Antibody-binding B7-H3 and uses thereof

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] The present application relates to antibodies that specifically bind to human B7-H3, and their preparation and uses, particularly in the treatment of B7-H3-associated diseases such as cancer. [Background technology]

[0002] T cell proliferation and activation depend on dual signals. The interaction between T cell receptor and peptides presented by MHC on antigen-presenting cells or tumor cells provides the first signal that is essential for T cell activation but does not induce cell proliferation or cytokine release. The second signal, the costimulatory signal, determines whether the T cell will be activated / proliferated, unresponsive, or undergo apoptosis. The B7-CD28 costimulatory signaling pathway plays an important role in the control of T cell activation and inhibition, where CD-28 molecules are expressed on T cells, while B7 molecules are generally present on activated antigen-presenting cells. To date, more than 10 B7 proteins have been discovered and are divided into three groups according to the signals and functions they transduce during T cell activation: i) costimulatory, ii) costimulatory, and iii) costimulatory / inhibitory.

[0003] B7-H3, also known as CD276, is a costimulatory / inhibitory molecule of the B7 family. When first discovered, it was considered a costimulatory 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, suppressing T cell activation / proliferation or the release of effector cytokines (mainly IFN-γ and IL-2), inhibiting NK cell activity, etc. (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, although only a few studies have been performed on the B7-H3 receptor.

[0004] B7-H3 is a type 1 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 motifs. In addition, human B7-H3 protein 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 predominant subtype found in human cells.

[0005] B7-H3 mRNA is widely expressed in normal tissues, whereas B7-H3 protein molecule is lowly 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, and in the basement membrane of stomach, gallbladder, prostate, cervix, and endometrium. B7-H3 protein is highly expressed by multiple malignant tumors. Various studies have shown that B7-H3 molecule is expressed in 33.0% of renal cell carcinoma patients and 91.8% of hepatocellular carcinoma patients. It should be noted that the proportion of patients with B7-H3 expression is very low in renal cell carcinoma and systemic hematological cancers (33.0% and 36.7%, respectively, p<0.001), while at least 52.3% of patients with other solid cancers 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%). Among all cancers, 59.5% (15877 / 26703) of patients are B7-H3 positive. On the other hand, it was found that pancreatic cancer patients with high B7-H3 expression had a better prognosis after surgery than those with low B7-H3 expression (Loos M. et al., (2009) BMC Cancer 9: 463). On the other hand, B7-H3 may contribute to an immunosuppressive tumor microenvironment, promoting the escape of cancer stem cells from immune surveillance, for example 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 initiation 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 provide energy to tumors (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 therapy. Notably, 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 eliminate even cancer cells with low B7-H3 expression.

[0007] Currently, various forms of B7-H3 targeted therapeutics are in clinical trials. Enoblituzumab is the first monoclonal antibody targeting the B7-H3 protein with an optimized Fc region, and it shows antitumor activity through both antibody-dependent cell-mediated cytotoxicity and enhancement of T cell immune function. According to data published in the 2018 Annual Report of the Society for Immunotherapy of Cancer (SITC), the objective response rate (ORR) of enoblituzumab and anti-PD-L1 antibody combination therapy was 33.3% in patients with squamous cell carcinoma of the 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%). Notably, patients previously treated with anti-PD-1 / L1 antibodies responded to such therapy (partial remission: PR), with disease suppression in a similar proportion of patients compared with patients undergoing anti-PD-1 / L1 therapy for the first time. 131 The I-labeled monoclonal antibody 8H9 (omburtamab, Y-mAbs) has been used to treat patients with metastatic central nervous system neuroblastoma (NCT00089245) and desmoplastic small cell tumors (NCT01099644) via intraperitoneal injection and has been well tolerated. 124 Monoclonal antibody 8H9 labeled with I has been tested in diffuse intrinsic pontine glioma (NCT01502917) and has also been well tolerated. Several other B7-H3 targeted therapeutics, including small molecule drugs, monospecific antibodies, bispecific antibodies, antibody-drug conjugates, CAR-T and the like, are being 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, desmoplastic small cell tumor, medulloblastoma and the like (Zhou WT, Jin WL. (2021) Front Immunol 12: 701006).

[0008] However, despite the broad antitumor effect of B7-H3 targeting therapy, these therapies are still in the early stage of clinical trials and show clinical efficacy for only a small proportion of patients. In addition, it should be noted that these therapies may initially show very good therapeutic effects in some patients, but resistance may develop later. Discovering the cause of the progression of non-responsiveness and resistance in certain patients, and then developing new therapeutic means and methods, is important for further cancer treatment. There is still a great demand in the cancer treatment field for more 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] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0010] The present application provides antibodies that have comparable or higher human / monkey B7-H3 binding affinity / avidity, comparable or higher B7-H3 binding affinity / avidity, and / or comparable or higher B7-H3 binding affinity / avidity compared to prior art antibodies such as enoblituzumab. + The present application 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 antibody dependent cell mediated cytotoxicity (ADCC), and equivalent or higher in vivo anti-cancer activity. The monoclonal antibodies of the present application are + It can be internalized by cells and its binding specificity for human B7-H3 is superior to prior art antibodies such as enoblituzumab.

[0011] The antibodies of the present application may have multiple applications, including the treatment of B7-H3-associated diseases, such as cancer.

[0012] Accordingly, in one aspect, the disclosure provides an isolated monoclonal antibody (e.g., a human antibody) or antigen-binding portion thereof that binds to B7-H3, comprising: 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, the VH CDR2 region, and the VH CDR3 region the CDR3 region may comprise: (1) the amino acid sequence 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 an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; 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, the VL CDR2 region, and the VL CDR3 region are identical to each other; The CDR3 region may comprise: (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) SEQ ID NOs:26, 27, and 28, respectively, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the above sequences.

[0013] The isolated monoclonal antibody, or antigen-binding portion thereof, of the present application can comprise a heavy chain variable region and a light chain variable region, wherein a VH CDR1 region, a VH CDR2 region, a VH CDR3 region, a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region. The CDR3 region may comprise the amino acid sequence 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 an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0014] The heavy chain variable region of the isolated monoclonal antibody, or antigen-binding portion thereof, of the disclosure may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:29, 31, 33, 35 or 37.

[0015] The light chain variable region of the isolated monoclonal antibody, or antigen-binding portion thereof, of the disclosure may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:30, 32, 34, 36 or 38.

[0016] The heavy chain variable region and the light chain variable region of the isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise 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 to the above sequences.

[0017] In another aspect, the disclosure provides an isolated monoclonal antibody (e.g., a human antibody) or antigen-binding portion thereof that binds to B7-H3, comprising: 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 the VH CDR1 region, the VH CDR2 region, and the VH CDR3 region have the identity of the VH CDR1 region, the VH CDR2 region, and the VH CDR3 region of a designated VH sequence; and / or ii) a light chain variable region, where the light chain variable region comprises a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, where the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region have the identity of the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region of a designated VL sequence. and (5) the designated VH sequences and designated VL sequences are one of: (1) the designated VH sequences and designated VL sequences set forth in SEQ ID NOs: 29 and 30, respectively; (2) the designated VH sequences and designated VL sequences set forth in SEQ ID NOs: 31 and 32, respectively; (3) the designated VH sequences and designated VL sequences set forth in SEQ ID NOs: 33 and 34, respectively; (4) the designated VH sequences and designated VL sequences set forth in SEQ ID NOs: 35 and 36, respectively; and (5) the designated VH sequences and designated VL sequences set forth in SEQ ID NOs: 37 and 38, respectively.

