or less is considered "specific" binding. KD can be measured using known methods, such as FACS or surface plasmon resonance. "Specific binding" does not exclude cross-reactivity with homologous antigens of other species (e.g., Macaca fascicularis, Pan troglodytes, or Callithrix jacchus). In view of this, "anti-B7-H3...," "specific binding to B7-H3...," refers to an anti-B7-H3 antibody or antigen-binding fragment thereof that is capable of specifically recognizing or binding to B7-H3 (or an epitope thereof).

[0270] "Affinity" is used to describe the strength of the noncovalent interaction between an anti-B7-H3 antibody or antigen-binding fragment thereof and its antigen or epitope thereof. Affinity is typically denoted by an 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) and is expressed as a molar concentration.

[0271] "Fully human antibody" includes an antibody having variable and constant regions of immunoglobulin sequences of the human germline.

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

[0273] "Chimeric antibody" is an antibody that fuses the variable region of an antibody of a first species (e.g., murine, rabbit) to the constant region of an antibody of another species (e.g., human).

[0274] "Antibody fragment" or "antigen-binding fragment" is a molecule other than an intact antibody that comprises 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 antibody, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0275] "Fab" consists of one light chain and the CH1 and variable regions of one heavy chain.

[0276] "Fab' fragment" 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 comprising two Fab' fragments linked by disulfide bridges at the hinge region.

[0278] “Fab'-SH” refers to an Fab' in which the cysteine residue of the constant region has 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 said antibody domains and said linker have one of the following orders from N- 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 said 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 combining sites, which comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) connected in the same polypeptide chain.

[0281] “Linear antibodies” comprise 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] “Fv fragment” has the VL and VH domains of one arm of an antibody.

[0283] “Single-domain antibody” is an antibody fragment that comprises all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.

[0284] Single-chain antibody (scFv) is an antibody formed by the connection of a heavy chain variable region and a light chain variable region through a linker.

[0285] “Fc” contains two heavy chain fragments comprising CH2 and CH3 domains. The two heavy chain fragments are held together by disulfide bonds and by hydrophobic interactions of the CH3 domains.

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

[0287] “Complementarity determining region” or “CDR” refers to a region within a variable region that primarily contributes to binding to an antigen. Each of VHand VLcomprises four framework regions (FRs) and three complementarity determining regions (CDRs). VHis comprised of three CDR regions: HCDR1, HCDR2, and HCDR3; VLis comprised of three CDR regions: LCDR1, LCDR2, and LCDR3. Each VHand VLis arranged, from N-terminus to C-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0288] “Framework” or “FR” refers to a variable domain residue other than a hypervariable region (CDR) residue. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences typically appear in the following order in a VHand (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

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

[0290] The boundaries of the amino acid sequences of the CDRs can be determined by various well-known schemes, such as: the Kabat numbering system (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), the Chothia numbering system, and the ImMunoGeneTics (IMGT) numbering system (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16;9:2278). Exemplarily, in the following table, the amino acid sequences of the “alternative” CDR1, CDR2, and CDR3 of the heavy chain variable region and the light chain variable region of 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 understood by those skilled in the art. In other words, when a CDR sequence under one numbering system and its position in an antibody is provided, the skilled person is able to determine the corresponding CDR sequence under another numbering system and its position in an antibody. The technical solutions corresponding to different numbering systems are considered as equivalent technical solutions.

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

[0295] A "cytotoxic agent" refers to a chemical molecule that has a destructive effect on growth of target cells. Cytotoxic agents can in principle kill cells at sufficient concentrations, but due to lack of specificity, they also cause apoptosis or death of normal cells, leading to side effects, when killing tumor cells. Cytotoxic agents include toxins (such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin), radioisotopes, chemotherapeutic drugs, antibiotics, nucleolytic enzymes.

[0296] The term "linker unit" or "linking fragment" or "linker" refers to a chemical moiety or bond that links one end to an antibody or antigen binding fragment thereof and the other end to a drug, or can be linked to another linker before being linked to a drug. The linker can be a degradable linker or a non-degradable linker. A degradable linker is typically susceptible to degradation in the intracellular environment, for example, the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptide-based linkers that are degradable by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, for example, glucuronide-containing linkers that are degradable by glucuronidases. Peptide-based linkers can 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 a pH less than 5.5, such as hydrazone linkers) and linkers that are degradable under reducing conditions (e.g., disulfide linker). A non-degradable linker is typically released to release the drug under conditions in which the antibody is hydrolyzed by proteases.

[0297] The linker has a reactive group capable of reacting with certain amino acid residues prior to attachment to the antibody, and attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodo, bromo or chloro); haloesters (e.g., iodo, bromo or chloro); halo methyl ketones (e.g., iodo, bromo or chloro), benzyl halides (e.g., iodo, bromo or chloro); vinyl sulfone, pyridyl disulfides; mercury derivatives such as 3,6-bis-(mercurymethyl) dioxane, and counterions are acetate, chloride or nitrate; and polymethylene dimethyl sulfide sulfonate. The linker can include, for example, a maleimide for attachment to the antibody via a thio-butyrimidate.

[0298] As an example, the linker of the present disclosure has the structure -L a -L b -L c -L d - wherein:

[0299] L a is selected from -(CH2)m-C(=0)-, -(CH2CH20)p-C(=0)- and -(CH2)q-C(=0)-, wherein -(succinimid-3-yl-N)-(CH2)m-C(=0)-, -(succinimid-3-yl-N)-(CH2CH20)p-C(=0)- and (CH2)q-C(=0)-, wherein represents the attachment site to Ab, and * represents the attachment site to 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;

[0300] L b is selected from -(CH2)r-C(=0)-, -NH-(CH2CH20)s-(CH2)t-C(=0)-, -NH-(CH2)w-C(=0)- 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 is a single bond; wherein the amino acids are selected from the group consisting of valine, citrulline, glycine, phenylalanine, alanine, proline, isoleucine, lysine, serine, glutamic acid and aspartic acid, which are unsubstituted or each independently substituted by one or more substituents, wherein each substituent is independently selected from the group consisting of halogen, hydroxyl, cyano, amino, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, C3-7 cycloalkyl and Where # represents the substitution site;

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

[0303] Key; where a * Indicates with L c The connection site, b * This indicates the connection site with D.

