Antibody-conjugated B7-H3 and its use

Abstract

The present application provides isolated monoclonal antibodies or antigen-binding portions thereof that specifically bind to B7-H3. Nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, and expression vectors, host cells, and methods for expressing the antibodies or antigen-binding portions thereof, are also provided. The present application further provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibodies or antigen-binding portions thereof, and methods of treatment using the disclosed antibodies or antigen-binding portions thereof.

Description

[Technical Field]

[0001] This application relates to an antibody that specifically binds to human B7-H3, and to the preparation and use of the same, in particular to its use in the treatment of B7-H3-related diseases such as cancer. [Background technology]

[0002] T cell proliferation and activation depend on dual signaling. The interaction between the T cell receptor and peptides presented by MHC on antigen-presenting cells or tumor cells provides the first signal, which is essential for T cell activation but does not induce cell proliferation or cytokine release. The second signal, namely the co-stimulatory signal, determines whether the T cell undergoes activation / proliferation, unresponsiveness, or apoptosis. The B7-CD28 co-stimulatory signaling pathway plays a crucial role in regulating T cell activation and suppression, where the CD-28 molecule is expressed on T cells, while the B7 molecule is generally present on activated antigen-presenting cells. To date, more than 10 B7 proteins have been discovered and can be divided into three groups according to the signaling and function they transduce during T cell activation: i) co-stimulatory, ii) co-inhibitory, and iii) co-stimulatory / inhibitory.

[0003] B7-H3, also known as CD276, is a co-stimulatory / inhibitory molecule of the B7 family. When it was first discovered, it was considered a co-stimulatory molecule that promotes T cell activation and IFN-γ production. However, recent studies suggest that B7-H3 plays an inhibitory role in adaptive immunity, for example, by suppressing T cell activation / proliferation or the release of effector cytokines (mainly IFN-γ and IL-2), and inhibiting NK cell activity (Prasad DVR. et al., (2004) J Immunol 173(4):2500-2506; Castriconi R. et al., (2004) Proc Natl Acad Sci USA 101(34):12640-12645). The seemingly contradictory regulatory activities of B7-H3 may be mediated by different receptors, but there has been little research on B7-H3 receptors.

[0004] B7-H3 is a type I transmembrane protein that shares 20%–27% homology with other B7 family members. It is encoded by a gene located on chromosome 15q24 and contains an extracellular domain, a transmembrane domain, and a short intracellular domain. The intracellular domain is very short and is not known to have any signaling motif. In addition, human B7-H3 proteins may contain one or two pairs of extracellular domains due to exon duplication, resulting in two subtypes, namely 2IgB7-H3 and 4IgB7-H3. The former subtype contains one pair of immunoglobulin variable region (IgV)-like and immunoglobulin constant region (IgC)-like extracellular domains, while the latter contains two pairs of identical IgV-like and IgC-like extracellular domains and is the main subtype found in human cells.

[0005] B7-H3 mRNA is widely expressed in normal tissues, while the B7-H3 protein molecule is low-expressed in only a few tissues (Flem-Karlsen K. et al., (2018) Trends Cancer 4(6):401-404). For example, weak staining was observed in the cytoplasm of salivary gland acinar cells, gastric epithelial cells, and adrenal cells, as well as in the basement membrane of the stomach, gallbladder, prostate, cervix, and endometrium. The B7-H3 protein is highly expressed in several malignancies. Various studies have shown that the B7-H3 molecule is expressed in 33.0% of patients with renal cell carcinoma and in 91.8% of patients with hepatocellular carcinoma. The proportion of patients with B7-H3 expression is very low in renal cell carcinoma and systemic hematological malignancies (33.0% and 36.7%, p<0.001, respectively), while at least 52.3% of patients with other solid tumors have B7-H3 expression. Among gastrointestinal cancers, the proportion of patients with B7-H3 expression is highest in hepatocellular carcinoma (91.8%) and lowest in gastric cancer (58.0%). Among malignant genitourinary cancers, the proportion of patients with B7-H3 expression is lowest in renal cell carcinoma and highest in ovarian cancer (88.3%). Of all cancers, 59.5% (15877 / 26703) of patients are B7-H3 positive. On the other hand, pancreatic cancer patients with high B7-H3 expression have been found to have a better postoperative prognosis than patients with low B7-H3 expression (Loos M. et al., (2009) BMC Cancer 9: 463). On the other hand, B7-H3 is thought to contribute to an immunosuppressive tumor microenvironment, for example, by promoting the evasion of cancer stem cells from immune surveillance in squamous cell carcinoma (Wang C. et al., (2021) Cell Stem Cell 28(9): 1597-1613; Jin MZ, Jin WL., (2020) Signal Transduct Target Ther 5(1): 166). B7-H3 also promotes cancer development and progression through multiple non-immune pathways.For example, it induces VEGFA expression in colorectal cancer cells, promotes epithelial-mesenchymal transition in colorectal cancer, and induces aerobic glycolysis to supply energy to the tumor (Wang RQ et al., (2020) Cell Death Dis (2020) 11(1):55; Jiang B. et al., (2016) Oncotarget 7(22): 31755-31771; Lim S. et al., (2016) Cancer Res 76(8): 2231-2242).

[0006] The in vivo expression pattern of B7-H3 makes it a promising target for cancer treatment. In particular, high B7-H3 expression is also found in stromal cells (fibroblasts) in the tumor microenvironment and tumor-associated vasculature (TAV), so B7-H3 targeted therapy may even eliminate cancer cells with low B7-H3 expression.

[0007] Currently, various forms of B7-H3 targeted therapies are in clinical trials. Enobrituzumab is the first monoclonal antibody to target the B7-H3 protein with an optimized Fc region, exhibiting antitumor activity through both antibody-dependent cell-mediated cytotoxicity and T-cell immune enhancement. According to data published in the 2018 annual report of the Society for Immunotherapy of Cancer (SITC), the objective response rate (ORR) of enobrituzumab in combination with an anti-PD-L1 antibody was 33.3% in patients with squamous cell carcinoma of head and neck (SCCHN) in clinical trials, and an ORR of 35.7% was achieved in patients with non-small cell lung cancer (NSCLC) with low PD-L1 expression (<1%). In particular, patients who had previously received anti-PD-1 / L1 antibody therapy responded to such therapy (partial remission: PR), and disease was suppressed in the same proportion of patients compared to patients who were first candidates for anti-PD-1 / L1 therapy. 131 The monoclonal antibody 8H9 (omblutamab, Y-mAbs) labeled with I has been used via intraperitoneal injection to treat patients with metastatic central nervous system neuroblastoma (NCT00089245) and fibrous small cell tumor (NCT01099644) and has shown good tolerability. Similarly, 124 A monoclonal antibody 8H9 labeled with I has been tested in diffuse endogenous pontine glioma (NCT01502917) and has shown good tolerability. Several other B7-H3 targeted therapies, including small molecule drugs, monospecific antibodies, bispecific antibodies, antibody drug conjugates, CAR-T and similar therapies, have been tested in clinical trials for solid tumors such as melanoma, prostate cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, central nervous system cell tumors, glioblastoma, fibroplastic small cell tumors, medulloblastoma and similar therapies (Zhou WT, Jin WL. (2021) Front Immunol 12: 701006).

[0008] However, despite the broad antitumor effects of B7-H3 targeted therapies, these therapies are still in the early stages of clinical trials and demonstrate clinical efficacy in only a small fraction of patients. Furthermore, it should be noted that while these therapies may initially show very good therapeutic effects in some patients, resistance may develop later. Identifying the causes of non-responsiveness and progression of resistance in specific patients, and then developing new therapeutic means and methods, is crucial for further cancer treatment. There remains a great demand in the field of cancer treatment for a wider range of anti-B7-H3 antibodies with different properties and efficacy characteristics, such as anti-B7-H3 antibodies with different binding affinities, blocking activities, and even different binding epitopes.

[0009] Any citation or specification of any document in this application does not constitute an endorsement that such document is available as prior art for the present invention. [Overview of the Initiative]

[0010] This application aims to provide an antibody that offers equivalent or higher human / monkey B7-H3 binding affinity / activity compared to conventional antibodies such as enobrituzumab, and equivalent or higher B7-H3 binding activity. + This invention provides isolated monoclonal antibodies, such as human or chimeric monoclonal antibodies, that bind to B7-H3 (e.g., human B7-H3, monkey B7-H3, or mouse B7-H3) and have cell-binding activity, equivalent or higher immune cell (e.g., NK cell) activation activity, equivalent or higher ability to induce regression-dependent cell-mediated cytotoxicity (ADCC), and equivalent or higher in vivo anticancer activity. The monoclonal antibodies of this application bind to B7-H3 + It can be taken up by cells, and its binding specificity to human B7-H3 is superior to that of conventional antibodies such as enobrituzumab.

[0011] The antibody of this invention may have multiple applications, including the treatment of B7-H3 related diseases such as cancer.

[0012] Therefore, in one aspect, the disclosure provides an isolated monoclonal antibody (e.g., a human antibody) or an antigen-binding moiety thereof that binds to B7-H3, i) a heavy chain variable region, where the heavy chain variable region comprises a VH CDR1 region, a VH CDR2 region and a VH CDR3 region, where VH CDR1 region, VH CDR2 region and VH The CDR3 region may include (1) the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; (2) SEQ ID NOs: 1, 7, and 8, respectively; (3) SEQ ID NOs: 12, 13, and 14, respectively; (4) SEQ ID NOs: 18, 19, and 20, respectively; or (5) SEQ ID NOs: 23, 24, and 25, respectively, or amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences; and / or ii) a light chain variable region, where the light chain variable region includes a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, where VL CDR1 region, VL CDR2 region, and VL The CDR3 region may include (1) the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; (2) SEQ ID NOs: 9, 10, and 11, respectively; (3) SEQ ID NOs: 15, 16, and 17, respectively; (4) SEQ ID NOs: 9, 21, and 22, respectively; or (5) the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively, or amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences.

[0013] The isolated monoclonal antibody of this application or its antigen-binding moiety may include a heavy chain variable region and a light chain variable region, where VH CDR1 region, VH CDR2 region, VH CDR3 region, VL CDR1 region, VL CDR2 region, and VL The CDR3 region may contain the amino acid sequences of (1) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (2) SEQ ID NOs: 1, 7, 8, 9, 10, and 11, respectively; (3) SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively; (4) SEQ ID NOs: 18, 19, 20, 9, 21, and 22, respectively; or (5) SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively, or amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences.

[0014] The isolated monoclonal antibody of this disclosure or the heavy chain variable region of its antigen-binding moiety may contain an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 29, 31, 33, 35, or 37.

[0015] The light chain variable region of the isolated monoclonal antibody or its antigen-binding moiety of the present disclosure may contain an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 30, 32, 34, 36, or 38.

[0016] The heavy chain variable region and light chain variable region of the isolated monoclonal antibody of the present invention or its antigen-binding portion may include the amino acid sequences of (1) SEQ ID NOs: 29 and 30, respectively; (2) SEQ ID NOs: 31 and 32, respectively; (3) SEQ ID NOs: 33 and 34, respectively; (4) SEQ ID NOs: 35 and 36, respectively; or (5) SEQ ID NOs: 37 and 38, respectively, or amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences.

[0017] In another embodiment, the disclosure provides an isolated monoclonal antibody (e.g., a human antibody) or an antigen-binding moiety thereof that binds to B7-H3, i) a heavy chain variable region, wherein the heavy chain variable region comprises a VH CDR1 region, a VH CDR2 region and a VH CDR3 region, wherein the VH CDR1 region, VH CDR2 region and VH CDR3 region have the identity of the VH CDR1 region, VH CDR2 region and VH CDR3 region of a specified VH sequence, and / or ii) a light chain variable region, wherein the light chain variable region comprises a VL CDR1 region, a VL CDR2 region and a VL CDR3 region, wherein the VL CDR1 region, VL CDR2 region and VL CDR3 region have the identity of the VL CDR1 region, VL CDR2 region and VL CDR3 region of a specified VL sequence. Having identity with the CDR3 region; where the specified VH sequence and specified VL sequence include (1) the specified VH sequence and specified VL sequence described in sequence numbers 29 and 30, respectively; (2) the specified VH sequence and specified VL sequence described in sequence numbers 31 and 32, respectively; (3) the specified VH sequence and specified VL sequence described in sequence numbers 33 and 34, respectively; (4) the specified VH sequence and specified VL sequence described in sequence numbers 35 and 36, respectively; and (5) one of the specified VH sequence and specified VL sequence described in sequence numbers 37 and 38, respectively.