[0018] In one embodiment, the isolated monoclonal antibody or antigen-binding portion thereof of the present disclosure may comprise 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. Further, the heavy chain constant region may be, for example, an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, or a functional fragment thereof. +To enhance ADCC against cells, the heavy chain constant region may be engineered 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 comprising the amino acid sequence of SEQ ID NO: 43. In one embodiment, the heavy chain constant region may be a human IgG1 constant region comprising 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 kappa constant region, for example, a human kappa constant region comprising the amino acid sequence of SEQ ID NO: 45, or a functional fragment thereof. The N-terminus of the heavy chain constant region is linked to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is linked to the C-terminus of the light chain variable region.

[0019] The antibodies or antigen-binding portions thereof of the present application can be recombinantly expressed, for example, in certain mammalian cell lines for afucosylation. Cell lines for expressing afucosylated antibodies or antigen-binding portions thereof include, but are not limited to, Slc35C1 knockout cell line, FUT8 knockout cell line, Lec13 cell line (mutated CHO cell line), rat myeloma cell line YB2 / 0, cell lines containing small interfering RNA specific for the gene FUT8, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II.

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

[0021] The present application further provides an immunoconjugate comprising the antibody or antigen-binding portion thereof of the present disclosure, wherein the antibody or antigen-binding portion thereof is linked to a therapeutic agent, such as a cytotoxin or anti-cancer drug. The present disclosure further provides a bispecific molecule comprising the antibody or antigen-binding portion thereof of the present disclosure, wherein the antibody or antigen-binding portion thereof is linked to a second functional moiety (e.g., a second antibody), wherein the second functional moiety has a different binding specificity than the antibody or antigen-binding portion thereof of the present disclosure. In another aspect, the antibody or antigen-binding portion thereof of the present disclosure can 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 antigen-binding portion thereof of the present disclosure can also be encoded by or carried by an oncolytic virus.

[0022] The present application further includes nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, or bispecific molecules of the disclosure, and expression vectors comprising the nucleic acids, and host cells comprising the expression vectors.

[0023] In one embodiment, an expression vector of the present disclosure comprises one or more of the nucleic acids referred to above. In another embodiment, an expression vector of the present disclosure comprises two of the nucleic acids referred to above, each encoding a VH and a VL that can both bind to B7-H3. In another embodiment, the present disclosure provides a pair of vectors, where each vector comprises one of the nucleic acids referred to above, and the pair of vectors both encoding a VH and a VL that can both bind to B7-H3.

[0024] The present disclosure further provides a method for preparing an antibody or antigen-binding portion thereof, or bispecific molecule of the disclosure using a host cell comprising the above expression vector, the method comprising: (i) expressing the antibody, antigen-binding portion thereof, or bispecific molecule in the host cell; and (ii) isolating the antibody, antigen-binding portion thereof, or bispecific molecule from the host cell or a culture thereof.

[0025] The present application further provides a pharmaceutical composition comprising the disclosed antibodies or antigen-binding portions thereof, immunoconjugates, bispecific molecules, immune cells, oncolytic viruses, nucleic acid molecules, expression vectors, or host cells and a pharma- ceutical acceptable carrier.

[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 administering to the subject a therapeutically effective amount of the disclosed pharmaceutical composition. The B7-H3-related disease can be cancer or an autoimmune disease. The cancer can be a solid tumor, 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, desmoplastic small cell tumor, diffuse pontine glioma, medulloblastoma, pancreatic cancer, liver cancer, colorectal cancer, non-Hodgkin's lymphoma, esophageal cancer, ovarian cancer, bladder cancer, small cell carcinoma, endometrial cancer, renal cancer, gastric cancer, acute myeloid leukemia, hepatocellular carcinoma, hypopharyngeal squamous cell carcinoma, urothelial cell carcinoma. The autoimmune disease includes, but is not limited to, asthma. In certain embodiments, the disclosed antibodies or antigen-binding portions thereof may be administered 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 another embodiment, the disclosed antibodies or antigen-binding portions thereof may be administered 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 another embodiment, the disclosed antibodies or antigen-binding portions thereof may be administered with a chemotherapeutic agent, which may be a cytotoxic agent. The disclosed antibodies may be, for example, human antibodies.

[0027] In one aspect, the disclosure provides a method of enhancing an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a disclosed antibody, or antigen-binding portion thereof. The enhanced immune response includes activation of immune cells, including NK cells and the like.

[0028] All documents cited or referenced in this application, including but not limited to all literature documents, patents, and published patent applications 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 for any products mentioned herein or in any documents incorporated by reference herein, are hereby incorporated by reference and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences referred to in this disclosure are incorporated by reference.

[0029] It should be noted that in this disclosure, and particularly in the claims, terms such as "comprise", "comprise" and the like may have the meaning given to them in Chinese patent law; terms such as "consist essentially of" have the meaning given to them in Chinese patent law, e.g., they permit elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of the invention.

[0030] Other features and advantages of the present disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, GenBank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. [Brief description of the drawings]

[0031] The following detailed description, given by way of example and not intended to limit the invention solely to the particular embodiments described, can be best understood in conjunction with the accompanying drawings, in which:

[0032] [Figure 1]Binding activity of fully human anti-B7-H3 antibodies to 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] [Diagram 2] Binding activity of fully human anti-B7-H3 antibodies to B7-H3-tumor cells, including Jurkat cells (A), Daudi cells (B) and Raji cells (C).

[0034] [Diagram 3] Binding activity of fully human anti-B7-H3 antibodies to B7-H3+ normal cells including human prostatic smooth muscle cells (A), human dermal fibroblasts (B), human aortic smooth muscle cells (C), and human dendritic cells (D) is shown.

[0035] [Figure 4] Binding activity of fully human anti-B7-H3 antibodies, i.e., 6B5-WT, 6B5-AF, and 6B5-Mut, with or without Fc engineering, to HUVEC cells (A) and NCI-H520 cells (C), and binding activity of 10D7-WT, 10D7-AF, and 10D7-Mut to HUVEC cells (B) and NCI-H520 cells (D) are shown.

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

[0037] [Figure 6]Shown is the ability of fully human anti-B7-H3 antibodies, i.e., 10D7-WT, 10D7-AF, and 10D7-Mut, with or without Fc engineering, 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] The ability of afucosylated 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) are shown.

[0039] [Figure 8] FIG. 1 shows the ability of afucosylated fully human anti-B7-H3 antibodies, 10D7-AF and 6B5-AF, to induce killing of mature dendritic cells by NK cells.

[0040] [Figure 9] 1 shows uptake of fully human anti-B7-H3 antibody by NCI-H520 cells (A) and A549 cells (B).

[0041] [Figure 10] FIG. 13 is a tree diagram derived from average linkage clustering of B7-H3 epitopes bound by fully human anti-B7-H3 antibodies.

[0042] [Figure 11] Binding affinity of 6B5 antibody to B7 family members by SPR.

[0043] [Figure 12] 1 shows the change in tumor size in mice bearing 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 of the afucosylated fully human anti-B7-H3 antibody 6B5-AF are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] In order to ensure that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0046] The term "B7-H3" refers to B7 homolog 3, also known as cluster of differentiation 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 for human B7-H3 may in certain cases cross-react with B7-H3 protein from another species, such as monkeys. In other embodiments, an antibody specific for human B7-H3 protein may be completely specific for human B7-H3 protein and show no cross-reactivity to other species or other forms, or may cross-react with B7-H3 protein from certain other species but not all other species.