[0304] In this context, the term "peptide residue" refers to a short peptide formed by the linear linkage of amino acid residues through amide bonds. The term "amino acid" refers to an organic compound whose molecular structure contains both amino and carboxyl groups, with both groups directly attached to the -CH- structure. When amino acids combine, some groups participate in the formation of amide bonds, resulting in the loss of a water molecule; therefore, the amino acid units in a peptide are called "amino acid residues."

[0305] The term "single bond" refers to a covalent bond between two atoms in a compound molecule that shares 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., C2). 1-20 Alkyl group. Alkyl groups can be substituted or unsubstituted. When substituted, they can be substituted at any usable connection point. The substituents are preferably selected from one or more of the following: D atom, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0307] Term "C" 1-6 Alkyl groups are lower alkyl groups 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, etc. Alkyl groups can be substituted or unsubstituted.

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

[0309] The term "cycloalkyl" refers to a cyclic hydrocarbon substituent that is saturated or partially unsaturated, either monocyclic or polycyclic. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, and cyclooctyl. Polycyclic cycloalkyl groups include spirocyclic, fused-ring, or bridged-ring cycloalkyl groups.

[0310] Term "C" 3-7 "Cycloalkyl" refers to a cycloalkyl group containing 3 to 7 cyclic carbon atoms, as defined above.

[0311] The term "alkoxy" refers to -O- (alkyl), where alkyl is defined as described above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy, etc. Alkoxy groups can be substituted or unsubstituted, and when substituted, they can be substituted at any usable link. Substituents are preferably selected from one or more of the following: D atom, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0312] Term "C" 1-6 "Alkoxy" refers to an alkoxy group containing 1 to 6 carbon atoms, as defined above.

[0313] The term "aryl" refers to a monocyclic all-carbon aromatic ring (i.e., monocyclic aryl) or a polycyclic aromatic ring system (i.e., polycyclic aryl) having a conjugated π-electron system, having 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., 6 to 14-membered aryl). The aryl is preferably an aryl having 6 to 10 ring atoms (i.e., 6 to 10-membered aryl). The monocyclic aryl is, for example, phenyl. Non-limiting examples of the polycyclic aryl include naphthyl, anthraceneyl, phenanthrene, etc. The polycyclic aryl further includes fusion of the phenyl with one or more heterocyclic groups or cycloalkyl groups, or fusion of the naphthyl with one or more heterocyclic groups or cycloalkyl groups, wherein the bonding point is on the phenyl or 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, non-limiting examples including:

[0314]

[0315] Aryl can be substituted or non-substituted, and when substituted, it can be substituted at any available point of attachment with one or more substituents preferably selected from D atoms, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, oxo, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0316] The term "heteroaryl" refers to a monocyclic heteroaromatic ring (i.e., monocyclic heteroaryl) or a polycyclic heteroaromatic ring system (i.e., polycyclic heteroaryl) having a conjugated pi-electron system, which contains at least one (e.g., 1, 2, 3, or 4) heteroatom selected from nitrogen, oxygen, and sulfur (said nitrogen can optionally be oxidized, i.e., form a nitro oxide; said sulfur can optionally be oxidized, i.e., form a sulfoxide or sulfone, but not -O-O-, -O-S-, or -S-S-) within the ring, which has 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., 5- to 14-membered heteroaryl). The heteroaryl group preferably has 5 to 10 ring atoms (i.e., 5- to 10-membered heteroaryl), more preferably 5 or 6 ring atoms (i.e., 5- or 6-membered heteroaryl).

[0317] The term "cycloalkyloxy" refers to cycloalkyl-O-, wherein 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 aryl-O-, wherein 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 alkyl-S-, wherein alkyl is as defined above.

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

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

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

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

[0326] The term "hydroxyl" refers to the -OH group.

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

[0328] The term "amino" means -NH2.

[0329] The term "cyano" means -CN.

[0330] N-ethyldiisopropylamine is abbreviated as DIEA.

[0331] N,N-dimethylformamide is abbreviated as DMF.

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

[0333] 1-hydroxybenzotriazole is abbreviated as HOBt.

[0334] 2-bromoacetic anhydride is abbreviated as BrAc20.

[0335] "Substituted" means that one or more hydrogen atoms, preferably 1 to 6, more preferably 1 to 3, of a group are independently of each other replaced with a corresponding number of substituents. Those skilled in the art are able to determine, without undue effort, experimentally or theoretically, what substitutions are possible or impossible. For example, an amino or hydroxyl group with a free hydrogen can not be stable when bound to a carbon atom with an unsaturated (e.g., olefinic) bond.

[0336] The term "drug-to-antibody ratio" (DAR) refers to the average number of cytotoxic agents conjugated per antibody in a population of antibody drug conjugates, and can also be expressed as the ratio of the amount of cytotoxic agent to the amount of antibody. The drug loading can range from 1-20, preferably 1-10 cytotoxic agents (D) per antibody (Ab). In embodiments of the disclosure, the drug loading is denoted as n, which can be, by way of example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or the average of the values between any two of these values (which can be integers or decimals). n is preferably an integer or decimal between 1-10, more preferably 1-5, or 2-5. Drug loading can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, monoclonal molecular size variant assays (CE-SDS), and HPLC profiling. While the ratio of cytotoxic agent to antibody has an exact value for a particular conjugate molecule, it will be understood that when used to describe a sample containing a population of molecules, the value will often be an average. In some embodiments, the DAR is between about 1 and about 6, and is typically 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 "… has at least 85% sequence identity…", the identity refers to the extent (percentage) of amino acid / nucleic acid in two sequences which are identical at equivalent positions when the two sequences are optimally aligned. Gaps are introduced, if necessary, to achieve 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 to SEQ ID NO: 1" means that the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 3, HCDR2 set forth in SEQ ID NO: 4, HCDR3 set forth in SEQ ID NO: 5, and amino acid mutations are allowed to be introduced in the regions outside of the CDRs so as to have at least 85% sequence identity to SEQ ID NO: 1.