[0018] In one embodiment, the isolated monoclonal antibody of the present disclosure or its antigen-binding moiety may include a heavy chain constant region and / or a light chain constant region. The heavy chain constant region may be an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a functional fragment thereof. Furthermore, the heavy chain constant region may be, for example, B7-H3 +To enhance ADCC against cells, it may be manipulated to have enhanced FcR binding activity. For example, the heavy chain constant region may be introduced with SI mutations (S239D and I332E mutations according to EU numbering) or VLPLL mutations (L235V, F243L, R292P, Y300L, and P396L according to EU numbering). In one embodiment, the heavy chain constant region may be a wild-type human IgG1 constant region containing the amino acid sequence of SEQ ID NO: 43. In one embodiment, the heavy chain constant region may be a human IgG1 constant region containing the amino acid sequence of SEQ ID NO: 44 (L235V, F243L, R292P, Y300L, and P396L according to EU numbering). The light chain constant region may be a κ constant region, for example, a human κ constant region containing the amino acid sequence of SEQ ID NO: 45, or a functional fragment thereof. The N-terminus of the heavy chain constant region is ligated to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is ligated to the C-terminus of the light chain variable region.

[0019] The antibody or its antigen-binding moiety of the present invention can be expressed by recombinant means, for example, in specific mammalian cell lines for afucosylation. Cell lines for expressing afucosylated antibodies or their antigen-binding moieties include, but are not limited to, Slc35C1 knockout cell lines, FUT8 knockout cell lines, Lec13 cell lines (mutant CHO cell lines), rat myeloma cell line YB2 / 0, cell lines containing small interfering RNAs specific to the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II.

[0020] In certain embodiments, the antibodies of this disclosure may comprise two heavy chains and two light chains, or consist of two heavy chains and two light chains, where each heavy chain comprises the heavy chain constant region, heavy chain variable region and / or CDR sequence mentioned above, and each light chain comprises the light chain constant region, light chain variable region and / or CDR sequence mentioned above. In certain embodiments, the antibodies of this disclosure may consist of a single-chain variable fragment or an antibody fragment such as a Fab or F(ab')2 fragment.

[0021] The present application further provides an immune complex comprising the antibody or its antigen-binding moiety, wherein the antibody or its antigen-binding moiety is linked to a therapeutic agent such as a cytotoxin or an anticancer agent. The present disclosure further provides a bispecific molecule comprising the antibody or its antigen-binding moiety, wherein the antibody or its antigen-binding moiety is linked to a second functional moiety (e.g., a second antibody), wherein the second functional moiety has a different binding specificity from the antibody or its antigen-binding moiety. In other words, the antibody or its antigen-binding moiety of the present disclosure may be part of a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). Immune cells comprising the above CAR and / or TCR, including T cells, NK cells, or the like, are also provided. The antibody or its antigen-binding moiety of the present disclosure may also be encoded by or harbored by an oncolytic virus.

[0022] The present application further includes a nucleic acid molecule encoding an antibody or an antigen-binding portion thereof, or a bispecific molecule, an expression vector containing said nucleic acid, and a host cell containing said expression vector.

[0023] In one embodiment, the expression vector of the Disclosure comprises one or more nucleic acids mentioned above. In another embodiment, the expression vector of the Disclosure comprises two nucleic acids mentioned above, each encoding VH and VL, respectively, which can bind together to B7-H3. In another embodiment, the Disclosure provides a pair of vectors, each vector comprising one of the nucleic acids mentioned above, and the pair of vectors together encoding VH and VL, which can bind to B7-H3.

[0024] The Disclosure further provides a method for preparing the antibody or its antigen-binding moiety or bispecific molecule using host cells containing the expression vector described above, the method comprising (i) expressing the antibody, its antigen-binding moiety or bispecific molecule in host cells, and (ii) isolating the antibody, its antigen-binding moiety or bispecific molecule from host cells or a culture thereof.

[0025] The present application further provides pharmaceutical compositions and pharmaceutically acceptable carriers comprising the disclosed antibody or its antigen-binding moiety, immune complex, bispecific molecule, immune cell, oncolytic virus, nucleic acid molecule, expression vector, or host cell.

[0026] In one aspect, the present application provides a method for treating or alleviating a B7-H3 related disease in a subject, the method comprising the step of administering a therapeutically effective amount of the disclosed pharmaceutical composition to the subject. The B7-H3 related disease may be cancer or an autoimmune disease. Cancer may be solid tumors, including, but not limited to, breast cancer, melanoma, prostate cancer, head and neck squamous cell carcinoma, lung cancer, non-small cell lung cancer, central nervous system neuroblastoma, glioblastoma, fibroplastic small cell tumor, diffuse pontine glioma, medulloblastoma, pancreatic cancer, liver cancer, colorectal cancer, non-Hodgkin lymphoma, esophageal cancer, ovarian cancer, bladder cancer, small cell carcinoma, endometrial cancer, kidney cancer, gastric cancer, acute myeloid leukemia, hepatocellular carcinoma, hypopharyngeal squamous cell carcinoma, and urothelial cell carcinoma. Autoimmune diseases may include, but are not limited to, asthma. In certain embodiments, the antibody or its antigen-binding moiety of the Disclosure may be administered together with at least one additional anti-cancer antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-4-1BB antibody, and the like. In other embodiments, the antibody or its antigen-binding moiety of the Disclosure may be administered together with a cytokine (e.g., IL-2 and / or IL-21) or a costimulatory antibody (e.g., an anti-CD137 antibody and / or an anti-GITR antibody). In other embodiments, the antibody or its antigen-binding moiety of the Disclosure may be administered together with a chemotherapeutic agent, which may be a cytotoxic agent. The antibody of the Disclosure may be, for example, a human antibody.

[0027] In one embodiment, the Disclosure provides a method for enhancing an immune response in a subject, the method comprising administering an effective amount of the antibody or its antigen-binding moiety to a subject in need thereof. The enhancement of the immune response includes the activation of immune cells, including NK cells and similar.

[0028] All documents cited or referenced herein (including, but not limited to, all documentary documents, patents, and patent application publications cited herein) ("Documents Cited herein"), all documents cited or referenced in the Documents Cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets relating to any product mentioned herein or in any document incorporated by reference herein, may be incorporated herein by reference and adopted in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated so as to be incorporated by reference. Any Genbank sequences mentioned herein are incorporated by reference.

[0029] In this disclosure, and in particular in the claims, terms such as “include,” “equip,” and similar terms may have the meanings given in Chinese patent law; terms such as “essentially constitute” may have the meanings given to them in Chinese patent law, and it should be noted that, for example, they permit elements not explicitly enumerated but exclude elements found in the prior art or that affect the fundamental or novel features of the present invention.

[0030] Other features and advantages of this disclosure are evident from the following detailed description and examples, but should not be construed as limitations. All references, GenBank entries, patents, and patent application publications cited throughout this application are expressly incorporated herein by reference. [Brief explanation of the drawing]

[0031] The following detailed description is given as an example, but is not intended to limit the invention exclusively to the specific embodiments described, and can be best understood in conjunction with the accompanying drawings.

[0032] [Figure 1]This shows the binding activity of a fully human anti-B7-H3 antibody against B7-H3+ tumor cells, including A549 cells (A), NCI-H520 cells (B), HepG2 cells (C), SK-OV-3 cells (D), and MCF-7 cells (E).

[0033] [Figure 2] This shows the binding activity of a fully human anti-B7-H3 antibody to B7-H3 tumor cells, including Jarcut cells (A), Daudi cells (B), and Large cells (C).

[0034] [Figure 3] This study demonstrates the binding activity of a fully human anti-B7-H3 antibody to B7-H3+ normal cells, including human prostate smooth muscle cells (A), human dermal fibroblasts (B), human aortic smooth muscle cells (C), and human dendritic cells (D).

[0035] [Figure 4] This study shows the binding activity of fully human anti-B7-H3 antibodies, namely 6B5-WT, 6B5-AF, and 6B5-Mut, with or without Fc manipulation, to HUVEC cells (A) and NCI-H520 cells (C), and the binding activity of 10D7-WT, 10D7-AF, and 10D7-Mut to HUVEC cells (B) and NCI-H520 cells (D).

[0036] [Figure 5] (A) The ability of fully human anti-B7-H3 antibodies with or without Fc manipulation, namely 10D7-WT, 10D7-AF, and 10D7-Mut, to induce NCI-H520 cell killing by NK cells and (C) to activate NK cells, and (B) the ability of 6B5-WT, 6B5-AF, and 6B5-Mut to induce NCI-H520 cell killing by NK cells and (D) to activate NK cells.

[0037] [Figure 6]This paper demonstrates the ability of fully human anti-B7-H3 antibodies, namely 10D7-WT, 10D7-AF, and 10D7-Mut, with or without Fc manipulation, to induce NCI-H520 cell killing by PBMCs (A), and the ability of 6B5-WT, 6B5-AF, and 6B5-Mut to induce NCI-H520 cell killing by PBMCs (B).

[0038] [Figure 7] This shows the ability of afcosylated fully human anti-B7-H3 antibodies, 10D7-AF and 6B5-AF, to induce HUVEC cell killing by NK cells (A) or PBMCs (B), and the release of IFN-γ by PBMCs during HUVEC cell killing (C).

[0039] [Figure 8] This demonstrates the ability of afcosylated fully human anti-B7-H3 antibodies, 10D7-AF and 6B5-AF, to induce NK cell-mediated killing of mature dendritic cells.

[0040] [Figure 9] This shows the uptake of fully human anti-B7-H3 antibody by NCI-H520 cells (A) and A549 cells (B).

[0041] [Figure 10] This is a tree diagram derived from average linkage clustering of B7-H3 epitopes bound by fully human anti-B7-H3 antibodies.

[0042] [Figure 11] This shows the binding affinity of the 6B5 antibody to B7 family members as determined by SPR.

[0043] [Figure 12] This shows changes in tumor size in mice with the human B7-H3 gene treated with fully human anti-B7-H3 antibodies 10F5-Mut(A), 6B5-Mut, and 15F11-Mut(B).

[0044] [Figure 13] Representative tissue staining results for the afucosylated fully human anti-B7-H3 antibody 6B5-AF are shown. [Modes for carrying out the invention]

[0045] To ensure that this disclosure is easier to understand, certain terms are defined first. Additional definitions are provided throughout the detailed description.

[0046] The term "B7-H3" refers to B7 homolog 3, also known as differentiation antigen group 276 (CD276), which can be divided into two subtypes, namely 4Ig and 2Ig, according to the extracellular domain structure. The term includes variants, homologs, orthologs, and paralogs. For example, an antibody specific to human B7-H3 may, in certain cases, cross-react with B7-H3 proteins from other species, such as monkeys. In other embodiments, an antibody specific to human B7-H3 protein may be completely specific to human B7-H3 protein and not cross-react with other species or other types, or it may cross-react with B7-H3 proteins from certain other species, but not with all other species.

[0047] The term "human B7-H3" refers to B7-H3 proteins having an amino acid sequence from humans, such as the B7-H3(4Ig) protein having the amino acid sequence with NCBI accession number NP_001019907.1 (Kanayama T. et al., (2021) Sci Rep 11(1): 18802), or the B7-H3(2Ig) protein having the amino acid sequence with NCBI accession number NP_001316557.1 (Fu M. et al., (2021) J Transl Med 19(1):404). The term "monkey B7-H3" refers to B7-H3 proteins having an amino acid sequence from monkeys, such as B7-H3 having the amino acid sequence with NCBI accession number XP_015308534.1. The term "mouse B7-H3" refers to B7-H3 proteins having an amino acid sequence from mice, such as the B7-H3 protein having the amino acid sequence with NCBI accession number NP_598744.1 (Cheng N. et al., (2021) Biochem Pharmacol 183: 114298).

[0048] As used herein, the term "antibody" is intended to include either a full-length antibody of IgG, IgA, IgD, IgE, and IgM, and an antigen-binding fragment thereof (i.e., the antigen-binding portion). A full-length antibody is a glycoprotein that may include at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain may include a heavy-chain variable region (abbreviated herein as V H and a heavy-chain constant region. The heavy-chain constant region may include three domains, C H1 C H2 and C H3 Each light chain may include a light-chain variable region (abbreviated herein as V L and a light-chain constant region. The light-chain constant region may include one domain, C L V H and V LThe region can be further subdivided into hyper-variable regions called complementary determination regions (CDRs), and more conservative regions called framework regions (FRs) are scattered throughout. H and V L The antibody consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the conventional complement system. A "functional fragment" of the antibody constant region refers to a fragment of the constant region that retains a specific required function, such as a fragment of the heavy chain constant region that retains FcR / complement system component binding activity.