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

[0048] The term "antibody" as used herein is intended to include any of the full-length antibodies, IgG, IgA, IgD, IgE, and IgM, and antigen-binding fragments (i.e., antigen-binding portions) thereof. Full-length antibodies are glycoproteins that may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region can include three domains, C H1 , C H2 and C H3 Each light chain may comprise a light chain variable region (referred to herein as V L The light chain constant region can include one domain, C L V H and V LThe regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. A "functional fragment" of an antibody constant region refers to a fragment of the constant region that retains a specific required function, such as a fragment of a heavy chain constant region that retains FcR / complement system component binding activity.

[0049] The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain 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 within the term "antigen-binding portion" of an antibody include (i) Fab fragments, V L , V H , C L , and C H1 (ii) a F(ab')2 fragment, a bivalent fragment that may include two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a V H and C H1 (iv) a Fd fragment consisting of the V domain of a single arm of an antibody; L and V H (v) dAb fragments (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs); and (viii) nanobodies, heavy chain variable regions comprising a single variable domain and two constant domains. L and V HThe two domains are encoded by separate genes, but can be linked using recombinant methods by a synthetic linker that allows them to be made into a single protein chain, where V L and V H The domains pair to form a monovalent molecule (also known as a single chain Fv (scFv); see, e.g., 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 encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0050] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificity. For example, an isolated antibody that specifically binds to B7-H3 protein is substantially free of antibodies that specifically bind to antigens other than B7-H3 protein. However, an isolated antibody that specifically binds to human B7-H3 protein may have cross-reactivity to other antigens, such as B7-H3 proteins from other species. Furthermore, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0051] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0052] The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline heavy chain-only antibody sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline antibody sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline antibody sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences.

[0053] The term "chimeric antibody" refers to an antibody made by combining genetic material from a non-human source with human genetic material, or more generally, a chimeric antibody is an antibody that has genetic material from one species with genetic material from another species.

[0054] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0055] As used herein, an antibody that "specifically binds to human B7-H3" is intended to refer to an antibody that binds to human B7-H3 protein (and B7-H3 from other non-human species) but does not substantially bind to non-B7-H3 proteins. Preferably, the antibody binds with "high affinity," i.e., greater than 5.0×10 -8 M or lower K D It binds to human B7-H3 protein.

[0056] The term "does not substantially bind" to a protein or cell means that the antibody does not bind or does not bind with high affinity to the protein or cell, i.e., does not bind more than 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0 x 10-3 M or higher, more preferably 1.0×10 -2 M or higher K D This means that the molecule binds to a protein or cell via the agonist.

[0057] The half maximal effective concentration (EC 50 The term "antibody concentration" refers to the concentration of antibody that gives half (50%) of the maximal response.

[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 cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which effector immune cells actively lyse target cells whose cell surface antigens are bound by an antibody, 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, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, with mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, being preferred.

[0061] The term "therapeutically effective amount" refers to an amount of an antibody of the present disclosure that is sufficient to prevent or alleviate symptoms associated with a disease or disorder (e.g., cancer). The therapeutically effective amount is relative to the disorder being treated, and the actual effective amount can be readily discerned by one of ordinary skill in the art.

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

[0063] The antibodies, or antigen-binding portions thereof, of the present disclosure are directed to B7-H3 +The disclosed monoclonal antibodies or antigen-binding portions thereof can bind to B7-H3 in human, monkey, and the like, including human cells, with binding activity / affinity equivalent to or better than that of prior art antibodies such as enoblituzumab. In addition, compared to prior art antibodies such as enoblituzumab, the disclosed antibodies or antigen-binding portions thereof have equivalent or higher immune cell (e.g., NK cell) activation capability, equivalent or higher antibody-dependent cell-mediated cytotoxicity (ADCC) induction capability, and equivalent or better in vivo anti-cancer activity. The disclosed monoclonal antibodies or antigen-binding portions thereof can bind to B7-H3 + It can be internalized by cells, and its binding specificity to human B7-H3 is better than prior art antibodies such as enoblituzumab.

[0064] Preferred antibodies of the present disclosure are monoclonal antibodies. The antibodies can be, for example, human or chimeric monoclonal antibodies.

[0065] Exemplary antibodies, or antigen-binding portions thereof, of the disclosure are structurally and chemically characterized below.

[0066] The heavy chain variable region CDRs and light chain variable region CDRs of the disclosed antibodies or antigen-binding portions thereof are defined by the Kabat numbering system. SEQ ID NOs of the CDR amino acid sequences and SEQ ID NOs of the heavy / light chain amino acid sequences are set forth in Table 1A. The heavy chain variable region CDRs and light chain variable region CDRs of the disclosed antibodies or antigen-binding portions thereof can also be determined by the Chothia numbering system, and SEQ ID NOs of the CDR amino acid sequences determined by such a system are set forth in Table 1B. Table 1A SEQ ID NOs of heavy / light chain variable region CDRs and heavy / light chain variable region amino acid sequences as determined by the Kabat numbering system [Table 1] Table 1B SEQ ID NOs of heavy / light chain variable region CDRs and heavy / light chain variable region amino acid sequences as determined by the Chothia numbering system [Table 2]

[0067] In addition to the above exemplary CDRs determined by the Kabat and Chothia numbering system, the heavy / light chain variable regions of the disclosed antibodies may contain 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 the heavy and light chain variable region CDRs may also be determined by, for example, the IMGT, AbM or Contact numbering systems / methods.

[0068] The disclosed antibodies can comprise a heavy chain constant region, such as a human IgG1 constant region, e.g., comprising the amino acid sequence of SEQ ID NO: 43, an IgG1 constant region. The constant region can be engineered to have enhanced FcR binding capability, such as a human IgG1 constant region comprising the amino acid sequence of SEQ ID NO: 44 (L235V, F243L, R292P, Y300L and P396L according to EU numbering). The light chain constant region can be a kappa constant region, such as a human kappa constant region, which can comprise 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 sequences (or CDR sequences) of the anti-B7-H3 antibodies of the present disclosure are H and V L Preferably, the V H and V L When chains (or CDRs within such chains) are mixed and combined, a particular V H / V L V from the pair H The sequence is structurally similar to V H Similarly, preferably, a particular V H / V L V from the pair L The sequence is structurally similar to V L It is replaced with an array.

[0070] Thus, in one embodiment, an antibody or antigen-binding portion thereof of the disclosure may include: (a) a heavy chain variable region comprising an amino acid sequence set forth in Table 1; and (b) a light chain variable region comprising an amino acid sequence set forth in Table 1, or the V of another anti-B7-H3 antibody. L , where the antibody specifically binds to human B7-H3.

[0071] In another embodiment, an antibody, or antigen-binding portion thereof, of the disclosure comprises: (a) CDR1, CDR2, and CDR3 of a heavy chain variable region as set forth in Table 1; and (b) CDR1, CDR2 and CDR3 of a light chain variable region set forth in Table 1, or the CDRs of another anti-B7-H3 antibody, where the antibody specifically binds to human B7-H3.

[0072] In yet another embodiment, the disclosed antibodies or antigen-binding portions thereof comprise the CDRs of another antibody that binds to human B7-H3, e.g., CDR1 and / or CDR3 from the heavy chain variable region, and / or the 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, it is known in the art that the CDR3 domain alone, independent of the CDR1 and / or CDR2 domains, can determine the binding specificity of an antibody to a similar antigen, 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. Patent Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185, which are incorporated herein by reference in their entireties.