[0339] An "amino acid mutation" includes an amino acid substitution, deletion, insertion, modification, or any combination thereof. Any combination of substitutions, deletions, insertions and modifications can be made to arrive at a final construct, so long as the final construct possesses a desired property, such as an amino acid substitution that reduces Fc region homodimerization. Amino acid substitutions can be introduced into an antibody of interest, and the products screened for a desired activity, such as retained / improved antigen binding, decreased immunogenicity. Amino acid sequence deletions and insertions include deletions and insertions at the amino- and / or carboxyl-terminus of a polypeptide chain. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., the replacement of one amino acid by another amino acid of different structure and / or chemical characteristics. Genetic or chemical methods known in the art can be used to generate the amino acid mutations. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like.

[0340] In the present disclosure, various ways can be used to indicate the same amino acid mutation. Illustratively, the amino acid residue at a particular position can be represented in the position + amino acid residue format, such as 366S, 368A, which indicates that the amino acid residue at position 366 is S, and the amino acid residue at position 368 is A.

[0341] As a supplement, in the anti-B7-H3 antibodies or antigen-binding fragments thereof of the present application, although the C-terminus of the Fc region is the complete C-terminus ending with PGK, it can also be a truncated C-terminus. Illustratively, in the truncated C-terminus, one or two C-terminal residues are removed (e.g., truncated C-terminus ending with PG). In a composition comprising antibodies, both antibodies with complete C-terminus and antibodies with truncated C-terminus can be included.

[0342] Exemplary anti-B7-H3 antibodies or antigen-binding fragments thereof

[0343] In particular embodiments, the anti-B7-H3 antibodies or antigen-binding fragments thereof of the present disclosure comprise 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 set forth in SEQ ID NO: 1, and

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

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

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

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

[0349] the amino acid sequence set forth in SEQ ID NO: 27;

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

[0351] the amino acid sequence set forth in SEQ ID NO: 28;

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

[0353] the amino acid sequence set forth in SEQ ID NO: 29;

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

[0355] the amino acid sequence set forth in SEQ ID NO: 30;

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

[0357] the amino acid sequence set forth in SEQ ID NO: 31;

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

[0359] the amino acid sequence set forth in SEQ ID NO: 32;

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

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

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

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

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

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

[0366] wherein the CDRs are determined according to the Kabat numbering system.

[0367] Exemplary antibody drug conjugates

[0368] In particular embodiments, the antibody drug conjugates of the present disclosure have the following structure:

[0369] wherein:

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

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

[0372] In particular embodiments, the antibody drug conjugates of the present disclosure have the following structure:

[0373] wherein:

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

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

[0376] In particular embodiments, the antibody drug conjugates of the present disclosure have the following structure:

[0377]

[0378] wherein:

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

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

[0381] In particular embodiments, the antibody drug conjugates of the present disclosure have the following structure:

[0382]

[0383] wherein:

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

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

[0386] In particular embodiments, the antibody drug conjugates of the present disclosure have the following structure:

[0387]

[0388] wherein:

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

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

[0391] In particular embodiments, the antibody drug conjugates of the present disclosure have the following structure:

[0392]

[0393] wherein:

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

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

[0396] The skilled artisan will note that in preferred antibody drug conjugates of the present disclosure, the anti-B7-H3 antibody or antigen binding fragment thereof and the L a The component forms two single bonds.

[0397] “Nucleic acid” is used interchangeably with “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The nucleic acids are synthetic, naturally occurring, and non-naturally occurring. Unless otherwise indicated, nucleic acid sequences also encompass conservatively modified variants (e.g., degenerate substitutions) and complementary sequences, as well as the sequence explicitly indicated. 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 has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV), into which additional DNA segments are ligated. Some vectors are capable of autonomous replication in a host cell, while others can be integrated into the host genome and replicated with the host genome.

[0399] "Host cell," "cell line" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced and the progeny of the cell, regardless of whether the progeny has undergone further mutation. The progeny can not be identical to the parent cell in nucleic acid content, but are still within the scope of the disclosure as long as the progeny cells have the same functional or activity as the original cell.

[0400] Host cells are not capable of developing into complete, adult organisms.

[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, canine, monkey, porcine, goat, bovine, equine, hamster cells. Exemplary host cells include, but are not limited to, CHO, NSO, COS, SP2 cells, HeLa cells, BHK cells, human hepatocarcinoma cells, A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast, such as, but not limited to, Pichia, Saccharomyces, Hansenula, Kluyveromyces.

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

[0403] In a particular 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 culture thereof).

[0404] "Pharmaceutical composition" means a mixture of one or more of the antibody drug conjugates, anti-B7-H3 antibodies or antigen-binding fragments thereof of the disclosure with other chemical components, such as a pharmaceutically acceptable carrier, diluent, buffer, or excipient.

[0405] "Pharmaceutically acceptable carrier, diluent, buffer, or excipient" means a component, other than the active ingredients, of a pharmaceutical formulation which does not itself induce the production of antibodies harmful 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 particular embodiments, the subject is a mammal, including, for example, a camel, a horse, a cow, a pig, a goat, a cat, a dog, a rabbit, a rat, a guinea pig, a mouse, a primate (e.g., a human). In particular embodiments, the subject is a human.

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

[0408] “Treatment” means providing to a subject an antibody drug conjugate, an anti-B7-H3 antibody or antigen binding fragment thereof, or a pharmaceutical composition thereof of the present disclosure. 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 population of subjects.

[0409] In particular embodiments, the selection of an effective amount can be determined by one of skill in the art based on considerations of a variety of factors, e.g., via clinical trials, including the disease to be treated, the symptoms involved, the route of administration, the severity of the disease, the weight of the subject, the immune status of the subject, other factors known to one of skill in the art.

[0410] An effective amount in a particular embodiment can be derived from dose- response curves derived from animal model test systems, and allows for judgment by the medical practitioner and circumstances of each subject. As an example, the amount of drug required for administration to a subject at one time can be conveniently determined by multiplying the weight of the subject by the unit weight dose required for that subject at one time. For example, in preparing a drug, an adult human is generally considered to weigh 50-70 kg, and the amount of drug for administration can initially be determined by an equivalent dose conversion relationship between the unit weight dose of an experimental animal and that of a human. For example, it can be determined according to the guidance opinions of drug regulatory agencies such as SFDA, FDA, etc. In some embodiments, the dose of a human and a mouse can be converted using a body surface area conversion coefficient of 0.0026 for a human and a mouse.