[0049] The term “antigen-binding portion” (or simply “antibody portion”) of an antibody, as used herein, refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., B7-H3 protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the term “antigen-binding portion” of an antibody include (i) Fab fragment, V L , V H , C L , and C H1 (ii) A monovalent fragment consisting of domains; (ii) A bivalent fragment that may include an F(ab')2 fragment and two Fab fragments linked by disulfide bridges in the hinge region; (iii) V H and C H1 Fd fragment consisting of domains; (iv) V of a single arm of the antibody L and V H (v) Fv fragment consisting of domains; (v) dAb fragment (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining region (CDR); and (viii) heavy chain variable region containing nanobodies, a single variable domain and two constant domains. Furthermore, the Fv fragment, V L and V HThe two domains are encoded by separate genes, but can be linked using a recombination method by a synthetic linker, which makes it possible to combine them into a single protein chain, where V L and V H The regions pair up to form a monovalent molecule (also known as a single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding portion" of the antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usability in the same manner as whole antibodies.

[0050] As used herein, “isolated antibody” is intended to refer to an antibody that substantially does not contain other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to the B7-H3 protein substantially does not contain antibodies that specifically bind to antigens other than the B7-H3 protein. However, an isolated antibody that specifically binds to the human B7-H3 protein may have cross-reactivity to other antigens, such as B7-H3 proteins from other species. Furthermore, an isolated antibody may substantially not contain other cellular material and / or chemical substances.

[0051] As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to a preparation of an antibody molecule in a single-molecule composition. Monoclonal antibody compositions exhibit single-binding specificity and affinity for a particular epitope.

[0052] As used herein, the term “human antibody” is intended to include antibodies having variable regions in which both the framework and CDR regions are derived solely from human germline heavy chain antibody sequences. Furthermore, if the antibody includes a constant region, the constant region is also derived from a human germline antibody sequence. The human antibodies of this disclosure may include amino acid residues not encoded by the human germline antibody sequence (e.g., mutations introduced by in vitro random or site-directed mutagenicity or in vivo somatic mutation). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species has been transplanted into a human framework sequence.

[0053] The term "chimeric antibody" refers to an antibody created by combining human genetic material with genetic material from a non-human source. More generally, a chimeric antibody is an antibody that contains genetic material from one species and genetic material from another species.

[0054] The terms "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are interchangeable in this specification with the term "antibody that specifically binds to an antigen."

[0055] As used herein, the term “antibody that specifically binds to human B7-H3” is intended to refer to an antibody that binds to the human B7-H3 protein (and B7-H3 from another non-human species) but substantially does not bind to non-B7-H3 proteins. Preferably, the antibody has “high affinity,” i.e., 5.0 × 10⁻⁶ -8 M or lower K D It then binds to the human B7-H3 protein.

[0056] The term "substantially unbound" to proteins or cells means that they do not bind to proteins or cells, or do not bind with high affinity, i.e., 1.0 × 10⁻⁶. -6 M or higher, comfortable 1.0×10 -5 M or higher, comfortable 1.0×10 -4 M or higher, 1.0 × 10-3 M or higher, comfortable 1.0×10 -2 M or higher K D This means binding to proteins or cells.

[0057] "EC" is also known as the semi-maximal effective concentration. 50 The term "50%" refers to the concentration of the antibody that gives half (50%) of the maximum reaction.

[0058] "I C 50 The term "half-maximal inhibitory concentration" refers to the concentration of a drug or inhibitor that inhibits a particular biological process by 50%.

[0059] The terms "antibody-dependent cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to cell-mediated immune defense in which effector immune cells actively lyse target cells to which cell surface antigens are bound by antibodies, such as the disclosed anti-B7-H3 antibody.

[0060] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, including mammals and non-mammals such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, but mammals such as non-human primates, sheep, dogs, cats, cattle, and horses are preferred.

[0061] The term "therapeutic dose" refers to the amount of the antibody of this disclosure that is sufficient to prevent or alleviate symptoms associated with a disease or illness (e.g., cancer). A person skilled in the art can easily identify the actual effective dose with respect to the disease being treated.

[0062] Various aspects of this disclosure are described in further detail below.

[0063] The antibody or its antigen-binding moiety in this disclosure is B7-H3 +The disclosed antibody or its antigen-binding moiety is capable of binding to B7-H3 in human, monkey, and similar organisms, including cells, and its binding activity / affinity is equivalent to or better than that of conventional antibodies such as enobrituzumab. In addition, compared to conventional antibodies such as enobrituzumab, the disclosed antibody or its antigen-binding moiety has equivalent or higher immune cell (e.g., NK cell) activation ability, equivalent or higher antibody-dependent cell-mediated cytotoxicity (ADCC) induction ability, and equivalent or better in vivo anticancer activity. The disclosed monoclonal antibody or its antigen-binding moiety is capable of binding to B7-H3 + It can be taken up by cells, and its binding specificity to human B7-H3 is better than that of conventional antibodies such as enobrituzumab.

[0064] The preferred antibodies of this disclosure are monoclonal antibodies. The antibodies may be, for example, human or chimeric monoclonal antibodies.

[0065] The exemplary antibodies or their antigen-binding moieties described herein are structurally and chemically characterized as follows.

[0066] The heavy chain variable region (CDR) and light chain variable region (CDR) of the disclosed antibody or its antigen-binding moiety are defined by the Kabat numbering system. The sequence numbers for the CDR amino acid sequences and the heavy / light chain amino acid sequences are listed in Table 1A. The heavy chain variable region (CDR) and light chain variable region (CDR) of the disclosed antibody or its antigen-binding moiety can also be determined by the Chothia numbering system, and the sequence numbers for the CDR amino acid sequences determined by such a system are listed in Table 1B. Table 1A The heavy / light chain variable region (CDR) and the sequence number of the heavy / light chain variable region (amino acid sequence) are determined by the Kabat numbering system. [Table 1] Table 1B The heavy / light chain variable region (CDR) and the sequence number of the heavy / light chain variable region (amino acid sequence) are determined by the Chothia numbering system. [Table 2]

[0067] In addition to the exemplary CDRs above determined by the Kabat and Chothia numbering systems, the heavy / light chain variable regions of the disclosed antibodies may include CDR regions determined by other numbering systems based on SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38. It is well known in the art that heavy chain variable region and light chain variable region CDRs can also be determined by, for example, the IMGT, AbM, or Contact numbering systems / methods.

[0068] The disclosed antibody may include a heavy chain constant region, such as a human IgG1 constant region, which may contain the amino acid sequence of SEQ ID NO: 43. The constant region may be manipulated to have enhanced FcR binding ability, such as a human IgG1 constant region containing the amino acid sequence of SEQ ID NO: 44 (EU numbering L235V, F243L, R292P, Y300L, and P396L). The light chain constant region may be a κ constant region, such as a human κ constant region which may contain the amino acid sequence of SEQ ID NO: 45.

[0069] V of other anti-B7-H3 antibodies that bind to human B7-H3 H and / or V L The sequence (or CDR sequence) is the V of the anti-B7-H3 antibody of this disclosure. H and V L It can be “mixed and combined” with the sequence (or CDR sequence). Preferably, V H and V L When chains (or CDRs within such chains) are mixed and combined, a certain V H / V L V from the pair H The arrays are structurally similar V H The sequence is replaced. Similarly, preferably a specific V H / V L V from the pair L The arrays are structurally similar V L It will be replaced with an array.

[0070] Therefore, in one embodiment, the antibody or antigen-binding moiety of the present disclosure may include the following: (a) Heavy chain variable region containing the amino acid sequence described in Table 1; and (b) Light chain variable region containing the amino acid sequence described in Table 1, or V of another anti-B7-H3 antibody L Here, the antibody specifically binds to human B7-H3.

[0071] In another embodiment, the antibody or antigen-binding moiety of the present disclosure comprises the following: (a) CDR1, CDR2 and CDR3 of the heavy chain variable region as described in Table 1; and (b) CDR1, CDR2, and CDR3 of the light chain variable region as described in Table 1, or the CDR of another anti-B7-H3 antibody, wherein the antibody specifically binds to human B7-H3.

[0072] In yet another embodiment, the disclosed antibody or its antigen-binding moiety includes a CDR of another antibody that binds to human B7-H3, e.g., CDR1 and / or CDR3 from the heavy chain variable region, and / or a heavy chain variable CDR2 region of an anti-B7-H3 antibody combined with CDR1, CDR2, and / or CDR3 from the light chain variable region of another anti-B7-H3 antibody.

[0073] In addition, in this technology, it is known that a CDR3 domain independent of the CDR1 and / or CDR2 domains can independently determine the binding specificity of antibodies against similar antigens, and that multiple antibodies with the same binding specificity can be predictively generated based on a common CDR3 sequence. For example, Klimka et al., British J. of Cancer 83(2):252-260 (2000);Beiboer et al., J. Mol. Biol. 296:833-849 (2000);Rader et al., Proc. Natl. Acad. Sci. USA 95:8910-8915 (1998);Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994);Barbas et al., Proc. Natl. Acad. Sci. USA 92:2529-2533 (1995);Ditzel et al., J. Immunol. 157:739-749 (1996);Berezov et al., BIAjournal 8: Scientific Review 8 (2001);Igarashi et al., J. See Biochem (Tokyo) 117:452-7 (1995); Bourgeois et al., J. Virol 72:807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. USA 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000). See also U.S. Patents No. 6,951,646; No. 6,914,128; No. 6,090,382; No. 6,818,216; No. 6,156,313; No. 6,827,925; No. 5,833,943; No. 5,762,905 and No. 5,760,185. These references are incorporated herein by reference as a whole.

[0074] In another embodiment, the antibody of disclosure may comprise a CDR2 of the heavy chain variable region of an anti-B7-H3 antibody, and at least a CDR3 of the heavy chain and / or light chain variable region of an anti-B7-H3 antibody, or a CDR3 of the heavy chain and / or light chain variable region of another anti-B7-H3 antibody, wherein the antibody is specifically capable of binding to human B7-H3. These antibodies preferably (a) compete for binding with B7-H3; (b) retain functional characteristics; (c) bind to the same epitope; and / or (d) have a binding affinity similar to that of the anti-B7-H3 antibody of this disclosure. In yet another embodiment, the antibody may further comprise a CDR2 of the light chain variable region of an anti-B7-H3 antibody, or a CDR2 of the light chain variable region of another anti-B7-H3 antibody, wherein the antibody is specifically capable of binding to human B7-H3. In another embodiment, the disclosed antibody may comprise the CDR1 of the heavy chain and / or light chain variable region of an anti-B7-H3 antibody, or the CDR1 of the heavy chain and / or light chain variable region of another anti-B7-H3 antibody, wherein the antibody is specifically capable of binding to human B7-H3.

[0075] In another embodiment, the antibody of the Disclosure may contain heavy and / or light chain variable region sequences of the CDR1, CDR2, and CDR3 sequences that differ from those of the anti-B7-H3 antibody of the Disclosure by one or more conservative modifications. In the Art of this, it is understood that certain conservative sequence modifications that do not exclude antigen binding may be made. For example, see Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem.32:6862-35; Adib-Conquy et al., (1998) Int. Immunol.10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0076] Therefore, in one embodiment, the antibody comprises a heavy chain variable region and / or a light chain variable region, where the heavy chain variable region and the light chain variable region each comprise CDR1, CDR2, and CDR3, where: (a) Heavy chain variable region CDR1 includes the sequences listed in Table 1 above, and / or their conservative modifications; and / or (b) Heavy chain variable region CDR2 includes the sequences listed in Table 1 above, and / or their conservative modifications; and / or (c) Heavy chain variable region CDR3 includes the sequences listed in Table 1 above, and their conservative modifications; and / or (d) Light chain variable regions CDR1 and / or CDR2 and / or CDR3 include the sequences and / or their conservative modifications listed in Table 1 above; and (e) The antibody specifically binds to human B7-H3.

[0077] The disclosed antibody exhibits high affinity and high binding specificity for human B7-H3, as well as enhanced FcR binding affinity, and therefore, B7-H3 + It may include one or more of the following functional features, such as an enhanced ability to induce ADCC in tumor cells:

[0078] In certain embodiments, the antibody or its antigen-binding moiety may be, for example, human or chimeric.