[0074] In another embodiment, the disclosed antibodies may comprise CDR2 of the heavy chain variable region of an anti-B7-H3 antibody and at least CDR3 of the heavy and / or light chain variable region of an anti-B7-H3 antibody, or CDR3 of the heavy and / or light chain variable region of another anti-B7-H3 antibody, where the antibody is capable of specifically 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 similar binding affinity as the anti-B7-H3 antibodies of the present disclosure. In yet another embodiment, the antibodies may further comprise CDR2 of the light chain variable region of an anti-B7-H3 antibody, or CDR2 of the light chain variable region of another anti-B7-H3 antibody, where the antibody is capable of specifically binding to human B7-H3. In another embodiment, the disclosed antibody can comprise CDR1 of the heavy and / or light chain variable region of an anti-B7-H3 antibody, or the CDR1 of the heavy and / or light chain variable region of another anti-B7-H3 antibody, wherein the antibody is capable of specifically binding to human B7-H3.

[0075] In another embodiment, the disclosed antibodies may comprise heavy and / or light chain variable region sequences of CDR1, CDR2 and CDR3 sequences that differ from the anti-B7-H3 antibodies of the present disclosure by one or more conservative modifications. It is understood in the art that certain conservative sequence modifications can be made that do not eliminate antigen binding. See, e.g., 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] Thus, in one embodiment, the antibody comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise CDR1, CDR2 and CDR3, wherein: (a) the heavy chain variable region CDR1 comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (b) the heavy chain variable region CDR2 comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (c) the heavy chain variable region CDR3 comprises a sequence listed in Table 1 above, or a conservative modification thereof; and / or (d) the light chain variable region CDR1 and / or CDR2 and / or CDR3 comprises a sequence listed in Table 1 above and / or a conservative modification thereof; and (e) The antibody specifically binds to human B7-H3.

[0077] The disclosed antibodies have high affinity and high binding specificity for human B7-H3, and enhanced FcR binding affinity and, therefore, B7-H3 + These may include one or more of the following functional characteristics, such as an enhanced ability to induce ADCC against tumor cells.

[0078] In certain embodiments, the antibody, or antigen-binding portion thereof, can be, for example, human or chimeric.

[0079] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can 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 one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with 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). Thus, one or more amino acid residues in the CDR regions of the disclosed antibodies can be substituted with other amino acid residues from the same side chain family, and the altered antibodies can be tested for retained function (i.e., the functions described above) using the functional assays described herein.

[0080] The disclosed antibodies include the V of the anti-B7-H3 antibodies of the present disclosure. H / V L An antibody having one or more of the sequences can be prepared as a starting material for engineering an engineered antibody. The antibody can have one or both variable regions (i.e., V H and / or V L ), for example, by modifying one or more residues in one or more CDR regions and / or one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant regions, for example to alter the effector functions of the antibody.

[0081] In certain embodiments, CDR grafting can be used to engineer the variable region of an antibody. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because the CDR sequences are responsible for the majority of antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors containing the CDR sequences from a particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., 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. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0082] Accordingly, another embodiment of the disclosure relates to isolated monoclonal antibodies, or antigen-binding portions thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the disclosure set forth above, and / or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the disclosure set forth above. These antibodies are similar to the V of the monoclonal antibodies of the disclosure. H and V L It contains the CDR sequences but may contain different framework sequences.

[0083] Such framework sequences can be obtained from public DNA databases or published references that contain 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 on the Internet 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 cited above, the contents of each of which 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 the HCo7 HuMAb mouse are available at attached Genbank accession numbers 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 at 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 compiled protein sequence databases using one of the sequence similarity searching methods known to those of skill in the art called Gapped BLAST (Altschul et al., (1997) supra).

[0085] Preferred framework sequences used in the disclosed antibodies are of similar structure to the framework sequences used by the disclosed antibodies. H The CDR1, CDR2, and CDR3 sequences can be grafted into framework regions having sequences identical to those found in a germline immunoglobulin gene (the framework sequences are derived from a germline immunoglobulin gene), or the CDR sequences can be grafted into framework regions that contain one or more mutations compared to the germline sequence. For example, in certain cases, it has been found to be beneficial to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0086] Another type of variable region modification is the V H and / or V L The amino acid residues in the CDR1, CDR2, and / or CDR3 regions are mutated to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effect on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays, as known in the art. Preferably, conservative modifications (known in the art) are introduced. The mutations can be amino acid substitutions, additions, or deletions, but are preferably substitutions. Furthermore, typically no more than 1, 2, 3, 4, or 5 residues in the CDR regions are altered.

[0087] Thus, in another embodiment, the disclosure provides a method for the preparation of a polypeptide comprising: (a) a V polypeptide having an amino acid sequence of the disclosure or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; H (b) a V CDR1 region comprising an amino acid sequence of the disclosure or having one, two, three, four, or five amino acid substitutions, deletions, or additions; H(c) a V CDR2 region comprising an amino acid sequence of the disclosure or having one, two, three, four, or five amino acid substitutions, deletions, or additions; H (d) a V sequence comprising an amino acid sequence of the disclosure or having one, two, three, four, or five amino acid substitutions, deletions, or additions; L (e) a V CDR1 region comprising an amino acid sequence of the disclosure or having one, two, three, four, or five amino acid substitutions, deletions, or additions; L and (f) a V sequence comprising the amino acid sequence of the present disclosure or having one, two, three, four, or five amino acid substitutions, deletions, or additions. L An isolated anti-B7-H3 monoclonal antibody, or antigen-binding portion thereof, is provided, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region including a CDR3 region.

[0088] The disclosed engineered antibodies can be modified, for example, by modifying the V H and / or V L Framework modifications include those in which modifications are made to framework residues within the CDR region. Framework modifications include the mutation of one or more residues in the framework region or even one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043.

[0089] In addition to, or instead of, modifications made in the framework or CDR regions, the disclosed antibodies can be engineered to include modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Further, the disclosed antibodies can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, which also alters one or more functional properties of the antibody.

[0090] In one embodiment, C H1The hinge region of is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This technique is further described in U.S. Pat. No. 5,677,425. H1 The number of cysteine ​​residues in the hinge region of the antibody is altered to, for example, facilitate 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 the antibody is mutated to increase or decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are made in the C of the Fc hinge fragment such that the antibody has weaker Staphylococcus protein A (SpA) binding compared to the SpA binding of the native Fc hinge domain. H2 -C H3 The domain interface region is introduced. This approach is described in further 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 can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such aglycosylation can increase the affinity of the antibody for an antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0093] Additionally or alternatively, antibodies can be made with altered glycosylation types, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase or decrease the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art, including, but not limited to, Slc35C1 knockout cell line, FUT8 knockout cell line, Lec13 cell line (mutated CHO cell line), rat myeloma cell line YB2 / 0, cell lines containing small interfering RNA specific for the gene fut8, 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 disclosed recombinant antibodies, thereby producing antibodies with altered glycosylation. Slc35C1 knockout cell lines can be prepared, for example, by adopting the fucose removal platform developed by MabWorks. See the CHO cell line described in U.S. Patent No. 10,377,833 B2, Accession No. CGMCC No. 14287.

[0094] Another modification of the antibodies herein is PEGylation. Antibodies may be PEGylated, for example, to increase the biological (e.g., blood) half-life of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to mono (C1-C 10It 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 PEGylating proteins are known in the art and can be applied to the disclosed antibodies. See, e.g., EP 0 154 316 and EP 0 401 384.

[0095] The disclosed antibodies can be characterized by various physical properties to detect and / or distinguish their different classes.

[0096] For example, an antibody may contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or alteration of 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 the N-X-S / T sequence. In some cases, it is preferable to have an anti-B7-H3 antibody that does not contain variable region glycosylation. This can be achieved either by selecting an antibody that does not contain glycosylation motifs in the variable region or by mutating residues within the glycosylation region.