[0411] An active ingredient (e.g., an antibody drug conjugate, an anti-B7-H3 antibody or antigen binding fragment thereof) can be administered at one time, or can be divided into a number of smaller unit doses to be administered at intervals of time. It will be appreciated that the dosages, duration, and intervals of treatment are a function of the disease being treated, and can be determined using animal or clinical trial data. The administration can include a single administration, or two or more administrations at appropriate intervals of time. Adjacent administrations can be separated by 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, 1.5 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.

[0412] It should be understood that, when a numerical range is disclosed, such as "A to B," "A to B" is intended to function as a shorthand way of referring individually to each numerical value within the range. For example, if a range is stated as "1 to 10," this is intended to disclose each individual number from 1 to 10, inclusive of the endpoints. It is specifically intended that the scope of the invention include all individual numerical values within the range even if this is not explicitly stated in the specification.

[0413] "and / or," such as "A and / or B" shall be understood to mean "A and B" or "A or B."

[0414] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. As used in the description and the claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0415] Examples

[0416] The following examples are provided to give a better understanding of the inventive subject matter. These examples should not be considered as limiting the subject matter described. Rather, it is understood that the examples and embodiments described herein are only meant to be illustrative and that various modifications or changes in addition or substitution thereof will occur to those skilled in the art and are intended to be included within the scope of the application.

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

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

[0419] Human B7-H3 protein (UniProt ID: Q5ZPR3) extracellular domain was first expressed as an immunogen, fusing the B7-H3 protein amino acid sequence (Leu29-Pro245) C-terminal to an antibody Fc sequence, and then cloned into an expression vector, transiently transfecting 293 cells with the expression vector, and collecting cell culture fluid after 7 days and purifying.

[0420] To prepare rabbit anti-human B7-H3 monoclonal antibody, New Zealand rabbits (purchased from Wuhan Wanchangjiaxing Biotechnology Co., Ltd.) were immunized with human B7-H3 extracellular region protein, a total of 5 times. After immunization, the B cells of the New Zealand rabbits were separated and monoclonal culture was carried out. The titer of the supernatant of the monoclonal B cells was detected by ELISA method. The immunogen B7-H3 protein was coated on the microplate at 1 μg / mL overnight at 4°C, then blocked with blocking solution at 37°C for 1 h, 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 for 5 minutes, then 2M H2SO4 was added to terminate the reaction. The absorbance was detected on the enzyme label instrument at 450 nm wavelength. According to the binding activity with B7-H3, the monoclonal 36F3 with high binding activity (the OD value measured at 450 nm wavelength was 2.3) was screened out, and the variable region sequences of 36F3 were shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0421] 36F3 VH: SEQ ID NO: 1

[0422]

[0423] 36F3 VL: SEQ ID NO: 2

[0424]

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

[0426] CDR Sequence Sequence 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 the light chain variable region of the 36F3 antibody were aligned 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 the light chain variable region of the rabbit-derived antibody were transplanted into the framework sequences of the matched 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 with the heavy chain constant region and the light chain constant region of human antibody to obtain the full-length sequence of 112-59 antibody. The sequence of 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. Mutations of 112-59 antibody

[0438] Mutating the light chain LCDR1 or LCDR3 of 112-59 to obtain a new light chain variable region, recombining the new light chain variable region 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. Sequences of LCDR1 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 sequences of the VL of 112-59 mutant antibodies are as follows:

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

[0447]

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

[0449]

[0450]

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

[0452]

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

[0454]

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

[0456]

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

[0458]

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

[0460]

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

[0462]

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

[0464]

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

[0466]

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

[0468]

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

[0470]

[0471]

[0472] The variable region sequences described above were linked to human heavy chain constant region and light chain constant region to obtain the full length sequence of the antibody. The sequences of exemplary antibodies are 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 Linker and Toxins

[0478] Toxins (Drug) used to prepare ADCs were pre-coupled with linkers to prepare linker-drug compounds LD-1 to LD-18; exemplary toxins are MMAE (purchased from MedChemExpress, Cat# HY-100374), Eribulin mesylate (MedChemExpress, Cat# HY-13442) and Exatecan mesylate (purchased from Advance ChemBlocks, Cat# 10484).

[0479] 1. Preparation of LD-1

[0480]

[0481] Corresponding ADC structure, Formula 1;

[0482]

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

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

[0485] 2. Preparation of LD-2:

[0486]

[0487]

[0488] Corresponding ADC structure, Formula 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] Corresponding ADC structure, Formula 3;

[0495]

[0496] To a solution of MC-GGFG-OH (purchased from Innopep San Diego, 16 mg, ) and exatecan mesylate (purchased from Advanced ChemBlocks Cat. No. 10484, 16 mg) in anhydrous DMF (1 mL), PyAOP ((7-Azabenzotriazol-l-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] Corresponding ADC structure, Formula 4;

[0501] LD-4 was prepared according to Example 43 in patent number US9808537B2.

[0502] 5. Preparation of LD-5:

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

[0504]

[0505] Corresponding ADC structure, Formula 5;

[0506] 6. Preparation of LD-6

[0507]

[0508] Corresponding ADC structure, Formula 6;

[0509]

[0510] To a solution of compound 6 (20 mg) in acetonitrile / water (6 / 4, v / v, 2 mL) was added saturated NaHC03(0.2 mL) and bromoacetic anhydride (9 mg). After the reaction mixture was stirred at room temperature (22 °C) for 10 min, the reaction mixture was directly purified by RP-HPLC to give compound 7 (17 mg) as a white solid, LD-6, after lyophilization.

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

[0512] 7. Preparation of LD-7:

[0513]

[0514] Corresponding ADC structure, Formula 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) was added saturated aqueous NaHC03(0.1 mL) followed by 2-bromoacetic anhydride (6 mg, Toronto Research Chemicals). The mixture was stirred at room temperature. After 10 min, the mixture was purified by RP-HPLC to give LD-7 (11 mg) as a white solid.