[0079] As used herein, the term “conservative sequence modification” is intended to refer to amino acid modifications that do not significantly affect, or alter, the binding properties of an antibody, including its amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications may be introduced into the disclosed antibodies by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the disclosed antibodies may be substituted with other amino acid residues from the same side chain family, and the modified antibodies may be tested for retained function (i.e., the function described above) using the functional assays described herein.

[0080] The disclosed antibody is the V anti-B7-H3 antibody of this disclosure. H / V L An antibody having one or more of the sequences can be prepared using an initiator for manipulating a modified antibody. The antibody has one or both of the variable regions (i.e., V H and / or V L For example, this can be manipulated by modifying one or more CDR regions and / or one or more residues in one or more framework regions. Additionally or alternatively, for example, to change the effector function of an antibody, an antibody may be manipulated by modifying residues within its constant region.

[0081] In certain embodiments, CDR implantation may be used to manipulate the variable region of an antibody. Antibodies interact with target antigens primarily through amino acid residues located in the heavy and light chain complementarity-determining regions (CDRs) of the six heavy and light chains. For this reason, the amino acid sequences within the CDRs exhibit greater diversity among individual antibodies than the sequences outside the CDRs. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors containing CDR sequences from specific naturally occurring antibodies transplanted onto framework sequences from different antibodies with different properties (see, for example, Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad; USA 86:10029-10033; U.S. Patents No. 5,225,539; No. 5,530,101; No. 5,585,089; No. 5,693,762 and No. 6,180,370).

[0082] Accordingly, another embodiment of the disclosure relates to an isolated monoclonal antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the Disclosure described above, and / or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the Disclosure described above, or to an antigen-binding moiety thereof. These antibodies are V of the monoclonal antibody of the Disclosure H and V L It contains CDR sequences, but may also contain sequences from different frameworks.

[0083] Such framework sequences can be obtained from public DNA databases or from publicly available references containing germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available online at www.mrc-cpe.cam.ac.uk / vbase) and in Kabat et al., (1991); Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836, as cited above; the contents of each of these are expressly incorporated herein by reference. As another example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in HCo7 HuMAb mice are available in the attached Genbank accessions 1-69 (NG--0010109, NT--024637&BC070333), 3-33 (NG--0010109&NT--024637), and 3-7 (NG--0010109&NT--024637). As another example, the following heavy chain germline sequences found in HCo12 HuMAb mice are available in the attached Genbank accession numbers: 1-69 (NG--0010109, NT--024637 & BC070333), 5-51 (NG--0010109 & NT--024637), 4-34 (NG--0010109 & NT--024637), 3-30.3 (CAJ556644), and 3-23 (AJ406678).

[0084] The antibody protein sequence is compared to a compiled protein sequence database using one of the sequence similarity search methods known to those skilled in the art, called Gapped BLAST (Altschul et al., (1997) above).

[0085] The preferred framework sequence used in the disclosed antibody has a structure similar to that of the framework sequence used by the disclosed antibody. H The CDR1, CDR2, and CDR3 sequences can be transplanted into a framework region having the same sequence as that found in germline immunoglobulin genes (the framework sequence is derived from the germline immunoglobulin gene), or the CDR sequences can be transplanted into a framework region containing one or more mutations compared to the germline sequence. For example, in certain cases, it has been found that mutating residues within the framework region is beneficial to maintain or enhance the antigen-binding ability of an antibody (see, for example, U.S. Patents 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0086] Another type of variable region modification is V H and / or V L The objective is to mutate amino acid residues in the CDR1, CDR2, and / or CDR3 regions, thereby improving one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis may be performed to introduce the mutation, and the effect on antibody binding, or other desired functional properties, may be evaluated by in vitro or in vivo assays, as known in the art. Preferably, conservative modifications (as known in the art) are introduced. The mutation may be an amino acid substitution, addition, or deletion, but substitution is preferred. Furthermore, typically, one, two, three, four, or five or fewer residues within the CDR region are altered.

[0087] Therefore, in another embodiment, the disclosure includes (a) the sequence of the present disclosure, or an amino acid sequence having amino acid substitutions, deletions or additions of 1, 2, 3, 4 or 5. H CDR1 region; (b) V containing the sequence of the present disclosure or an amino acid sequence having amino acid substitutions, deletions, or additions of 1, 2, 3, 4, or 5 HCDR2 region; (c) V containing the sequence of the present disclosure or an amino acid sequence having amino acid substitutions, deletions, or additions of 1, 2, 3, 4, or 5 H CDR3 region; (d) V containing the sequence of the present disclosure or an amino acid sequence having amino acid substitutions, deletions, or additions of 1, 2, 3, 4, or 5 L CDR1 region; (e) V containing the sequence of the present disclosure or an amino acid sequence having amino acid substitutions, deletions, or additions of 1, 2, 3, 4, or 5 L (f) the CDR2 region, and (f) the sequence of the present disclosure, or an amino acid sequence having amino acid substitutions, deletions, or additions of 1, 2, 3, 4, or 5. L The present invention provides an isolated anti-B7-H3 monoclonal antibody or its antigen-binding moiety, comprising a heavy chain variable region including a CDR3 region and a light chain variable region.

[0088] The manipulated antibodies in the disclosure are, for example, modified to improve the properties of the antibody. H and / or V L This includes modifications to framework residues within the molecule. Framework modifications include mutations in one or more residues in the framework region or, furthermore, in one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This technique is also known as “deimmunization” and is described in more detail in U.S. Patent Publication No. 20030153043.

[0089] In addition to, or instead of, modifications made within the framework or CDR region, the disclosed antibodies may be manipulated to include modifications within the Fc region to alter one or more functional properties of the antibody, such as blood half-life, complement binding, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, the disclosed antibodies may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter their glycosylation, thereby similarly altering one or more functional properties of the antibody.

[0090] In one embodiment, C H1The hinge region is modified so that the number of cysteine ​​residues in the hinge region is changed, for example, by increasing or decreasing it. This technique is further described in U.S. Patent No. 5,677,425. H1 The number of cysteine ​​residues in the hinge region is altered, for example, to facilitate the assembly of the light and heavy chains, or to increase or decrease the stability of the antibody.

[0091] In another embodiment, the Fc hinge region of an antibody is mutated to increase or decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are made to the C of the Fc hinge fragment such that the antibody has weaker SpA binding compared to the native Fc hinge domain of Staphylococcus protein A (SpA). H2 -C H3 It is introduced into the domain interface area. This technique is described in more detail in U.S. Patent No. 6,165,745.

[0092] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody may be produced (i.e., the antibody lacks glycosylation). Glycosylation may be modified, for example, to increase the affinity of the antibody to an antigen. Such hydrocarbon modifications may be achieved, for example, by altering one or more sites of glycosylation in the antibody sequence. For example, one or more amino acid substitutions may be made that result in the removal of one or more variable region framework glycosylation sites, thereby removing glycosylation at those sites. Such aglycosylation may increase the affinity of the antibody to an antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0093] Additionally or alternatively, antibodies with altered glycosylation patterns can be produced, such as low-fucosylated antibodies with reduced fucosyl residue levels, or antibodies with increased bipartite GlcNac structures. Such altered glycosylation patterns have been shown to increase or decrease the ADCC capacity of antibodies. Such hydrocarbon modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the Art, but are not limited to, Slc35C1 knockout cell lines, FUT8 knockout cell lines, Lec13 cell lines (mutant CHO cell lines), rat myeloma cell lines YB2 / 0, cell lines containing small interfering RNAs specific to the fut8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. They can be used as host cells to express the recombinant antibodies of the disclosure, thereby producing antibodies with altered glycosylation. Slc35C1 knockout cell lines can be prepared, for example, by employing a fucose removal platform developed by MabWorks. See the CHO cell line described in U.S. Patent No. 10377833B2, accession number CGMCC 14287.

[0094] Another modification of antibodies as used herein is PEGylation. Antibodies may be PEGylated, for example, to increase their biological (e.g., blood) half-life. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" means mono(C1-C1) 10) is intended to encompass any form of PEG used to derivatize other proteins, such as alkoxy or aryloxy polyethylene glycol or polyethylene glycol maleimide. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylated proteins are known in the Art and may be applied to the antibodies disclosed. See, for example, EP 0 154 316 and EP 0 401 384.

[0095] The disclosed antibodies may feature a variety of physical properties for detecting and / or distinguishing their different classes.

[0096] For example, an antibody may contain one or more glycosylation sites in either the light chain or heavy chain variable region. Such glycosylation sites can lead to increased immunogenicity of the antibody or a change in the antibody's pK due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation is known to occur in motifs containing N-X-S / T sequences. In some cases, it is preferable to have an anti-B7-H3 antibody that does not contain glycosylation in the variable region. This can be achieved by either selecting an antibody that does not contain a glycosylation motif in the variable region, or by mutating residues within the glycosylation region.

[0097] In preferred embodiments, the antibody does not contain an asparagine isomerized site. Deamidation of asparagine may occur at the NG or DG sequence, resulting in the formation of isoaspartic acid residues that introduce links into the polypeptide chain and reduce its stability (isoaspartic acid effect).

[0098] Each antibody generally has an inherent isoelectric point (pI) that falls within the pH range of 6 to 9.5. The pI of IgG1 antibodies typically falls within the pH range of 7 to 9.5, while the pI of IgG4 antibodies typically falls within the pH range of 6 to 8. Antibodies with pIs outside the normal range are thought to exhibit some unfolding and instability under in vivo conditions. Therefore, it is preferable to have an anti-B7-H3 antibody with a pI value within the normal range. This can be achieved by either selecting an antibody with a pI within the normal range or by mutating the charged surface residue.

[0099] In another embodiment, the disclosure provides nucleic acid molecules encoding the heavy and / or light chain variable regions or CDRs of the antibody or its antigen-binding moiety. The nucleic acid molecules may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. The nucleic acid molecules are “isolated” or “substantially pure” when purified by standard techniques from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins. The nucleic acid molecules of the Disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid molecule is a cDNA molecule.

[0100] The nucleic acid molecules of this disclosure can be obtained using standard molecular biological techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from genetically modified mice possessing human immunoglobulin genes, as further described below), the cDNA encoding the light and heavy chains of the antibodies produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), the nucleic acid molecules encoding such antibodies can be recovered from the gene library.

[0101] The preferred nucleic acid molecule to be disclosed is the V of the B7-H3 monoclonal antibody. H and V L Includes those that encode sequences or CDRs. V H and V L Once DNA fragments encoding the segment are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, V L or V H A DNA fragment encoding a protein is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. When used in this context, the term "operably linked" is intended to mean that the two DNA fragments are connected in such a way that the amino acid sequences encoded by both fragments remain in frame.

[0102] V H The isolated DNA encoding the region is V H The DNA encoding the heavy chain constant region (C H1 , C H2 , and C H3It can be converted into a full-length heavy-chain gene by operably linking it to another DNA molecule encoding H The DNA encoding can be operably linked to another DNA molecule encoding only the heavy-chain C H1 constant region.

[0103] V L The isolated DNA encoding the region can be converted into a full-length light-chain gene by operably linking the DNA encoding V L to another DNA molecule encoding the light-chain constant region C L . The sequences of human light-chain constant region genes are known in the art, and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light-chain constant region can be a κ or λ constant region.

[0104] To generate the scFv gene, the DNA fragments encoding V H and V L are operably linked to another fragment encoding a flexible linker, whereby the V H and V L sequences can be expressed as a continuous single-chain protein in which the V L and V H regions are connected by a flexible linker (see, for example, Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0105] The monoclonal antibodies of this disclosure may be prepared using somatic cell hybridization (hybridoma) techniques as described in Kohler and Milstein (1975) Nature 256: 495. Other embodiments for preparing monoclonal antibodies include virus or carcinogen-mediated B lymphocyte conversion and phage display techniques. Chimeric or humanized antibodies are well known in the art. See, for example, U.S. Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

[0106] The disclosed antibodies may also be produced by a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods (e.g., Morrison, S. (1985) Science 229:1202), as is well known in the art. In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biological techniques is inserted into one or more expression vectors. As a result, the gene is operably ligated to transcriptional and translational regulatory sequences. In this context, the term “operably ligated” is intended to mean that the antibody gene is ligated to the vector such that the transcriptional and translational regulatory sequences in the vector perform the intended function of controlling the transcription and translation of the antibody gene.