[0097] In a preferred embodiment, the antibody does not contain an asparagine isomerism site. Deamidation of asparagine can occur at NG or DG sequences, 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 has a unique isoelectric point (pI) that generally falls in the pH range of 6 to 9.5. The pI of an IgG1 antibody typically falls in the pH range of 7 to 9.5, and the pI of an IgG4 antibody typically falls in the pH range of 6 to 8. It is believed that antibodies with a pI outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, it is preferable to have an anti-B7-H3 antibody that contains a pI value that falls in the normal range. This can be achieved either by selecting an antibody with a pI in the normal range or by mutating charged surface residues.

[0099] In another aspect, the disclosure provides a nucleic acid molecule encoding the heavy and / or light chain variable region or CDR of the disclosed antibody or antigen-binding portion thereof. The nucleic acid molecule may be present in a whole cell, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid molecule is "isolated" or "substantially pure" when it has been purified by standard techniques from other cellular components or other contaminants, such as other cellular nucleic acids or proteins. The nucleic acid molecule of the present 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 the present disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from genetically modified mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies made by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), nucleic acid molecules encoding such antibodies can be retrieved from gene libraries.

[0101] A preferred nucleic acid molecule of the disclosure is the V H and V L V includes sequences encoding the V sequences or CDRs. H and V L Once DNA fragments encoding the segments have been obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L Or V H The DNA fragment encoding is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are connected such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0102] V H The isolated DNA encoding the region was H The DNA encoding the heavy chain constant region (C H1 , C H2 , and C H3The heavy chain constant region may be converted to a full-length heavy chain gene by operably linking to another DNA molecule encoding the V H The DNA encoding the heavy chain C H1 It may be operably linked to another DNA molecule encoding only the constant region.

[0103] V L The isolated DNA encoding the region was L The DNA encoding the light chain constant region C L The light chain constant region may be converted to a full-length light chain gene by operably linking it to another DNA molecule encoding the light chain constant region. 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 a preferred embodiment, the light chain constant region may be a kappa or lambda constant region.

[0104] To generate the scFv gene, H and V L The DNA fragment encoding the V is operably linked to another fragment encoding a flexible linker, thereby H and V L The sequence is V L and V H The regions can be expressed as a contiguous single-chain protein connected by a flexible linker (see, e.g., 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 the present 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 viral or carcinogen-mediated B lymphocyte transformation, and phage display techniques. Chimeric or humanized antibodies are well known in the art. See, e.g., U.S. Patent Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

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

[0107] The term "control sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such control sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred control 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), adenovirus, e.g., the adenovirus major late promoter (AdMLP) and the polyoma virus enhancer. Alternatively, non-viral control sequences, such as the ubiquitin promoter or the β-globulin promoter, can be used. Furthermore, regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

[0108] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In a preferred embodiment, the variable regions are used to create full-length antibody genes of any antibody isotype. They are inserted into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype. The resulting V H The segment is located in the vector H operably connected to the V segment; L The segment is located in the vector LThe antibody chain segment is operably linked to the antibody segment. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0109] In addition to the antibody chain genes and control sequences, the disclosed recombinant expression vectors may carry additional sequences, such as sequences that control replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin or methotrexate, on the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0110] For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the disclosed antibodies in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, with mammalian host cells being most preferred, is most preferred because such eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete properly folded, immunoreactive antibodies compared to prokaryotic cells.

[0111] Preferred mammalian host cells for expressing recombinant antibodies of the disclosure include Slc35C1 knockout cell lines, FUT8 knockout cell lines, Lec13 cell lines (mutated CHO cell lines), rat myeloma cell line YB2 / 0, cell lines containing small interfering RNA specific for the gene fut8, 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, e.g., used with the DHFR selectable marker as described in 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 a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or, more preferably, secretion of the antibody into the culture medium that the host cell is grown in. Antibody can be recovered from the culture medium using standard protein purification methods.

[0112] The antibodies or antigen-binding portions thereof of the present disclosure may be conjugated with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, 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 an ADC, 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 Publication Nos. WO02 / 096910; WO07 / 038,658; WO07 / 051,081; WO07 / 059,404; WO08 / 083,312; and WO08 / 103,693; U.S. Patent Publication Nos. 20060024317; 20060004081; and 20060247295.

[0113] In another aspect, the disclosure features bispecific molecules that can include one or more of the disclosed antibodies linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody, or a ligand for a receptor), 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 with three or more specificities.

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

[0115] Also provided herein is a chimeric antigen receptor comprising an anti-B7-H3 scFv, the anti-B7-H3 scFv comprising the CDRs and heavy / light chain variable regions described herein.

[0116] The anti-B7-H3 chimeric antigen receptor can comprise: (a) an extracellular antigen-binding domain comprising an 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 antigen-binding portions thereof of the present disclosure can be used in conjunction with oncolytic viruses. Alternatively, oncolytic viruses encoding the disclosed antibodies or antigen-binding portions thereof can be introduced into the human body.

[0118] In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more of the disclosed antibodies or antigen-binding portions thereof, vectors encoding the antibodies or antigen-binding portions, immunoconjugates, immune cells, bispecific antibodies, and / or oncolytic viruses, formulated with a pharma- ceutically acceptable carrier. The composition may optionally contain one or more additional pharma- ceutical active ingredients, such as antitumor antibodies, or antibodies for immune enhancement, or non-antibody antitumor or immune enhancement agents. The pharmaceutical composition of the present disclosure may be used, for example, in combination with antitumor agents or agents for immune enhancement.

[0119] Pharmaceutical compositions may contain any number of excipients.The excipients that can be used include carriers, surface active agents, thickeners or emulsifiers, solid binders, dispersing or suspending aids, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents, and combinations thereof.The selection and use of preferred excipients is 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 phrase "parenteral administration" refers to a method of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibody of the present disclosure may be administered other than parenteral routes, such as topical, epidermal or mucosal routes of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0121] The pharmaceutical compositions can be in the form of a sterile aqueous solution or dispersion, or they can be formulated as microemulsions, liposomes, or other ordered structures suitable to high drug concentration.

[0122] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular method of administration, but will generally be that amount of the composition which produces a therapeutic effect. Generally, this amount will range from about 0.01% to about 99% of the active ingredient combined with a pharma- ceutically acceptable carrier out of one hundred percent.

[0123] The dosage regimen is adjusted to provide the optimum 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 as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated. Each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required.

[0124] For administration of antibodies, dosages may range from about 0.001 to 100 mg / kg. An exemplary treatment regime involves administration once per week. A preferred means of administration of anti-B7-H3 includes intravenous administration.

[0125] A "therapeutically effective dose" of an anti-B7-H3 antibody of the present disclosure results in a reduction in the severity of disease symptoms and an increase in the frequency and length of disease symptom-free periods. For example, when treating a subject with a tumor, a "therapeutically 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 even more preferably at least about 80%, compared to an untreated subject. An effective amount of a therapeutic antibody may reduce tumor size or alleviate symptoms in the subject. The subject may be a human or another mammal.

[0126] The pharmaceutical composition can be a sustained release formulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, etc.See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0127] The pharmaceutical compositions may be administered via medical devices such as: (1) needleless hypodermic syringes (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion devices (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0128] In certain embodiments, the antibodies of the present disclosure can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of the disclosure cross the blood-brain barrier, they can be formulated in liposomes, which can additionally contain targeting moieties that enhance 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 Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

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

[0130] Considering the ability of the disclosed pharmaceutical composition to inhibit tumor cell proliferation and survival, the present application provides a method for inhibiting tumor cell proliferation in a subject, comprising administering the disclosed pharmaceutical composition to the subject, such that tumor proliferation is inhibited in the subject.Non-limiting examples of tumors that can be treated by the disclosed antibody 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, desmoplastic small cell tumor, diffuse pontine glioma, medulloblastoma, pancreatic cancer, liver cancer, colorectal cancer, non-Hodgkin's 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, urothelial cell carcinoma.Refractory or recurrent malignant tumors can also be treated by the disclosed pharmaceutical composition.The disclosed pharmaceutical composition can also treat autoimmune diseases such as asthma.