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

[0518] 8. Preparation of LD-8

[0519]

[0520] Corresponding ADC structure, Formula 8;

[0521]

[0522] To a solution of compound 12 (65 mg, prepared as per WO2022026915) and MMAE (72 mg) in dry DMF (2 mL), DIEA (0.002 mL) was added followed by HOBt (3 mg). After stirring the reaction mixture at room temperature (22 °C) for 18 h, it was diluted with water (20 mL). The reaction mixture was extracted with diethyl ether (40 mL) and the organic phase was dried over NaS04and concentrated to dryness under reduced pressure to obtain crude compound 13 which was finally dissolved in methanol (2 mL). Zinc dust (200 mg) was added to the methanolic solution of compound 13 followed by formic acid (0.2 mL). The reaction was allowed to stir at room temperature for 30 min. The solids were removed by filtration and the filtrate was directly purified by RP-HPLC to obtain compound 15 (72 mg) as a white solid 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) and NaOH (1 M, 0.3 mL) was added. The reaction mixture was stirred at room temperature (22 °C) to obtain compound 16. After 8 h, to the crude of compound 16, hydrochloric acid (1 M, 0.12 mL) was added followed by bromoacetic anhydride (14 mg). The crude reaction mixture was directly purified by RP-HPLC to obtain compound 17 (46 mg) as a white solid, i.e. LD-8 after lyophilization.

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

[0526] 9. Preparation of LD-9

[0527]

[0528] Corresponding ADC structure, Formula 9;

[0529]

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

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

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

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

[0534] To a solution of compound 34 (0.12 g) and MMAE (0.072 g) in DMF (2 mL) was added DIEA (0.018 mL) and the reaction was stirred at room temperature for 24 hours. The mixture was then diluted with ethyl acetate (30 mL) and washed with hydrochloric acid (0.5 M, 30 mL). The organic layer was washed with water (20 mL), dried (Na2S04) and evaporated to dryness under reduced pressure. The residue was re-dissolved in MeOH (3 mL) and MeONa (4.4 M in MeOH, 0.1 mL) was added. The reaction mixture was stirred at room temperature for 2 hours, then neutralised with 1 N hydrochloric acid (0.5 mL). The mixture was directly purified by RP-HPLC to give compound 35 as a white solid (TFA salt, 72 mg).

[0535] Compound 35 (14 mg, TFA salt) was dissolved in acetonitrile / water (6 / 4, 1 mL), saturated aqueous NaHC03(0.1 mL) was added, 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 give LD-9 as a white solid (12 mg).

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

[0537] 10. Preparation of LD-10

[0538]

[0539] The corresponding ADC structure, Formula 10;

[0540]

[0541] Compound 24 (10 mg, synthesis 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, followed by DIEA (0.005 mL). 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) sequentially. The organic layer was concentrated under reduced pressure to give a residue, which was suspended in acetonitrile / water (6 / 4, 2 mL). LiOH (1 N, 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 give compound 25 as a white powder, which was dissolved in acetonitrile / water (6 / 4, 1 mL), saturated aqueous NaHC03(0.06 mL) was added, followed by 2-bromoacetic anhydride (4 mg, Toronto Research Chemicals). The mixture was stirred at room temperature. After 10 minutes, 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, Formula 11;

[0546]

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

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

[0549]

[0550] To a solution of tert-butyl 2-[[tert-butoxycarbonyl-(tert- butyloxycarbonylamino)amino]methyl]prop-2-enoate (A1) (204 mg, 0.55 mmol) in acetic acid (AcOH) (8.0 mL) was added 3,4-dibromo furan-2,5-dione (140 mg, 0.55 mmol). The mixture was stirred under argon atmosphere at reflux for 11 days, then concentrated to 3 mL and purified with preparative HPLC to give 43 mg of A2 as a white solid (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 dry 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 with RP-HPLC to give compound 4 as a white solid (337 mg) after lyophilization.

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

[0555]

[0556] To a solution of compound 6 (31 mg, purchased from MedChemExpress, Cat# HY-100374) in DMF (1 mL) was added compound 4 (12 mg) 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 give compound 8 (17 mg) as a white solid, LD-11 after lyophilization.

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

[0558] 12. Preparation of LD-12

[0559]

[0560] The corresponding ADC structure, Formula 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 salt, 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 stirred at room temperature for 15 minutes. The reaction mixture was directly purified by RP-HPLC to give Compound 3 (110 mg, trifluoroacetate salt) as a white solid after lyophilization.

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

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

[0565] 13. Preparation of LD-13

[0566]

[0567]

[0568] corresponding ADC structure, Formula 13;

[0569]

[0570] Compound 20 (60 mg, purchased from InnoPep) and exatecan mesylate salt (Compound 21, 53 mg, purchased from Advance ChemBlocks, Cat# 10484) were dissolved in anhydrous N,N-dimethylformamide (2 mL), N-ethyldiisopropylamine (0.05 mL) was added followed by O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg). 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 to give Compound 22 (67 mg, trifluoroacetate salt) as a yellow powder after lyophilization.

[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 h, the crude mixture was purified by RP-HPLC to yield compound 23, LD-13 as a yellow solid (42 mg) after lyophilization.

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

[0573] 14. Preparation of LD-14

[0574]

[0575] Corresponding ADC structure, Formula 14;

[0576]

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

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

[0579] 15. Preparation of LD-15

[0580]

[0581]

[0582] Corresponding ADC structure, Formula 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 h and then purified by RP-HPLC to yield compound LD-15 as a white powder (11 mg).

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

[0586] 16. Preparation of LD-16

[0587]

[0588]

[0589] corresponding ADC structure, Formula 16;

[0590]

[0591] To a solution of compound 27 (TFA salt, 10 mg, synthesized according to patent number: WO 2022 / 048883 Al) in DMF (1 mL) was added compound 4 (as described above, 6 mg) followed by DIEA (0.007 mL). The mixture was stirred at room temperature for 2 hours, then purified by RP-HPLC to give 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] corresponding ADC structure, Formula 17;

[0596]

[0597] Compound 6 (0.18 mmol) was dissolved in 1 mL of anhydrous DMF, followed by the addition of DIEA (0.44 mmol) and glutaric anhydride (0.21 mmol) 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 rotary evaporated 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] corresponding ADC structure, Formula 18;

[0602]

[0603] To a solution of compound 35 (14 mg, TFA salt) in DMF (6 / 4, 1 mL) was added compound 36 (synthesized from adipic acid, 20 mg), followed by DIEA (0.005 mL). The mixture was stirred at room temperature. After 30 minutes of reaction, the mixture was purified by RP-HPLC to give 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 of the disclosure was reacted with a drug linker (LD) to prepare an ADC drug, and the DAR value of the prepared ADC was determined.