[0107] The term “regulatory sequence” is intended to include promoters, enhancers, and other expression regulatory elements that control the transcription or translation of antibody genes (e.g., polyadenylation signals). Such regulatory sequences are described, for example, by Goddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian virus 40 (SV40), and adenoviruses, e.g., adenovirus major late promoter (AdMLP) and polyomavirus enhancers. Alternatively, non-viral regulatory sequences such as ubiquitin promoters or β-globulin promoters may be used. Furthermore, the regulatory elements consist of sequences from different sources, such as the SRα promoter system, which includes sequences from the SV40 early promoter and long terminal repeat sequences from human T-cell leukemia type 1 virus (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). The expression vector and expression control sequences are selected to suit the expression host cells used.

[0108] The antibody light chain gene and antibody heavy chain gene can be inserted into the same or separate expression vector. In a preferred embodiment, the variable region is used to create a full-length antibody gene of any antibody isotype. They are inserted into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype. As a result, V H The segment is C in the vector. H The segment is operably connected, V L The segment is C in the vector. LThe segment is operably ligated. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes the secretion of antibody chains from host cells. The antibody chain gene may be cloned into the vector such that the signal peptide is ligated in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterogeneous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0109] In addition to antibody chain genes and regulatory sequences, the disclosed recombinant expression vectors may contain additional sequences, such as sequences that control vector replication in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665 and 5,179,017). For example, selectable marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (used in DHFR host cells in conjunction with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0110] For the expression of light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE dextran transfection, and similar methods. While it is theoretically possible to express the disclosed antibodies in either prokaryotic or eukaryotic host cells, antibody expression in eukaryotic cells (mammalian host cells being the most preferred) is most preferred because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunoactive antibodies.

[0111] Preferred mammalian host cells for expressing the recombinant antibodies of this disclosure include Slc35C1 knockout cell lines, FUT8 knockout cell lines, Lec13 cell lines (mutant CHO cell lines), rat myeloma cell line YB2 / 0, cell lines containing small interfering RNAs specific to the fut8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II, Chinese hamster ovary (CHO cells) (including dhfr-CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, and used with a DHFR-selectable marker as described in, for example, RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient to allow antibody expression in the host cells, or more preferably, the secretion of the antibody into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods.

[0112] The antibodies or antigen-binding moieties of the present disclosure may be conjugated with therapeutic agents to form immune complexes such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear transport inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and mitotic inhibitors. In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker such as a peptidyl linker, a disulfide linker, or a hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO02 / 096910; WO07 / 038,658; WO07 / 051,081; WO07 / 059,404; WO08 / 083,312; and WO08 / 103,693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295.

[0113] In another embodiment, the Disclosure features a bispecific molecule which may include one or more antibodies of the Disclosure linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor), in order to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, “bispecific molecule” includes molecules having three or more specificities.

[0114] Bispecific molecules can exist in many different formats and sizes. At one end of the size range, bispecific molecules retain the conventional antibody format but, instead of having two binding arms with identical specificity, have two binding arms, each with different specificities. Another extreme example is the so-called Bs(scFv)2 structure, a bispecific molecule consisting of two single-chain antibody fragments (scFv) linked by a peptide chain. Medium-sized bispecific molecules contain two different F(ab) fragments linked by a peptidyl linker. These and other formats of bispecific molecules can be prepared by genetic engineering, somatic hybridization, or chemical methods. See, for example, Kufer et al., Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al., Immunology Today, 21 (8), 391-397 (2000), cited above.

[0115] This specification also provides chimeric antigen receptors including anti-B7-H3 scFv, which include the CDR and heavy / light chain variable region described herein.

[0116] The anti-B7-H3 chimeric antigen receptor may comprise (a) an extracellular antigen-binding domain containing anti-B7-H3 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0117] Oncolytic viruses preferentially infect and kill cancer cells. The antibodies or their antigen-binding moieties of this disclosure may be used in conjunction with oncolytic viruses. Alternatively, oncolytic viruses encoding the antibodies or their antigen-binding moieties of this disclosure may be introduced into the human body.

[0118] In another embodiment, the Disclosure provides a pharmaceutical composition comprising one or more antibodies or their antigen-binding moieties, vectors encoding antibodies or antigen-binding moieties, immune complexes, immune cells, bispecific antibodies, and / or oncolytic viruses, which are formulated together with a pharmaceutically acceptable carrier. The composition may optionally comprise one or more additional pharmaceutically active ingredients, such as antitumor antibodies, or antibodies for immunoenhancing, or non-antibody antitumor agents or immunoenhancing agents. The pharmaceutical compositions of the Disclosure may be used, for example, in combination with antitumor agents or immunoenhancing agents.

[0119] A pharmaceutical composition may contain any number of excipients. Possible excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, solubilizers, colorants, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of preferred excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0120] The pharmaceutical composition is preferably suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, the term "parenteral administration" means a method of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, sub-articular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Alternatively, the antibodies of this disclosure may be administered via routes other than parenteral administration, such as topical, epidermal, or mucosal, for example, intranasal, oral, vaginal, rectal, sublingual, or topical.

[0121] Pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They may also be formulated as microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0122] The amount of active ingredient that can be combined with a carrier substance to produce a single-dose formulation varies depending on the target being treated and the specific method of administration, and is generally the amount of the composition that produces the therapeutic effect. Generally, this amount ranges from about 0.01% to about 99% of the active ingredient combined with a pharmaceutically acceptable carrier, out of 100 percent.

[0123] The administration plan is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased in response to instructions due to an emergency in the treatment situation. Formulating parenteral compositions in unit dose forms is particularly advantageous for facilitating administration and standardizing dosages. As used herein, unit dose forms refer to physically distinct units suitable as unit doses for the subject being treated. Each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect in combination with the necessary pharmaceutical carrier. Alternatively, antibodies may be administered as sustained-release formulations, in which case the frequency of administration required is reduced.

[0124] Regarding antibody administration, the dosage may range from approximately 0.001 to 100 mg / kg. An exemplary treatment plan involves administration once per week. Preferred means of anti-B7-H3 administration include intravenous administration.

[0125] As a result of the “therapeutably effective dose” of the anti-B7-H3 antibody of this disclosure, the severity of disease symptoms is reduced, and the frequency and length of disease-free periods are increased. For example, when treating a subject with a tumor, the “therapeutably effective dose” preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%, compared to an untreated subject. An effective dose of the therapeutic antibody may reduce the size of the tumor or alleviate the symptoms of the subject. The subject may be human or another mammal.

[0126] The pharmaceutical composition may be a sustained-release formulation including an implant and a microencapsulation delivery system. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0127] Pharmaceutical compositions may be administered via medical devices such as (1) needleless subcutaneous syringes (e.g., U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinjection pumps (U.S. Patents 4,487,603); (3) transdermal devices (U.S. Patents 4,486,194); (4) injectors (U.S. Patents 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patents 4,439,196 and 4,475,196), the disclosures thereof being incorporated herein by reference.

[0128] In certain embodiments, the antibodies of the Disclosure may be formulated to ensure proper in vivo distribution. For example, to ensure that the therapeutic antibodies of the Disclosure cross the blood-brain barrier, they may be formulated in liposomes which may additionally include a targeted moiety that enhances selective transport to specific cells or organs. For example, U.S. Patent Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; (1995) FEBS Lett.357:140;M. Owais et al., (1995) Antimicrob. Agents Chemother. 39:180;Briscoe et al., (1995) Am. J. Physiol. 1233:134;Schreier et al., (1994) J. Biol. Chem. 269:9090;Keinanen and Laukkanen (1994) FEBS See Lett. 346:123 and Killion and Fidler (1994) Immunomethods 4:273.

[0129] The pharmaceutical compositions of this disclosure may have many in vitro and in vivo applications, such as cancer treatment, or more generally, enhancement of the immune system in patients with diseases such as cancer. The pharmaceutical compositions may be administered to human subjects, for example, to inhibit tumor growth in vivo.

[0130] Given the ability of the disclosed pharmaceutical compositions to inhibit tumor cell proliferation and survival, the present application provides a method for inhibiting tumor cell proliferation in a subject, comprising the step of administering the disclosed pharmaceutical composition to a subject such that tumor proliferation is inhibited in the subject. Non-limited examples of tumors that can be treated with the antibodies disclosed include, but are not limited to, primary or metastatic breast cancer, melanoma, prostate cancer, head and neck squamous cell carcinoma, lung cancer, non-small cell lung cancer, central nervous system neuroblastoma, glioblastoma, fibroplastic small cell tumor, diffuse pontine glioma, medulloblastoma, pancreatic cancer, liver cancer, colorectal cancer, non-Hodgkin lymphoma, esophageal cancer, ovarian cancer, bladder cancer, small cell carcinoma, endometrial cancer, kidney cancer, gastric cancer, acute myeloid leukemia, hepatocellular carcinoma, hypopharyngeal squamous cell carcinoma, and urothelial cell carcinoma. Refractory or recurrent malignancies can also be treated with the disclosed pharmaceutical compositions. The disclosed pharmaceutical compositions can also treat autoimmune diseases such as asthma.

[0131] The disclosed pharmaceutical compositions may be used, for example, to enhance the immune response in a subject by activating immune cells such as NK cells and T cells.

[0132] This application provides a combination therapy in which the disclosed pharmaceutical composition is administered together with one or more other antibodies or non-antibody therapeutic agents capable of effectively inhibiting tumor growth in a subject. In one embodiment, this application provides a method for inhibiting tumor growth in a subject, the method comprising the step of administering the disclosed pharmaceutical composition and one or more other antibodies, such as an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-4-1BB antibody, to a subject. In a particular embodiment, the subject is a human. In another embodiment, this application provides a method for treating cancer in which the disclosed pharmaceutical composition is administered together with a chemotherapeutic agent, the chemotherapeutic agent may be a cytotoxic agent. Other therapies that may be combined with the disclosed pharmaceutical composition include, but are not limited to, administration of immunogens, administration of interleukin-2 (IL-2), radiotherapy, surgery, or hormone deprivation.

[0133] The therapeutic agent combinations described herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously as separate compositions with each agent in a pharmaceutically acceptable carrier. In another embodiment, the therapeutic agent combination may be administered sequentially.

[0134] Furthermore, if more than one dose of the combination therapy is administered sequentially, the order of sequential administration may be reversed or maintained at each point in time of administration, and sequential administration may be combined with simultaneous administration or any combination thereof.

[0135] The disclosure is further illustrated in the following examples, but this should not be construed as a further limitation. All figures and references, Genbank sequences, patents and patent application publications cited throughout this application are expressly incorporated herein by reference in their entirety. example Example 1 Production of anti-human B7-H3 monoclonal antibodies

[0136] To generate anti-human B7-H3 antibodies, B7-H3 RenMab KO mice were immunized with recombinant human B7-H3(4Ig) protein (His-tagged human B7-H3(4Ig) / B7-H3b protein, Cat#:B7B-H52E7, ACROBiosystems) and / or recombinant human B7-H3(2Ig) protein (His-tagged (MALS certified) human B7-H3 / CD276 protein, Cat#:B73-H52E2, ACROBiosystems). RenMab mice contain the locus of humanized immunoglobulin heavy chains and the locus of humanized immunoglobulin κ chains. The immunoglobulin heavy chain locus contains a region with genes encoding antibody heavy chains, and this locus contains the genes for IGHV (variability), IGHD (variability), IGHJ (connection), and the heavy chain constant region. The immunoglobulin κ chain locus contains the gene encoding the antibody light chain (κ chain). The immunoglobulin κ chain locus contains the genes for IGKV (variable), IGKJ (connected), and the light chain constant region. A detailed description of RenMab mice can be found in PCT / CN2020 / 075698, which is incorporated herein in whole by reference. B7-H3RenMab KO mice were obtained by knocking out the B7-H3 gene in RenMab mice, which did not express the functional B7-H3 protein. Mouse immunization

[0137] Immunization was performed four times in B7-H3RenMab KO mice using recombinant human B7-H3(4Ig) protein. The protein was mixed with an adjuvant in a 1:1 v / v ratio and then emulsified. Each animal was injected into the heel and neck. The adjuvant used for the first immunization was complete Freund's adjuvant (CFA), and the adjuvant used for the second to fourth immunizations was incomplete Freund's adjuvant (IFA). Two injections should be spaced at least 14 days apart.