[0131] The disclosed pharmaceutical compositions can be used to enhance an immune response in a subject, for example, to activate immune cells such as NK cells and T cells.

[0132] The present application provides combination therapy in which the disclosed pharmaceutical composition is administered with one or more other antibody or non-antibody therapeutic agents that can effectively inhibit tumor growth in a subject. In one embodiment, the present application provides a method for inhibiting tumor growth in a subject, the method comprising administering to the subject 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. In certain embodiments, the subject is a human. In another aspect, the present application provides a method of cancer treatment in which the disclosed pharmaceutical composition is administered with a chemotherapeutic agent, where the chemotherapeutic agent can be a cytotoxic agent. Other therapies that can be combined with the disclosed pharmaceutical composition include, but are not limited to, administration of an immunogen, administration of interleukin 2 (IL-2), radiation therapy, surgery, or hormone ablation.

[0133] The combination of therapeutic agents described herein may be administered simultaneously as a single composition in a pharma- ceutically acceptable carrier, or may be administered simultaneously as separate compositions with each agent in a pharma- ceutically acceptable carrier, hi another embodiment, the combination of therapeutic agents may be administered sequentially.

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

[0135] The present disclosure is further illustrated in the following examples, which should not be construed as further limiting. The figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference in their entirety. example Example 1 Generation 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 (human B7-H3(4Ig) / B7-H3b protein with His tag, Cat#: B7B-H52E7, ACROBiosystems) and / or recombinant human B7-H3(2Ig) protein (human B7-H3 / CD276 protein with His tag (MALS certified), Cat#: B73-H52E2, ACROBiosystems). RenMab mice contain a humanized immunoglobulin heavy chain locus and a humanized immunoglobulin kappa chain locus. The immunoglobulin heavy chain locus includes a region with genes encoding antibody heavy chains, which includes IGHV (variable), IGHD (diversity), IGHJ (joining), and heavy chain constant region genes. The immunoglobulin kappa chain locus includes genes encoding antibody light chains (kappa chains). The immunoglobulin kappa chain locus includes IGKV (variable), IGKJ (joining) and light chain constant region genes. A detailed description of RenMab mice can be found in PCT / CN2020 / 075698, which is incorporated herein by reference in its entirety. B7-H3RenMab KO mice were obtained by knocking out the B7-H3 gene in RenMab mice and did not express functional B7-H3 protein. Mouse immunization

[0137] Immunizations were performed four times in B7-H3RenMab KO mice using recombinant human B7-H3(4Ig) protein. The protein was mixed with adjuvant in a 1:1 v / v ratio and then emulsified. Each animal was injected in 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). The two injections should be separated by at least 14 days.

[0138] In another study carried out in parallel, immunizations were alternatively performed using recombinant human B7-H3(4Ig) and recombinant human B7-H3(2Ig) proteins. Proteins and adjuvants were mixed in a 1:1 v / v ratio and then emulsified. Each animal was injected in 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). The two injections should be separated by at least 14 days.

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

[0140] A 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 expressing the B7-H3 antigen on their surface. Antibody Discovery

[0141] Four days after the last immunization, 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 antigen-specific monoclonal antibody-secreting plasma cells. Antibody variable region sequences were obtained by reverse transcription and PCR, and the antibody heavy / light chain variable regions were cloned into vectors encoding human IgG constant regions and human kappa constant regions to express antibodies, and their binding specificity to B7-H3 was tested by FACS. Exemplary fully human antibodies obtained include 6B5, 10D7, 10F5, 10F7, and 15F11, with heavy and light chain variable region and CDR sequences and SEQ ID NOs listed in Tables 1 and 9. Antibody construction, expression and purification

[0142] Fragments encoding the variable region and human IgG1 / κ constant region (amino acid sequences of the heavy and light chain constant regions set forth in SEQ ID NOs: 43 and 45, respectively) were inserted between the XhoI / BamHI restriction sites in pCDNA3.1 (Invitrogen, USA) to construct an antibody expression vector.

[0143] The expression vectors obtained as above were used to transfect HEK-293F cells (Cobioer, China) with PEI. In particular, 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 with polyethylenimine (PEI), the ratio of DNA to PEI was 1:3, where the amount of DNA was 1.5 μg per milliliter of cell culture medium. The transfected 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 min, and filtered through a 0.22 μm membrane to remove cell debris. Monoclonal antibodies were concentrated and purified by passing through a pre-equilibrated Protein A affinity column (Cat#: 17040501, GE, USA) and eluted using elution buffer (20 mM citric acid, pH 3.0-pH 3.5). The antibodies were then stored in PBS (pH 7.0) and the antibody concentration was measured in 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) provided with a pre-immobilized protein A sensor chip. Briefly, anti-human B7-H3 antibodies (1 μg / mL) were injected at 10 μL / min for 20 seconds into the Biacore T200 biosensor to achieve the desired protein density (100±30 RU). Antigen proteins were then 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 30 μL / min for 180 seconds and dissociation was monitored for 600 seconds. Enoblituzumab, 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 set forth in SEQ ID NOs: 39 and 40, respectively, the heavy chain constant region is an IgG1 constant region with five point mutations, F243L / R292P / Y300L / L235V / P396L, to enhance ADCC, and the light chain constant region is a kappa constant region with sequences set forth in SEQ ID NOs: 44 and 45, respectively) was used as a positive control.

[0145] The generated data was analyzed using Biacore Insight V2.0.1 5.12933 using a one-to-one Langmuir binding model. Association rates (k on ) and dissociation rate (k off ) were obtained simultaneously. The affinity was calculated using two kinetic rate constants (K D =k off / k on ) was calculated as the quotient of

[0146] All of the disclosed antibodies bound to the two splice variants of human B7-H3 and to the monkey B7-H3 protein. Antibodies 6B5 and 15F11 also bound to the mouse B7-H3 protein. Affinity test results for representative disclosed antibodies 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 antibodies 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 antibodies 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 + As tumor cells, Jurkat cells (Cat#:CBP60520, COBIOER), Daudi cells (Cat#:CBP60262, COBIOER), and Raji cells (Cat#:CBP60272, COBIOER) were used as representative 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 PBMC) as representative normal cells.

[0148] A549, NCI-H520, Jurkat, Daudi and Raji 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 and SK-OV-3 cells were cultured in MEM medium (Cat#: 12561-056, Gibco) and McCoy's 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 specific medium (Cat#: CM-0149, ProCell), 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 subsequent in vitro induction. Briefly, PBMCs (Cat#:PB004F-C, ALLCELLS) were resuspended in RPMI1640 medium and incubated in a 37°C incubator for 2 hours. The cells that adhered to the wall, i.e., isolated monocytes, were harvested. Monocytes were cultured in RPMI1640 medium supplemented with 100ng / ml recombinant human GM-CSF (Cat#:7954-GM, R&D), 100ng / 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 one. On day 6 of culture, the culture medium was replaced with medium containing 100ng / ml recombinant human GM-CSF, 100ng / ml recombinant human IL-4, 10ng / ml rhTNF-α (Cat#: 210-TA-100, R&D), 1000U / ml rhIL-6 (Cat#: 7270-IL-025, R&D), 1μg / ml PEG2 (Cat#: 363-24-6, TOCRIS), and 10ng / ml IL-1β (Cat#: 210-LB-025, R&D). After incubating the cells for another 2 days, mature human dendritic cells were obtained.