[0607] 1. The antibody was treated 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). Thus, the partially or completely reduced antibody was reacted with a drug linker or linker reagent with an electrophilic functional group (such as a maleimide or bromoacetyl) to ultimately prepare an ADC.

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

[0609] 2. DAR value determination method of ADC:

[0610] The DAR value of the ADC of the present application was analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). The ADC was separated in the chromatographic column using a MabPac HIC-Butyl analytical column (4.6x100mm, 5μm, item number 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 as the initial condition, using a linear gradient of 85% A and 15% B to 5% A and 95% B for 30 minutes, and using 5% A and 95% B for an additional 5 minutes. The flow rate and temperature were set to 0.5mL / min and 25°C. The ADC drug distribution was detected at 214 and 280nm for DAR value calculation, with the formula as follows:

[0611]

[0612] 3. Preparation of MC linker ADC:

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

[0614] 4. Preparation of 4. Br linker ADC

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

[0616] 5. Preparation of 5. DiBr linker ADC

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

[0618] 6. Preparation of 6. Pfp linker ADC

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

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

[0621] Table 4

[0622]

[0623] 8. Preparation of control antibodies:

[0624] MGA017 antibody reference Mol Cancer Ther (2020) 19(11): 2235-2244 was prepared, the sequence of which 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 from the sequences of No. 51 and No. 53 in the reference EP2703486A1 patent, the sequence of which is as follows:

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

[0631]

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

[0633]

[0634] Test Example 1. Affinity detection of antibodies

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

[0636] Table 5. Affinity of antibodies

[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 for detecting binding of antibodies to antigens

[0641] ELISA method was used to detect the binding activity of 112-59 point mutant antibodies and antigens. B7-H3-His solution (manufacturer: ACRO; item number: B73-H52E2) was diluted with PBS buffer to a concentration of 2 μg / mL, and 100 μL / well was added to the enzyme-labeled plate wells for 4°C coating for 16 h. Then the plate was washed with PBST for 3 times, and the residual liquid was patted dry. 3% BSA blocking solution was added to the enzyme-labeled plate wells at a volume of 200 μL / well, and incubated at 37°C for 2 h. The blocked enzyme-labeled plate was taken out, and the plate was washed with PBST for 3 times. The primary antibody, i.e., the antibody to be tested, was added to the corresponding wells of the coated plate at a volume of 100 μL / well, with the first well at a concentration of 15 μg / mL and 5-fold gradient dilution, and incubated at 37°C for 1.5 h. The plate was washed 5 times, and the secondary antibody Anti-Human IgG (Fc-specific) was added at a dilution of 3000 times. The secondary antibody was added to the enzyme-labeled plate at a volume of 100 μL / well, and incubated at 37°C for 1 h. The plate was washed 5 times and the residual liquid was patted dry. 100 μL of TMB color developing solution was added to each well, and the reaction was carried out at 37°C for 20 min in the dark. The stop solution (2M HCL) was added to the enzyme-labeled plate at a volume of 100 μL / well to stop the reaction, and the OD450 absorbance value was read. The experimental results are shown in the following table and Figure 1

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

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

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

[0645] Test Example 3. Binding of antibodies to B7-H3-expressing cells

[0646] Flow cytometry was used to analyze the binding activity of antibodies to cells. The target cells Calu-6 (Shanghai Cell Bank) were treated, the activity was measured and the cells were counted, and the cells were divided into 2×10 5 ​​488 Goat anti-human IgG (H+L) Vendor: Thermo Cat# A11013) : 1200 rpm centrifugation for 5 min, washing 3 times, secondary antibody dilution 750 times, volume 100-200 μL, blank control PBS, 4°C incubation for 30 min. 1200 rpm centrifugation for 5 min, washing 3 times; machine: 150 μL staining buffer constant volume to round bottom 96-well plate, fluorescence intensity analysis of live cells, select Green fluorescence channel.

[0647] Results are shown in Tables 8-1, 8-2 and Figures 2-3 The binding activity EC50values of the mutated molecules to Calu-6 cells were comparable to 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. Antibody binding experiment to cells expressing B7-H3

[0653] The cell lines selected for this experiment were: 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 carcinoma cells), RH41 (DSMZ ACC 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, MFE-280 cells were cultured in 45% EMEM, 45% RPMI-1640 mixed medium containing 10% fetal bovine serum, 2 mM L-glutamine, 1x insulin-transferrin-selenium medium supplement, in a 37°C, 5% CO2incubator.

[0655] The experimental procedure is as follows:

[0656] Cells in logarithmic growth phase were dissociated with 0.25% trypsin and resuspended in staining buffer (Biolengend) for washing, cell counting, and adjusting to 4.5 x 10 5 cells / 100 μL staining buffer, and added to a 96-well plate, 45 μL per well. 5 μL of human TruStain (BioLegend) receptor blocking agent was added to each well to prevent non-specific binding of antibodies to human tumor cells. The antibody to be tested was diluted, with an initial concentration of 200 nM, and diluted 3-fold, to obtain 11 concentration points including 200 nM and zero. 50 μL of the diluted antibody to be tested was added to each well of the 96-well plate, with a final concentration of 100 nM at the highest point. After mixing, the reaction was carried out at 4°C for 15 minutes. After the reaction, the cells were washed in staining buffer, and then the PE-labeled constant region (Fc)-specific antibody (rabbit anti-human IgG PE conjugate, BioLegend, 410707) was suspended in 5 μL / 2 x 10 5 cells / 100 μL staining buffer, and reacted at 4°C for 15 minutes. After the cell reaction, the cells were washed in staining buffer, and the cells were resuspended in 100 μL of staining buffer. The Novocyte 3000 (Agilent) device was used to analyze the reading of single cells in the PE channel. The antibody protein concentration was used as the abscissa, and the corresponding PE channel reading was used as the ordinate. The dose-response curve was plotted using the Sigmoidal, 4PL four-parameter equation, and the EC 50 value was generated after analysis. The equation is:

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

[0658] MFI fold = highest PE reading of the antibody-treated experimental group / PE reading of the group without antibody; the FACS binding activity results of the anti-hB7-H3 antibody test substance with 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, and the binding ability is higher. In addition, on the same cell line, the anti-B7-H3 antibody 112-59 in the present application can recognize and mark a higher total amount of antigen than the control antibodies MGA017 and 112cDa, which is manifested as a significant increase in the MFI ratio.