[0138] In a separate study conducted in parallel, immunization was alternatively performed using recombinant human B7-H3(4Ig) protein and recombinant human B7-H3(2Ig) protein. The protein and adjuvant were mixed in a 1:1 v / v ratio and then emulsified. Each animal was injected into the heel and neck. The adjuvant used for the first immunization was complete Freund's adjuvant (CFA), and the adjuvant for the second to fourth immunizations was incomplete Freund's adjuvant (IFA). Two injections should be spaced at least 14 days apart.

[0139] Peripheral blood (serum) was collected, and antibody titers were measured by fluorescence-activated cell sorting (FACS) or ELISA.

[0140] The final boost immunization was performed at least 14 days after the fourth immunization by intraperitoneal injection of B7-H3(4Ig) protein and tail vein injection of CHO cells expressed on the surface of the B7-H3 antigen. Antibody discovery

[0141] Four days after the final immunization, the mice were sacrificed. Antigen-positive B cells were directly isolated from the mice and subjected to single-cell sorting (Beacon® Optofluidic System, Berkeley Lights Inc.) to obtain plasma cells secreting antigen-specific monoclonal antibodies. Antibody variable region sequences were obtained by reverse transcription and PCR, and the heavy / light chain variable regions of the antibodies were cloned into vectors encoding the human IgG and human κ constant regions to express the antibodies, and their binding specificity to B7-H3 was tested by FACS. The exemplary fully human antibodies obtained included 6B5, 10D7, 10F5, 10F7, and 15F11, and their heavy and light chain variable regions, CDR sequences, and sequence numbers are listed in Tables 1 and 9. Antibody construction, expression, and purification

[0142] The antibody expression vector was constructed by inserting fragments encoding the variable region and the human IgG1 / κ constant region (the amino acid sequences of the heavy chain constant region and light chain constant region described in SEQ ID NOs: 43 and 45, respectively) between the restriction sites XhoI / BamHI in pCDNA3.1 (Invitrogen, USA).

[0143] The expression vectors obtained as described above were used to transfect HEK-293F cells (Cobioer, China) with PEI. Specifically, HEK-293F cells were cultured in Free Style® 293 expression medium (Cat#:12338-018, Gibco, USA), and each expression vector was transfected into the cells using polyethyleneimine (PEI) with a DNA-to-PEI ratio of 1:3, where the amount of DNA was 1.5 μg per milliliter of cell culture medium. After transfection, HEK-293F cells were cultured at 120 rpm in an incubator shaker at 37°C and 5% CO2. The cell culture supernatant was collected after 10–12 days, centrifuged at 3500 rpm for 5 minutes, and filtered through a 0.22 μm membrane to remove cell debris. Monoclonal antibodies were concentrated and purified by passing them through a pre-equilibriumized protein A affinity column (Cat#:17040501, GE, USA), and eluted using elution buffer (20 mM citrate, pH 3.0-3.5). The antibodies were then stored in PBS (pH 7.0), and the antibody concentration was measured using a NanoDrop spectrophotometer. Example 2 Affinity measurement of fully human anti-B7-H3 antibodies

[0144] Fully human anti-human B7-H3 antibodies were tested for binding affinity to human B7-H3 (4Ig), human B7-H3 (2Ig), monkey B7-H3 (monkey B7-H3 / CD276 protein, His-tagged, Cat#:B73-C52Ha, ACROBiosystems), and mouse B7-H3 protein (mouse B7-H3 / CD276 protein, His-tagged, Cat#:B73-M52H4, ACROBiosystems) by surface plasmon resonance (SPR) using a Biacore T200 biosensor (Biacore, USA) equipped with a pre-immobilized protein A sensor chip. Briefly, anti-human B7-H3 antibody (1 μg / mL) was injected into the Biacore T200 biosensor at 10 μL / min over 20 seconds to achieve the desired protein density (100 ± 30 RU). Next, the antigen protein was injected at concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, and 0 nM at a rate of 30 μL / min over 180 seconds, and dissociation was monitored for 600 seconds. Enobrituzumab, a humanized anti-B7-H3 antibody with Fc optimization developed by MacroGenics (see US8802091B2 for antibody sequence; heavy and light chain variable region sequences are described in SEQ ID NOs: 39 and 40, respectively; the heavy chain constant region is an IgG1 constant region with five point mutations to enhance ADCC, F243L / R292P / Y300L / L235V / P396L; the light chain constant region is a κ constant region with sequences described in SEQ ID NOs: 44 and 45, respectively), was used as a positive control.

[0145] The generated data was analyzed using a one-to-one Langmuir coupling model with Biacore Insight V2.0.1 5.12933. The association rate (k on ) and dissociation rate (k off ) were obtained simultaneously. Affinity is obtained by the two velocity constants (K D =k off / k on It was calculated as the quotient of ).

[0146] All disclosed antibodies bound to two splicing variants of human B7-H3, as well as to monkey B7-H3 protein. Antibodies 6B5 and 15F11 also bound to mouse B7-H3 protein. Affinity test results for representative antibodies disclosed are shown in Tables 2 and 3. Table 2 Binding affinity of anti-B7-H3 antibodies to human B7-H3(4Ig) and human B7-H3(2Ig) [Table 3] Table 3 Binding affinity of anti-B7-H3 antibodies to monkey B7-H3 and mouse B7-H3 [Table 4] Example 3 B7-H3 + Tumor cells, B7-H3 - Tumor cells, and B7-H3 + Binding activity of fully human anti-B7-H3 antibody to normal cells

[0147] Different types of B7-H3 + Tumor cells, B7-H3 - Tumor cells, and B7-H3 + To determine the binding activity of the disclosed fully human anti-B7-H3 antibody to normal cells, A549 cells (Cat#:CBP60084, COBIOER), NCI-H520 cells (Cat#:CBP60139, COBIOER), HepG2 cells (Cat#:CBP60199, COBIOER), SK-OV-3 cells (Cat#:CBP60291, COBIOER), and MCF-7 cells (Cat#:CL-0149, ProCell) were used as representative B7-H3 cells. + Representative tumor cells include Jurcutt cells (Cat#:CBP60520, COBIOER), Daudi cells (Cat#:CBP60262, COBIOER), and Large cells (Cat#:CBP60272, COBIOER) in B7-H3. -FACS was performed using tumor cells (according to the literature, these three cells are B7-H3 mRNA negative), and human prostate smooth muscle cells (Cat#: CC-2587, Lonza), human dermal fibroblasts (Cat#: CC-2511, Lonza), human aortic smooth muscle cells (Cat#: H-6080, Cell Biologics), and human dendritic cells (derived from human PBMCs) as representative normal cells.

[0148] A549 cells, NCI-H520 cells, Jarcutt cells, Dowdie cells, and Large cells were cultured in RPMI1640 medium (Cat#: 12-115F, Lonza) supplemented with 10% FBS (Cat#: FND500, Excell) and 1% penicillin-streptomycin (Cat#: SV30010, Hyclone). HepG2 cells and SK-OV-3 cells were cultured in MEM medium (Cat#: 12561-056, Gibco) and McCoy 5A medium (Cat#: 16600-082, Hyclone), respectively, both supplemented with 10% FBS (Cat#: FND500, Excell) and 1% penicillin-streptomycin (Cat#: SV30010, Hyclone). MCF-7 cells were cultured in MCF7 cell medium (Cat#:CM-0149, ProCell), and human prostate smooth muscle cells were cultured in SmGM®-2 smooth muscle cell growth medium BulletKit® (Cat#:CC-3182, Lonza) according to the manual. Human dermal fibroblasts were cultured in FGM®-2 fibroblast growth medium BulletKit® (Cat#:CC-3132, Lonza) according to the manual, and human aortic smooth muscle cells were cultured in complete smooth muscle cell medium (Cat#:M2268, Cell Biologics) according to the manual.

[0149] Human dendritic cells were obtained by isolating PBMCs and subsequently in vitro inducing them. Briefly, PBMCs (Cat#:PB004F-C, ALLCELLS) were resuspended in RPMI1640 medium and incubated in a 37°C incubator for 2 hours. Cells adhering to the wall, i.e., isolated monocytes, were collected. The monocytes were cultured in RPMI1640 medium supplemented with 100 ng / ml recombinant human GM-CSF (Cat#:7954-GM, R&D), 100 ng / ml recombinant human IL-4 (Cat#:6507-IL, R&D), and 10% FBS. After 3 days, half of the culture medium was replaced with fresh medium. On day 6 of culture, the culture medium was replaced with a medium containing 100 ng / ml recombinant human GM-CSF, 100 ng / ml recombinant human IL-4, 10 ng / ml rhTNF-α (Cat#:210-TA-100, R&D), 1000 U / ml rhIL-6 (Cat#:7270-IL-025, R&D), 1 μg / ml PEG2 (Cat#:363-24-6, TOCRIS), and 10 ng / ml IL-1β (Cat#:210-LB-025, R&D). After incubating the cells for a further 2 days, mature human dendritic cells were obtained.

[0150] The binding activity of the anti-B7-H3 antibody to the above cells was tested. Briefly, in 100 μl of culture medium, 10 5 Cells were seeded on a 96-well plate and 50 μl of serially diluted anti-B7-H3 antibody was added. After incubation at 4°C for 1 hour, the 96-well plate was washed three times with PBST. Next, PE-goat-anti-mouse IgG (Cat#:PA1-86078, Invitrogen) diluted 1:500 was added to the plate. After incubation at 4°C for 1 hour, the 96-well plate was washed three times with PBS and then subjected to cell fluorescence assay using a FACS machine (BD). Enobrituzumab was used as a positive control. Representative anti-B7-H3 antibody test results are shown in Figures 1-3.

[0151] As shown in Figure 1, 6B5 and 10D7 are various types of B7-H3 + It binds to tumor cells, and the binding activity of both is significantly higher than that of enobrituzumab. B7-H3 mRNA - Figure 2 shows that there was almost no binding of 6B5 or 10D7 to tumor cells, and that the binding activity was significantly lower than that of enobrituzumab. All of these suggest that the disclosed antibody has better B7-H3 binding specificity. As shown in Figure 3, multiple normal human B7-H3 + The binding ability of 6B5 to cells was slightly higher than that of enobrituzumab, while the binding activity of 10D7 was comparable to that of enobrituzumab. Example 4 Fc operation is B7-H3 + It did not affect the binding of fully human anti-B7-H3 antibodies to cells.

[0152] Fragments encoding the heavy / light chain variable regions of 10D7 and 6B5, as well as the human IgG1 / κ constant region (SEQ ID NOs: 43 and 45), were cloned into pCDNA3.1 (Invitrogen, USA), and expression vectors were constructed by transfecting CHO-K1 cells and slc35c1 knockout CHO-K1-AF cells. slc35c1 knockout CHO-K1-AF cells were generated by Beijing MabWorks Biotech Inc., and the detailed generation method can be found in US10377833B2. Proteins expressed by such cell lines are essentially fucosylated. 10D7 and 6B5 were transiently expressed in CHO-K1 and CHO-K1-AF cells, purified, and the resulting antibodies were designated as 10D7-WT, 6B5-WT, 10D7-AF(afcosylated), and 6B5-AF(afcosylated). Meanwhile, fragments encoding the heavy / light chain variable regions of 10D7 and 6B5, as well as five point mutations in the human IgG1 Fc / κ constant region (SEQ ID NOs: 44 (EU numbering L235V, F243L, R292P, Y300L, and P396L) and 45), were cloned into pCDNA3.1. Expression vectors were constructed by transfecting CHO-K1 cells for transient expression, and the resulting antibodies were designated as 10D7-Mut and 6B5-Mut. Point mutations and afucosylation in the Fc region were used to enhance ADCC. Antibody expression and purification were performed according to the protocol of Example 1.

[0153] These anti-B7-H3 antibodies were tested by FACS for their binding ability to human B7-H3-expressing HUVEC cells (Cat#: C-12200, PromoCell, Germany) and NCI-H520 cells. HUVEC cells were cultured in EGM®-2 Endothelial Cell Growth Medium-2 BulletKit® (Cat#: CC-3162, Lonza) and subcultured according to the manual. For details on NCI-H520 cell culture and FACS testing, please refer to Example 3.

[0154] As shown in Figure 4, changes, afucosylation, or point mutations in the Fc region are B7-H3 + It does not affect the binding of anti-B7-H3 antibodies to cells. 10D7-AF, 10D7-WT, and 10D7-Mut showed completely identical binding activity to HUVEC (B) and NCI-H520 cells (D), while 6B5-AF, 6B5-WT, and 6B5-Mut showed nearly identical binding activity to HUVEC (A) and NCI-H520 cells (C). Example 5 Activity of a fully human anti-B7-H3 antibody that induces ADCC and activates immune cells.