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

[0151] As shown in Figure 1, 6B5 and 10D7 bind to various types of B7-H3 + Both bind to tumor cells and the binding activity of both is significantly higher than that of enoblituzumab. B7-H3 mRNA - It can be seen from FIG. 2 that there was almost no binding of 6B5 or 10D7 to tumor cells, and the binding activity was significantly lower than that of enoblituzumab. All of this suggests that the disclosed antibodies have better B7-H3 binding specificity. As shown in FIG. 3, multiple normal human B7-H3 bindings were observed. + The binding ability of 6B5 to cells was slightly higher than that of enoblituzumab, while the binding activity of 10D7 was comparable to that of enoblituzumab. Example 4 Fc manipulation, B7-H3 + Did not affect binding of fully human anti-B7-H3 antibodies to cells

[0152] Fragments encoding the heavy / light chain variable regions and human IgG1 / κ constant regions (SEQ ID NO: 43 and 45) of 10D7 and 6B5 were cloned into pCDNA3.1 (Invitrogen, USA) to construct expression vectors that were transfected into CHO-K1 cells and slc35c1 knockout CHO-K1-AF cells. slc35c1 knockout CHO-K1-AF cells were produced by Beijing MabWorks Biotech Inc., and the detailed production method can be found in US10377833B2, and the proteins expressed by such cell lines are essentially free of fucosylation. 10D7 and 6B5 were transiently expressed and purified in CHO-K1 and CHO-K1-AF cells, and the obtained antibodies were designated as 10D7-WT, 6B5-WT, 10D7-AF (afucosylated) and 6B5-AF (afucosylated). Meanwhile, expression vectors were constructed in which fragments encoding the heavy / light chain variable regions of 10D7 and 6B5, and the five-point mutated human IgG1 Fc / κ constant region (SEQ ID NO: 44 (L235V, F243L, R292P, Y300L and P396L according to EU numbering) and 45) were cloned into pCDNA3.1 and transfected into CHO-K1 cells for transient expression, and the obtained 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 in Example 1.

[0153] These anti-B7-H3 antibodies were tested for their binding ability to HUVEC cells (Cat#: C-12200, PromoCell, Germany) and NCI-H520 cells expressing human B7-H3 by FACS. 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 of NCI-H520 cell culture and FACS testing, please refer to Example 3.

[0154] As shown in FIG. 4, changes in the Fc region, afucosylation or point mutations, enhance the expression of B7-H3 + The binding of anti-B7-H3 antibody to cells was not affected. 10D7-AF, 10D7-WT, and 10D7-Mut showed completely identical binding activity to HUVEC (B) and NCI-H520 cells (D), and 6B5-AF, 6B5-WT, and 6B5-Mut showed almost identical binding activity to HUVEC (A) and NCI-H520 cells (C). Example 5 Fully human anti-B7-H3 antibody activity in inducing ADCC and activating immune cells

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

[0156] Briefly, NCI-520 cells, HUVEC cells and DCs were cultured at 1.0 × 10 6 The cells were resuspended in complete RPMI medium at a cell density of 100 / ml. The cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, Cat#;C34554I, Invitrogen). Specifically, the cells were incubated with 2.5 μM CFSE for 10 min at 37°C. After labeling, the 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 min. 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 about 90% according to the cell count results.5 50 μl of target cells (NCI-H520 cells, HUVEC cells or DCs) at a cell density of 10 / ml and 2 × 10 6 50μl of NK92MI-CD16a cells at a cell density of 100 / ml were added to each well of a 96-well plate, with an effector to target ratio of 5:1. Antibodies to be tested were added to each well at different concentrations, with a starting final concentration of 50000ng / ml, and other final concentrations were diluted 5-fold, resulting in a total of 10 concentrations. The 96-well plate was incubated at 37°C for 4 hours, and the cells on the plate were washed 3 times with PBS, and then incubated with LIVE / DEAD fixable dead cell stain for 30 minutes at 37°C. The cells on the plate were washed 3 times with PBS, and 2μl of PE mouse anti-human CD69 antibody (Cat#: 555531, BD) was added. After incubation at room temperature for 30 minutes, the plate was centrifuged, washed 3 times with PBS, and subjected to FACS. CFSE + The cell death rate of cells (NCI-H520 cells, HUVEC cells, or DCs) was determined, and CFSE - The mean PE staining fluorescence intensity of cells (NK92MI-CD16a cells) was calculated. Enoblituzumab was used as a positive control.

[0157] Anti-B7-H3 antibody was further tested for its ability to induce human PBMC-mediated ADCC against NCI-H520 and HUVEC cells, where NCI-H520 and HUVEC cells were labeled using carboxyfluorescein succinimidyl ester (CFSE, Cat#: C34554I, Invitrogen). Human PBMCs were obtained by density gradient centrifugation using lymphocyte separation medium and cultured overnight in medium (RIPM1640+10% FBS+300IU IL-2). ADCC test was performed using LIVE / DEAD fixable dead cell staining kit (Thermo Fisher, USA, Cat#: L34964). Target and effector cells (PBMCs) were centrifuged at 1200 rpm for 5 min. Cells were resuspended in ADCC test medium (RIPM1640 medium+1% FBS) and cell viability was about 90% according to cell count results. 4×10 5 50 μl of target cells at a cell density of 10 / ml and 4 × 10 6 50 μl of PBMCs with a cell density of 100 / ml were added to each well of a 96-well plate, with an effector:target ratio of 10:1. Antibodies to be tested were added to each well, starting at a final concentration of 50000ng / ml, and the other final concentrations were diluted 5-fold, resulting in a total of 10 concentrations. The 96-well plate was incubated at 37°C for 24 hours, and the supernatant from each well was harvested to measure IFN-γ concentration by ELISA (Cat#: SIF50, R&D) according to the manufacturer's manual. The cells on the plate were washed 3 times with PBS and incubated with LIVE / DEAD fixable dead cell stain for 30 minutes at 37°C. The cells were washed 3 times with PBS and subjected to FACS testing. CFSE + The cell death rate of cells (NCI-H520 cells or HUVEC cells) was determined. Enoblituzumab was used as a positive control. The test results are shown in Figures 5 to 8.

[0158] As shown in Figure 5, 10D7-AF and 10D7-Mut have significantly higher abilities to induce NK92-mediated ADCC against NCI-H520 tumor cells than 10D7-WT, and their abilities are also higher than enoblituzumab, the positive control (A); 10D7-AF induces higher levels of the activation marker CD69 on the NK cell surface, thus indicating a higher NK cell activation ability than 10D7-Mut (C). Similarly, the abilities of 6B5-AF and 6B5-Mut to induce ADCC are significantly higher than 6B5-WT, and also higher than enoblituzumab, the positive control (B); in addition, 6B5-AF induces higher levels of CD69, thus indicating a higher NK cell activation ability than 6B5-Mut (D). In addition, the positive control enoblituzumab showed a clear hook effect in inducing NK92-mediated NCI-H520 killing at high doses. That is, as the antibody concentration was increased after reaching the designated value, the killing effect detected decreased, an effect that was not observed with 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 is significantly higher than that of 10D7-WT and also higher than that of the positive control enoblituzumab (A). Such a trend was also observed for 6B5 in ADCC induction, i.e., the ADCC induced by 6B5-AF and 6B5-Mut against NCI-H520 tumor cells is more potent than that of 6B5-WT and also higher than that of the positive control enoblituzumab (B). Similarly, the positive control enoblituzumab showed a clear hook effect in inducing PBMC-mediated NCI-H520 death at high doses. This effect was not observed in 10D7 or 6B5.