[0662] Test Example 5. Antibody endocytosis experiment of the antibody and B7-H3 expressing cells

[0663] The cell lines and culture methods selected for the experiment are consistent with those in Test Example 4, and the experimental process is as follows:

[0664] The cells in the logarithmic growth phase were dissociated with 0.25% trypsin, resuspended in cell culture medium, and centrifuged at 200g for 5min. The precipitated cells were resuspended in cell culture medium and counted. Prepare a cell suspension with a concentration of 5000 cells / 50μL of culture medium, and add 50μL of the prepared cell suspension to each well of a 96-well plate. The 96-well plate containing the cells was cultured overnight in a 37°C incubator to allow the cells to grow adherently. 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 the lysosome after endocytosis of the antibody, in an environment with a pH of 4.5-5.5. The specific method for fluorescently labeling the antibody is as follows: Mix the antibody to be tested with the antibody labeling reagent with a molecular weight of 1:3 in the cell culture medium, and incubate at 37°C for 15min after mixing well. Add 50μl of the labeled antibody to each well of the 96-well plate cultured overnight, with a final concentration of 3μg / mL. Place the 96-well plate in the Incucyte machine Select the adherent cell mode, and take pictures every 1-2 hours within 24 hours using a 20x objective lens in the orange fluorescence and visible light channels, and take pictures at three different positions in each well.

[0665] After the experiment, the The integrated image analysis tool uses the Cell-by-cell method to analyze the pictures, to cut and measure the cell background signal and the fluorescent signal, and to minimize the influence of background fluorescence. After the cell-by-cell analysis is completed, the software gives the average cell fluorescence intensity cumulative value that reflects the enhancement of the fluorescent signal: OCU x pm2. The Classification function is then used to analyze the endocytosis-positive cell ratio that represents the increase in fluorescent area: cells with fluorescent signal positive / all cells %. The threshold value for background fluorescence positive is selected as OCU = 0.05 during 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 anti-hB7-H3 antibody test substances on different cell lines are shown in Table 10.

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

[0667]

[0668] - indicates not tested.

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

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

[0671] In this experiment, the anti-proliferation effect of the drug was evaluated using CellTiterGlo2 (Promega) reagent. The selected cell lines and culture methods in the experiment were consistent with those in Test Example 4. The experimental process was as follows:

[0672] Cells were seeded at a density of 2 x 10 3 - 5 x 10 3 cells per well in a 96-well plate, 50 μL / well, and cultured for 24 h. Then different concentrations of ADC molecules diluted in culture medium were added, 100 μL / well, with duplicate wells for each concentration, and solvent controls and cell-free medium wells of the corresponding concentration were set. After incubation at 37°C in a 5% CO2 incubator for 96 h (Calu-6) or 144 h (HCC-1954, OVCAR3, MFE-280), 100 μL of CellTiterGlo2 was added to each well, and mixed on an orbital shaker at room temperature for 15 min. The luminescence value was determined, and the IC 50Values (nM). Results are shown in the table below.

[0673] Table 11-1. IC of different antibody conjugate ADCs on Calu-6 cell proliferation inhibition 50 Values (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 of different antibody conjugate ADCs on HCC-1954 cell proliferation inhibition 50 Values (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 of different antibody conjugate ADCs on OVCAR3 cell proliferation inhibition 50 Values (nM)

[0678]

[0679] Table 11-4. IC of different antibody conjugate ADCs on cell proliferation inhibition 50 Values (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 effect on several different B7-H3 expression tumor cells. It is shown that the ADC molecules described in the present application can specifically kill positive cells through B7-H3 mediated endocytosis. And there is obvious inhibition on cancer cell lines from breast cancer, ovarian cancer, lung cancer, endometrial cancer and melanoma, respectively, which shows that the obtained ADC has good inhibition effect on various types of solid tumors.

[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 x 10 6 cells per mouse of human B7-H3 expressing breast cancer cell line HCC1954 were transplanted subcutaneously into female severe immunodeficient NSG mice (purchased from Jackson Laboratories). After transplantation, when the tumor size reached an average of 150 mm 3 (120 mm 3 - 180 mm 3At day 0 (interval), mice were divided into groups and administered once intravenously 1.0 mg / kg, 3.0 mg / kg, or 10.0 mg / kg ADC-6, ADC-7, or ADC-17 prepared in Example 3. In the control group, mice were administered 4 mL / kg PBS intravenously. Tumor size and body weight were measured 28 days later.

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

[0686] T d and C d To measure the mean tumor volume of animals in the treatment and control groups on the first day of drug administration, T0 and C0 represent the mean tumor volumes of animals in the treatment and control groups on the first day of drug administration. Results are shown below. Figure 4 And Table 12.

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

[0688]

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

[0690] 2. ADC efficacy testing in a mouse model transplanted with the HCC1806 breast cancer cell line

[0691] 5×10 6 One cell / human B7-H3-expressing breast cancer cell line, HCC1806, was transplanted subcutaneously into female immunodeficient Athymic Nude mice (purchased from Charles River Laboratories). Following transplantation, when the tumor size reached an average of 150 mm... 3 (120mm 3 -180mm 3 At day 0, mice were divided into groups and administered once intravenously 1.0, 3.0, or 10.0 mg / kg ADC-6, ADC-7, or ADC-17 prepared in Example 3. In the control group, mice were administered 4 mL / kg PBS intravenously. Tumor size and body weight were measured 14 days later.

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

[0693] T d and Cd To measure the average tumor volume of the treatment group and the control group animals on the day, T0 and C0 are the average tumor volumes of the treatment group and the control group animals on the first day of administration. The results are shown in Table 1 and Figure 1. Figure 5 and Table 13.

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

[0695]

[0696] The results show that the ADC-17 of the present application can significantly inhibit the growth of HCC1806 xenografts in mice.