[0155] The disclosed anti-B7-H3 antibody further contains B7-H3 + NCI-H520 tumor cells, B7-H3 + The ability to induce antibody-dependent cytotoxicity (ADCC) against human umbilical vein endothelial cells (HUVECs) and mature dendritic cells (DCs), and to activate NK cells and PBMCs was tested, where dendritic cells were derived, differentiated, and matured from isolated PBMCs (see details in Example 3).

[0156] In short, NCI-520 cells, HUVEC cells, and DCs are 1.0 × 10⁶ 6 Cells were resuspended in complete RPMI medium at a cell density of / ml. Cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, Cat#;C34554I, Invitrogen). Specifically, cells were incubated with 2.5 μM CFSE at 37°C for 10 minutes. After labeling, cells were resuspended in complete RPMI medium (RPMI + 10% FBS). NCI-H520 cells, HUVEC cells, DCs, and NK92MI-CD16a (effector cells, Huabo Bio) were centrifuged at 1200 rpm for 5 minutes. These cells were suspended in ADCC test medium (MEM medium, Cat#:12561-056, Gibco; 1% FBS, Cat#:FND500, EX cells; 1% BSA, Cat#:V900933-1KG, VETEC), and the cell viability was approximately 90% according to the cell count results. 4×105 50 μl of target cells (NCI-H520 cells, HUVEC cells, or DCs) with a cell density of 1 / ml and 2 × 10⁶ 6 50 μl of NK92MI-CD16a cells at a cell density of 1 / ml were added to each well of a 96-well plate, with an effector-to-target ratio of 5:1. The antibodies to be tested were added to each well at different concentrations, with a starting final concentration of 50,000 ng / ml, and other final concentrations being 5-fold dilutions for a total of 10 concentrations. The 96-well plates were incubated at 37°C for 4 hours, and the cells on the plates were washed three times with PBS and then incubated at 37°C for 30 minutes using LIVE / DEAD fixable dead cell staining. The cells on the plates were washed three times with PBS and 2 μl of PE mouse anti-human CD69 antibody (Cat#: 555531, BD) was added. After incubation at room temperature for 30 minutes, the plates were centrifuged, washed three times with PBS, and subjected to FACS. CFSE + The cell death rate of the cells (NCI-H520 cells, HUVEC cells, or DCs) is determined, and CFSE - The mean PE staining fluorescence intensity of cells (NK92MI-CD16a cells) was calculated. Enobrituzumab was used as a positive control.

[0157] The anti-B7-H3 antibody was further tested for its ability to induce human PBMC-mediated ADCC in NCI-H520 and HUVEC cells, where NCI-H520 and HUVEC cells were labeled with carboxyfluorescein succinimimidyl ester (CFSE, Cat#:C34554I, Invitrogen). Human PBMCs were obtained by density gradient centrifugation using lymphocyte isolation medium and cultured overnight in medium (RIPM1640 + 10% FBS + 300 IU IL-2). The ADCC test was performed using a LIVE / DEAD fixable dead cell staining kit (Thermo Fisher, USA, Cat#:L34964). Target cells and effector cells (PBMCs) were centrifuged at 1200 rpm for 5 minutes. Cells were resuspended in ADCC test medium (RIPM1640 medium + 1% FBS), and cell viability was approximately 90% according to cell count results. 4 x 10 5 50 μl of target cells with a cell density of 4 × 10⁶ / ml, and 4 × 10⁶ 6 50 μl of PBMCs with a cell density of 1 / ml was added to each well of a 96-well plate, with an effector:target ratio of 10:1. The antibody to be tested was added to each well, with an initial final concentration of 50,000 ng / ml, and other final concentrations were diluted 5-fold to a total of 10 concentrations. The 96-well plate was incubated at 37°C for 24 hours, and the supernatant from each well was collected and the IFN-γ concentration was measured by ELISA (Cat#: SIF50, R&D) according to the manufacturer's manual. Cells on the plate were washed three times with PBS and incubated at 37°C for 30 minutes using LIVE / DEAD fixable dead cell staining. Cells were washed three times with PBS and subjected to FACS testing. CFSE + The cell death rate of cells (NCI-H520 cells or HUVEC cells) was determined. Enobrituzumab was used as a positive control. The test results are shown in Figures 5-8.

[0158] As shown in Figure 5, 10D7-AF and 10D7-Mut were significantly more effective than 10D7-WT in inducing NK92-mediated ADCC in NCI-H520 tumor cells, and their abilities were also higher than enobrituzumab, the positive control (A); 10D7-AF induced higher levels of the activation marker CD69 on the NK cell surface, thus demonstrating higher NK cell activation ability than 10D7-Mut (C). Similarly, the ability of 6B5-AF and 6B5-Mut to induce ADCC was significantly higher than 6B5-WT, and also higher than enobrituzumab, the positive control (B); in addition, 6B5-AF induced higher levels of CD69, thus demonstrating higher NK cell activation ability than 6B5-Mut (D). Furthermore, enobrituzumab, the positive control, showed a clear hook effect at high doses in inducing NK92-mediated NCI-H520 killing. In other words, when the antibody concentration increased after reaching the specified value, the detected lethal effect decreased. This effect was not observed in 10D7 or 6B5.

[0159] As shown in Figure 6, the ability of 10D7-AF and 10D7-Mut to induce PBMC-mediated NCI-H520 tumor cell death was significantly higher than that of 10D7-WT and also higher than that of the positive control enobrituzumab (A). A similar trend was also observed for 6B5 in ADCC induction; that is, ADCC induced by 6B5-AF and 6B5-Mut against NCI-H520 tumor cells was more potent than that of 6B5-WT and also more potent than that of the positive control enobrituzumab (B). Similarly, the positive control enobrituzumab showed a clear hook effect in inducing PBMC-mediated NCI-H520 death at high doses. This effect was not observed with 10D7 or 6B5.

[0160] As shown in Figure 7, the ability of 6B5-AF and 10D7-AF to induce NK92-mediated HUVEC cell death was somewhat higher than that of enobrituzumab and the positive control (A). However, in a reaction system involving PBMCs as effector cells, none of 6B5-AF, 10D7-AF, or enobrituzumab induced clear HUVEC cell death (B) or clear IFN-γ release (C).

[0161] As shown in Figure 8, the ability of 6B5-AF and 10D7-AF to induce DC killing by NK92 cells was equivalent to or lower than that of the positive control, enobrituzumab. Example 6 Uptake of fully human anti-B7-H3 antibody

[0162] To test whether the disclosed anti-B7-H3 antibody could be taken up by cells, A549 cells and NCI-H520 cells were used for uptake, and enobrituzumab-IgG1-Mut and ombrutamab-IgG1 were used as positive controls. Ombrutamab is a radioactive iodine-labeled anti-human B7-H3 monoclonal antibody developed by Y-mAbs Therapeutics, and ombrutamab-IgG1 contains the heavy and light chain variable regions of SEQ ID NOs: 41 and 42, with the heavy chain constant region being wild-type IgG1 and the light chain constant region being the κ constant region, and containing the amino acid sequences of SEQ ID NOs: 43 and 45, respectively.

[0163] The anti-human B7-H3 antibody to be tested was homogeneously mixed with pHAb-labeled goat anti-human IgG1 Fc (Cat#:SSA015, Sino Biological, labeled with pHAb) in a 1:1 concentration ratio. This mixture was homogeneously mixed with 10,000 A549 cells, and the final concentrations of both the anti-B7-H3 antibody and the secondary antibody were 25 μg / mL. Human IgG1 protein was used in a blank control well. The mixture was incubated on ice in the dark for 1 hour, washed three times by centrifugation using pre-cooled FACS buffer (90% DMEM + 10% FBS) (the supernatant was discarded), and incubated together in a 5% CO2 incubator at 37°C with pre-heated complete culture medium. Cell cultures were harvested at 0, 3, 6, and 9 hours and stored on ice in the dark. After all samples were collected, they were centrifuged at 1200 rpm at low temperature for 3 minutes to discard the supernatant, and washed once with PBS buffer. Fluorescence signals from the cell surface were read. For cells incubated with different antibodies, raw data was collected from flow cytometry, and the MFI of fluorescence emitted from cells that had taken up different antibodies by binding to cell surface B7-H3 was analyzed and determined based on the raw data. MFI curves were plotted based on the amount of anti-B7-H3 antibody taken up.

[0164] Following the protocol above, the uptake of anti-human B7-H3 antibody was also tested using NCI-H520 cells.

[0165] The results are shown in Figure 9. During the overall study, no uptake occurred when cells alone, cells and secondary antibodies alone, or cells, secondary antibodies, and human IgG1 protein alone were incubated. However, when cells were incubated with 6B5, 10D7, 10F5, 10F7, enobrituzumab, or ombrutamab-IgG1, the MFI tended to increase over time. In particular, a clear increase in MFI was observed for 10D7, 10F7, and ombrutamab-IgG1. The data indicate that 6B5, 10D7, 10F5, 10F7, enobrituzumab, and ombrutamab-IgG1 are all capable of being taken up by NCI-H520 and A549 cells, where 10D7, 10F7, and ombrutamab-IgG1 were taken up at relatively high rates. Example 7 Analysis of epitopes to which fully human anti-B7-H3 antibodies bind.

[0166] To determine the relationships between epitopes to which anti-human B7-H3 antibodies bind, the inhibitory effects of each anti-human B7-H3 antibody on antigen binding of other antibodies were tested using surface plasmon resonance (SPR) in a serial configuration. A total of four anti-human B7-H3 antibodies were tested: 6B5 ("Ab6B5"), 10D7 ("Ab10D7"), 10F5 ("Ab10F5"), and enobrituzumab (abbreviated as "AbEno").

[0167] HBS-EP + A buffer (10 mM 4-(2-hydroethyl)-1-piperazine ethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P2O, pH 7.4) was used as the electrophoresis buffer. Anti-His antibody was amine-bonded to the surface of the Series S sensor tip CM5 to generate an anti-His tip. Next, 1 M ethanolamine (pH 8.5) was injected to block any remaining active carboxyl groups on the tip surface, and the tip was HBS-EP. +The proteins were equilibrated in buffer for 2 hours. Recombinant human B7-H3(4Ig) protein with a His tag (Cat#:B7B-H52E7, Acrobiosystems) was injected into a Biacore8K biosensor at a rate of 30 μL / min over 50 seconds and captured by an anti-His tip until the protein density reached approximately 50 RU.

[0168] A pair of antibodies (both at a concentration of 800 nM) were injected into the chip at a rate of 30 μL / min. The binding time for the first injected antibody (analyte 1) was 300 seconds, and the binding time for the second injected antibody (analyte 2) was also 300 seconds. During each analysis cycle, the chip was regenerated twice by using glycine buffer (pH 1.7, 30 μL / min, 30 seconds) after antibody injection. The same test steps were performed for each pair of monoclonal antibodies to obtain the inhibitory effect of each monoclonal antibody on antigen binding of the other antibody. The binding values ​​for each antibody were obtained using Biacore Insight. To quantify how one antibody interferes with antigen binding of the other antibody, the kinetic speeds of each pair of antibodies were determined and compared. Cluster analysis was performed using statistical software.

[0169] As shown in Figure 10, the four anti-human B7-H3 antibodies were classified into three epitope clusters. In particular, 6B5 (Ab6B5) and 10F5 (Ab10F5) can bind to the same epitope, or their epitopes may overlap; 10D7 (Ab10D7) binds to a unique epitope; the epitopes to which 6B5 (Ab6B5), 10F5 (Ab10F5), and 10D7 (Ab10D7) bind, and the epitopes to which enobrituzumab (AbEno) binds, may overlap or be different. Example 8 Binding of anti-human B7-H3 antibodies to B7 family proteins

[0170] Anti-human B7-H3 antibodies were tested for their binding ability to B7 family proteins by SPR using a Biacore T200 biosensor (Biacore, USA) with a pre-immobilized protein A sensor chip.

[0171] In particular, the tip captures 1 μg / mL anti-human B7-H3 antibody at a flow rate of 10 μL / min and then, over 180 seconds, at a flow rate of 30 μL / min, recombinant human CD80 protein (human B7-1 / CD80 protein, His tagged, Cat#:B71-H5228, Acrobiosystems), CD86 (human B7-2 / CD86 protein, His tagged, Cat#:CD6-H5223, Acrobiosystems), and PDL2 (human PD-L2 / B7-DC protein, His tagged) in concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, or 0 nM at a rate of 30 μL / min. PD1 (Human PD-L1 / B7-H1 protein, His tagged, Cat#:PD2-H5220, Acrobiosystems), B7-H2 (Human B7-H2 / ICOSLG protein, His tagged, Cat#:B72-H5221, Acrobiosystems), B7-H3 (4Ig) (Cat#:B7B-H52E7, Acrobiosystems), B7-H4 (Human B7-H4 protein, His tagged (MALS certified), Cat#:BH7-HM174, Kactus) Acrobiosystems injected B7-H5 (human B7-H5 / Gi24 / VISTA protein, His-tagged (MALS certified), Cat#:B75-H52H0), B7-H6 (human B7-H6 / NCR3LG1 protein, His-tagged, Cat#:B71-H5228), and B7-H7 (human B7-H7 / HHLA2 protein, His-tagged (MALS certified), Cat#:B77-H52H5). Dissociation was monitored for 600 seconds. Data were analyzed using Biacore8k evaluation software version 3.0 to obtain binding responses, plot protein concentration-binding response curves, and determine the cross-reactivity of anti-human B7-H3 antibodies to B7 family proteins.

[0172] The results are summarized in Table 4. Similar to enobrituzumab, antibodies 6B5, 10D7, and 10F5 did not bind to recombinant human CD80, CD86, PDL2, PDL1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, or B7-H7 proteins, but specifically bound to recombinant human B7-H3 protein. The results of 6B5 binding to recombinant human proteins are shown in Figure 11. The binding response of each antibody to recombinant human proteins at 200 nM is summarized in Table 4 below. Table 4 Conjugation of fully human anti-B7-H3 antibodies to B7 family proteins [Table 5] Example 9 In vivo anticancer activity of fully human anti-B7-H3 antibodies

[0173] The in vivo antitumor activity of antibodies 6B5, 10D7, 10F5, and the positive control enobrituzumab was tested. To better demonstrate the biological effect of the antibody variable region, all antibodies were enhanced with ADCC by selecting human IgG1 as the Fc region into which five point mutations (according to EU numbering, L235V, F243L, R292P, Y300L, and P396L, SEQ ID NO: 44) were introduced.

[0174] In the first experiment, mice possessing the human B7-H3 gene (Cat#: 110028, Biocytogen) had 2 × 10⁶ cells overexpressing human B7-H3 protein on one side of their abdomen. 5 EL4 cells were injected subcutaneously. The tumor was 90-120 mm in size. 3 Upon reaching a certain stage, mice were randomly assigned to one of three groups (n=8 / group) and administered PBS (G1), enobrituzumab (G2), and 10F5-Mut (G3) intraperitoneally twice weekly at a dose of 10 mg / kg, for a total of six doses. The day of grouping was designated as day 0, and administration began on this day.

[0175] In another experiment, the tumor was 90-120 mm 3Upon reaching maturity, B7-H3 humanized mice were randomly assigned to four groups (n=8 / group) and administered intraperitoneally with PBS (G1), enobrituzumab (G2), 6B5-Mut (G3), and 15F11-Mut (G4) at the same doses and frequencies as above. The day of grouping was designated as day 0, and administration began on this day.

[0176] The weight of the mice was measured during the experiment. The size of the tumor was measured twice a week, and the tumor size was given by V = (length × width). 2 It was calculated as ) / 2. Tumor growth inhibition was calculated as TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100, where T i T0 refers to the average tumor volume of the treatment group on day i, and V refers to the average tumor volume of the treatment group on day 0. i V0 is the mean tumor volume of the control group on day i, and V0 is the mean tumor volume of the control group on day 0.

[0177] Throughout the entire study, all animal groups survived in good health. Compared to the control group, the treated mice did not show any significant change in body weight (P>0.05). In particular, the mean body weight for each group was 19.3–19.5 on day 0 when grouping took place, and was 19.8–20.9 at the end of the study (day 17 after grouping in the first experiment and day 20 after grouping in the second experiment), so the change in body weight was between 103.2% and 111.1%. The results showed that the tested anti-human B7-H3 antibodies were well-tolerated by mice, meaning that all antibodies were non-toxic to the mice.

[0178] Figure 12 shows the changes in tumor size in mice from each group. Compared to the control group, all anti-human B7-H3 antibodies slowed or inhibited tumor growth to some extent. In particular, 10F5-Mut(A), 6B5-Mut, and 15F11-Mut(B) showed higher antitumor efficacy than the positive control, enobrituzumab.

[0179] The date of grouping, tumor size on day 7 or 14 after grouping, tumor volume at the end of the experiment, mouse survival rate, and tumor (volume) inhibition rate (TGI) were recorded. TV The main data and analysis results, including the percentage and statistical significance (P-value) between the treatment group and the control group, are presented in Tables 5 and 6. The data showed that 6B5-Mut, 15F11-Mut, and 10F5-Mut all significantly inhibited tumor growth, with 10F5-Mut having the highest antitumor effect, followed by 15F11-Mut. Table 5 Summary of the in vivo anticancer activity of the fully human anti-human B7-H3 antibody 10F5-Mut [Table 6] Table 6 Summary of the in vivo anticancer effects of fully human anti-human B7-H3 antibodies 6B5-Mut and 15F11-Mut [Table 7] Example 10 Cross-reaction staining of normal human tissue with fully human anti-human B7-H3 antibody.

[0180] The cross-reactivity of the fully human anti-human B7-H3 antibody 6B5-AF against 35 types of frozen normal human tissues (each derived from three individuals) was tested using streptavidin-biotin binding immunohistochemical staining, with enobrituzumab used as the control antibody. The human tissues are listed in Table 7.

[0181] Immunohistochemical staining was performed as follows: Frozen sections were dried at room temperature and immersed in PBST for 5-10 minutes; 0.3% hydrogen peroxide / anhydrous methanol was added and reacted at room temperature for 10-15 minutes, followed by three immersions in PBST; avidin was added and reacted at room temperature for 10-15 minutes, followed by three immersions in PBST; d-biotin solution was added and reacted at room temperature for 10-15 minutes, followed by three immersions in PBST; sheep serum (working solution) was added for blocking and reacted at room temperature for 10-15 minutes, then the solution was discarded; primary antibody (E Noblitzumab-biotin, 6B5-AF-biotin, human IgG1 / κ isotype control-biotin) or PBST was added and reacted at room temperature for 10-15 minutes, followed by three dips in PBST; streptavidin-peroxidase was added and incubated at room temperature for 10-15 minutes, followed by three dips in PBST; stained with DAB, counterstained with hematoxylin, dehydrated with alcohol and then xylene while increasing the concentration; culture medium was added, covered with a coverslip, and dried in the air for microscopic examination. Table 7 35 types of frozen normal human tissue [Table 8]

[0182] Staining results were observed via microscope, and the level of antibody bound to the tissue was determined by staining. Staining results were scored according to staining intensity and the percentage of stained cells (Positive cell staining intensity: 0 = no cell staining; 1 = weak or ambiguous staining; 2 = weak positive staining; 3 = moderate positive staining; 4 = strong positive staining; M = tissue loss or not applicable. Percentage of positive cells in the same cell type: 0 = no cell staining; 1 = positive cells account for less than 25% of cells of the same cell type; 2 = positive cells account for 25% to 50% of cells of the same cell type; 3 = positive cells account for 50% to 75% of cells of the same cell type; 4 = positive cells account for more than 75% of cells of the same cell type; M = tissue loss or not applicable). Tissues without any positive staining from the primary antibody (enobrituzumab-biotin, 6B5-AF-biotin, human IgG1 / κ isotype control-biotin) or PBST were excluded from statistics, and the scores for positively stained tissues are summarized in Table 8.

[0183] Table 8 shows that the tissue staining specificity of the fully human anti-B7-H3 antibody 6B5-AF is consistent with that of enobrituzumab. Specifically, of the 35 normal human tissues tested, 24 showed negative staining, while only 11 showed weak staining. On the other hand, due to differences in the B7-H3 antigen epitopes between 6B5-AF and enobrituzumab, the staining intensity differed slightly in some tissues that exhibited positive staining. As shown in Table 8, compared to enobrituzumab, 6B5-AF showed relatively strong staining in the pituitary gland, but slightly weaker staining in the thymus, cervix, and endometrium.

[0184] Figure 13 shows the staining results of representative tissues, where 1-1, 2-1, 3-1, and 4-1 show staining with enobrituzumab in the spleen, skeletal muscle, adrenal gland, and bladder, and 1-2, 2-2, 3-2, and 4-2 show staining with 6B5-AF in the spleen, skeletal muscle, adrenal gland, and bladder. Table 8 Summary of results for tissues with positive staining [Table 9] a: Positive cell staining intensity - score based on the percentage of positive cells in the same type of cell: Positive cell staining intensity: 0 = no cell staining; 1 = weak or ambiguous staining; 2 = weak positive staining; 3 = moderate positive staining; 4 = strong positive staining; M = tissue loss or not applicable; Percentage of positive cells in the same type of cell: 0 = no cell staining; 1 = positive cells make up less than 25% of the same type of cell; 2 = positive cells make up 25% to 50% of the same type of cell; 3 = positive cells make up 50% to 75% of the same type of cell; 4 = positive cells make up more than 75% of the same type of cell; M = tissue loss or not applicable.

[0185] The sequences mentioned in this application are summarized in Table 9. Table 9. Array [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0186] Although the present invention has been described using one or more embodiments, it should be understood that the present invention is not limited to these embodiments, and that the above description is intended to include all other alternative forms, modifications, and equivalents that fall within the spirit and scope of the appended claims. All references referenced herein are incorporated by reference in their entirety.

Claims

1. i) Heavy chain variable region, wherein the heavy chain variable region includes the VH CDR1 region, the VH CDR2 region, and the VH CDR3 region; and ii) Light chain variable region, wherein the light chain variable region includes a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, the VH CDR3 region, the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region each contain (1) the amino acid sequences of sequence numbers 1, 2, 3, 4, 5, and 6, respectively; or (2) the amino acid sequences of sequence numbers 18, 19, 20, 9, 21, and 22, respectively. An isolated monoclonal antibody or its antigen-binding moiety capable of binding to B7-H3, comprising the above.

2. The isolated monoclonal antibody or antigen-binding moiety according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 29 or 35.

3. The isolated monoclonal antibody or antigen-binding moiety according to claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 30 or 36.

4. The isolated monoclonal antibody or antigen-binding moiety according to claim 2, wherein the heavy chain variable region and the light chain variable region each contain an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with (1) SEQ ID NOs: 29 and 30, respectively, or (2) SEQ ID NOs: 35 and 36, respectively.

5. The isolated monoclonal antibody or its antigen-binding moiety according to claim 1, comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is a human IgG1 constant region and the light chain constant region is a human κ constant region.

6. The isolated monoclonal antibody or antigen-binding moiety according to claim 5, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 44, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:

45.

7. The isolated monoclonal antibody or antigen-binding moiety according to claim 1, which is an afucosylated monoclonal antibody or an antigen-binding moiety thereof.

8. An isolated monoclonal antibody according to any one of claims 1 to 7, or a nucleic acid molecule encoding the antigen-binding portion thereof.

9. An expression vector comprising the nucleic acid molecule described in claim 8.

10. A host cell comprising the expression vector described in claim 9.

11. A pharmaceutical composition comprising an isolated monoclonal antibody or an antigen-binding moiety thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition according to claim 11, used for treating B7-H3 related diseases.

13. The aforementioned B7-H3 related disease is B7-H3 ＋ A pharmaceutical composition for use according to claim 12, wherein the patient is a tumor.

14. The pharmaceutical composition for use according to claim 13, wherein the tumor is selected from the group consisting of breast cancer, melanoma, prostate cancer, squamous cell carcinoma of the head and neck, lung cancer, non-small cell lung cancer, neuroblastoma of the central nervous system, glioblastoma, fibroplastic small cell tumor, diffuse pontine glioma, medulloblastoma, pancreatic cancer, liver cancer, colorectal cancer, non-Hodgkin lymphoma, esophageal cancer, ovarian cancer, bladder cancer, small cell carcinoma, endometrial cancer, kidney cancer, gastric cancer, acute myeloid leukemia, hepatocellular carcinoma, hypopharyngeal squamous cell carcinoma, and urothelial cell carcinoma.

15. An isolated monoclonal antibody or antigen-binding moiety thereof according to any one of claims 1 to 7, used to enhance an immune response.

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