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

[0698] 1 x 10 7 The lung cancer cell line Calu-6 expressing human B7-H3 was transplanted into the subcutaneous of female immunodeficient CD1 Nude mice (purchased from Charles River Laboratories) at 1 x 10 3 (120mm 3 -180mm 3 interval) (day 0), the mice were grouped, and 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. Twenty-four 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)] x 100%, wherein:

[0700] T d and C d To measure the average tumor volume of the treatment group and the control group animals on the day, T0 and C0 are the average tumor volumes of the treatment group and the control group animals on the first day of administration. The results are shown in Table 1 and Figure 1. Figure 6 and Table 14.

[0701] Table 14. Inhibition rate of ADC molecules on Calu-6 lung cancer xenografts in CD1 Nude female mice (Day 24)

[0702]

[0703] The results show that the ADC-17 of the present application can significantly inhibit the growth of Calu-6 xenografts in mice.

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

[0705] 5×10 6 One cell / human B7-H3-expressing ovarian cancer cell line, OVCAR3, was transplanted subcutaneously into female severely immunodeficient NSG mice (purchased from Charles River Laboratories). Following transplantation, when the tumor size reached an average of 150 mm... 3 (120mm 3 -180mm 3 At day 0 (interval), mice were divided into groups and administered once intravenously 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. In the control group, mice were administered 4 mL / kg PBS intravenously. Tumor size and body weight were measured 21 days later.

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

[0707] T d and C d To measure the mean tumor volume of animals in the treatment and control groups on the first day of drug administration, T0 and C0 represent the mean tumor volumes of animals in the treatment and control groups on the first day of drug administration. Results are shown below. Figure 7 And Table 15.

[0708] Table 15. Inhibition rate of ADC molecules on OVCAR3 ovarian cancer xenografts in NSG female mice (Day 21)

[0709]

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

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

[0712] 2×10 6 One cell / human B7-H3-expressing prostate cancer cell line, PC-3, was transplanted subcutaneously into female immunodeficient Athymic Nude mice (purchased from Charles River Laboratories). Following transplantation, when the tumor size reached an average of 150 mm... 3 (120mm 3 -180mm3 On the day (Day 0) when the interval (TGI) was measured, the mice were grouped, and 3.0 mg / kg of ADC-6, ADC-7, 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. Twenty-four days thereafter, the tumor size transplanted into the mice and the body weight of the mice were measured.

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

[0714] T d and C d T0and C0are the average tumor volumes of the treatment group and the control group animals on the day of measurement, and T1and C1are the average tumor volumes of the treatment group and the control group animals on the day of measurement.

[0715] The results are shown in Figure 8 and Table 16.

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

[0717]

[0718] The results show that the ADC-17 of the present application can significantly inhibit the growth of PC-3 transplanted tumor in mice.

Claims

1. An anti-B7-H3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region; 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 includes 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 includes 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 includes 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 includes LCDR1 shown in any of the sequences SEQ ID NO: 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 includes 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 includes LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in any of the sequences SEQ ID NO: 13, 14, 15 and 16; The amino acid sequences of HCDR and LCDR are determined according to the Kabat numbering system.

2. An anti-B7-H3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region; wherein: The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 52, HCDR2 shown in SEQ ID NO: 53, and HCDR3 shown in SEQ ID NO: 5; and The light chain variable region includes LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8; The amino acid sequences of the HCDR and LCDR are determined according to the Chothia numbering system.

3. An anti-B7-H3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region; wherein: The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 45, HCDR2 shown in SEQ ID NO: 46, and HCDR3 shown in SEQ ID NO: 47; and The light chain variable region includes LCDR1 shown in SEQ ID NO: 48, LCDR2 shown in SEQ ID NO: 49, and LCDR3 shown in SEQ ID NO: 8; The amino acid sequences of the HCDR and the LCDR are determined according to the IMGT numbering system.

4. The anti-B7-H3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the anti-B7-H3 antibody or its antigen-binding fragment is a rabbit antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a fully human antibody or its antigen-binding fragment, or a chimeric antibody or its antigen-binding fragment.

5. The anti-B7-H3 antibody or its antigen-binding fragment 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 contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 10 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 10; 2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 1 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 2 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 2; 3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 27 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 27; 4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 28 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 28; 5) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 29 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 29; 6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 30 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 30; 7) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 31 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 31; 8) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 32 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 32; 9) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 33 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 33; 10) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 34 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 34; 11) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 35 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 35; 12) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 36 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 36; 13) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 37 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 37; and 14) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 38 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO:

38.

6. The anti-B7-H3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from any one of: Fab, scFv, Fv, Fab', F(ab′)2 and scFab.

7. The anti-B7-H3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, further comprising a heavy chain constant region and a light chain constant region; The heavy chain constant region is selected from human IgG1, human IgG2, human IgG3, and human IgG4. The light chain constant region is selected from the λ and κ light chain constant regions.

8. The anti-B7-H3 antibody or its antigen-binding fragment according to claim 7, wherein 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.

9. The anti-B7-H3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein: The heavy chain contains an amino acid sequence as shown in SEQ ID NO: 39 or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

39. The light chain contains an amino acid sequence as shown in SEQ ID NO: 40 or contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO:

40.

10. An antibody-drug conjugate having a structure selected from any of the following: Formula 1; Formula 6; Formula 7; Formula 11; Equation 12; Formula 16; in: Ab is an anti-B7-H3 antibody, which contains the heavy chain shown in SEQ ID NO: 39 and the light chain shown in SEQ ID NO: 40; n is 4.

11. A pharmaceutical composition comprising: 1) A therapeutically effective amount of the antibody-drug conjugate of claim 10, and 2) One or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

12. An isolated nucleic acid that encodes: The anti-B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 to 9.

13. An expression vehicle that expresses: The anti-B7-H3 antibody or its antigen-binding fragment as described in any one of claims 1 to 9.

14. A host cell comprising: The isolated nucleic acid as described in claim 12, or The expression vector according to claim 13.

15. Use of the antibody-drug conjugate of claim 10 in the preparation of a medicament for the treatment of cancer; The cancers mentioned are selected from: prostate cancer, melanoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, and endometrial cancer.

16. Use of the antibody-drug conjugate of claim 10 in the preparation of a medicament, wherein the medicament is used to inhibit the growth or proliferation of cancer cells; wherein the cancer is selected from: prostate cancer, melanoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, and endometrial cancer.

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