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

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

[0162] To test whether the disclosed anti-B7-H3 antibodies can be internalized into cells, A549 cells and NCI-H520 cells were used for internalization, and enoblitutamab-IgG1-Mut and omburtamab-IgG1 were used as positive controls. Omburtamab is a radioiodine-labeled anti-human B7-H3 monoclonal antibody developed by Y-mAbs Therapeutics, and omburtamab-IgG1 comprises the heavy and light chain variable regions of SEQ ID NOs: 41 and 42, the heavy chain constant region is wild-type IgG1, the light chain constant region is a kappa constant region, and comprises the amino acid sequences of SEQ ID NOs: 43 and 45, respectively.

[0163] The anti-human B7-H3 antibody to be tested was mixed homogeneously with pHAb-labeled goat anti-human IgG1 Fc (Cat#:SSA015, Sino Biological, labeled with pHAb) at a concentration ratio of 1:1. This mixture was mixed homogeneously with 10000 A549 cells, the final concentration of both anti-B7-H3 antibody and secondary antibody was 25μg / mL, and human IgG1 protein was used in blank control wells. The mixture was incubated on ice in the dark for 1 hour, washed three times using centrifugation with pre-cooled FACS buffer (90%DMEM+10%FBS) (supernatant was discarded), and pre-heated complete culture medium was added and incubated together in a 37℃ 5%CO2 incubator. The 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 for 3 min in low temperature to discard the supernatant, and washed once by adding PBS buffer. The fluorescence signals from the cell surface were read. For cells incubated with different antibodies, raw data were collected from flow cytometry, and the MFI of the fluorescence emitted from cells that had internalized different antibodies by binding to cell surface B7-H3 was analyzed and determined based on the raw data. The MFI curves were plotted based on the amount of anti-B7-H3 antibody internalized.

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

[0165] The results are shown in Figure 9. During the entire study, no uptake occurred when cells alone, cells and secondary antibody alone, or cells, secondary antibody and human IgG1 protein alone were incubated, but the MFI tended to increase with time when cells were incubated with 6B5, 10D7, 10F5, 10F7, enobrituzumab or omburtamab-IgG1. In particular, a clear increase in MFI was observed for 10D7, 10F7, and omburtamab-IgG1. The data showed that 6B5, 10D7, 10F5, 10F7, enobrituzumab and omburtamab-IgG1 could all be taken up by NCI-H520 and A549 cells, where 10D7, 10F7 and omburtamab-IgG1 were taken up at a relatively high uptake rate. Example 7 Analysis of epitopes bound by fully human anti-B7-H3 antibodies

[0166] To determine the relationship between the epitopes bound by the anti-human B7-H3 antibodies, the inhibitory effect of each anti-human B7-H3 antibody on the antigen binding of the other antibodies was tested using surface plasmon resonance (SPR) in a tandem manner. A total of four anti-human B7-H3 antibodies were tested: 6B5 ("Ab6B5"), 10D7 ("Ab10D7"), 10F5 ("Ab10F5"), and enoblituzumab (simply referred to as "AbEno").

[0167] HBS-EP + Buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4) was used as the running buffer. Anti-His antibodies were amine-coupled to the surface of a Series S SensorChip CM5 to generate an anti-His chip. Next, 1 M ethanolamine (pH 8.5) was injected to block the remaining active carboxyl groups on the chip surface, and the chip was then loaded onto HBS-EP +Equilibrated with buffer for 2 hours. Recombinant human B7-H3(4Ig) protein with His tag (Cat#: B7B-H52E7, Acrobiosystems) was injected into the Biacore8K biosensor at a rate of 30 μL / min for 50 seconds and captured by the anti-His chip to reach a protein density of approximately 50 RU.

[0168] A pair of antibodies (both at a concentration of 800 nM) was injected into the chip at a rate of the order 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 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 the antigen binding of the other antibody. The binding values ​​of each antibody were obtained using Biacore Insight. Kinetic velocities were determined to compare each pair of antibodies to quantify how one antibody interferes with the antigen binding of another antibody. 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) may bind to the same epitope or their epitopes may overlap; 10D7 (Ab10D7) binds to a unique epitope; the epitopes bound by 6B5 (Ab6B5), 10F5 (Ab10F5) and 10D7 (Ab10D7) and the epitope bound by enoblituzumab (AbEno) 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) provided with a pre-immobilized protein A sensor chip.

[0171] In particular, the chip captured 1 μg / mL anti-human B7-H3 antibody at a flow rate of 10 μL / min and injected recombinant human CD80 protein (human B7-1 / CD80 protein, His-tagged, Cat#: Cat#: B71-H5228, Acrobiosystems), CD86 (human B7-2 / CD86 protein, His-tagged, Cat#: CD6-H5223, Acrobiosystems), PDL2 (human PD-L2 / B7-DC protein, His-tagged, Cat#: ... s-tagged, Cat#:PD2-H5220, Acrobiosystems), PDL1 (human PD-L1 / B7-H1 protein, His-tagged, Cat#:PD1-H5229, 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 Biosystems), B7-H5 (human B7-H5 / Gi24 / VISTA protein, His-tagged (MALS certified), Cat#: B75-H52H0, Acrobiosystems), B7-H6 (human B7-H6 / NCR3LG1 protein, His-tagged, Cat#: B71-H5228, Acrobiosystems), and B7-H7 (human B7-H7 / HHLA2 protein, His-tagged (MALS certified), Cat#: B77-H52H5, Acrobiosystems) were injected into the 50-well plate, respectively. Dissociation was monitored for 600 seconds. Data was analyzed to obtain binding responses using Biacore8k evaluation software version 3.0, and protein concentration-binding response curves were plotted to determine cross-reactivity of anti-human B7-H3 antibody against B7 family proteins.

[0172] The results are summarized in Table 4. Similar to enoblituzumab, 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 Binding of fully human anti-B7-H3 antibodies to B7 family proteins [Table 5] Example 9 In vivo anti-cancer activity of fully human anti-B7-H3 antibodies

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

[0174] In the first experiment, mice carrying the human B7-H3 gene (Cat#: 110028, Biocytogen) were implanted with 2 × 10 5 EL4 cells were injected subcutaneously. Tumors were 90-120 mm 3 When mice reached 10 mg / kg, they were randomly assigned to three groups (n=8 / group) and intraperitoneally injected with PBS (G1), enoblituzumab (G2), and 10F5-Mut (G3) at a dose of 10 mg / kg twice a week for a total of six doses. The day of grouping was designated as day 0, and treatment began on this day.

[0175] In another study, tumors between 90 and 120 mm 3When the mice reached maturity, the B7-H3 humanized mice were randomly assigned to four groups (n=8 / group) and intraperitoneally injected with PBS (G1), enoblituzumab (G2), 6B5-Mut (G3), and 15F11-Mut (G4) at the same doses and frequency as above. The day of grouping was designated as day 0, and dosing began on this day.

[0176] Mice were weighed throughout the study. Tumor size was measured twice weekly and was calculated as V = (length × width). 2 Tumor growth inhibition was calculated as TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100, where T i refers to the mean tumor volume of the treatment group on day i, T0 is the mean tumor volume of that treatment group on day 0, and V i 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] During the entire study, each group of animals survived in good health. Compared with the control group, the mice in the treatment group did not show obvious changes in body weight (P>0.05). In particular, the average body weight of each group was 19.3-19.5 on day 0 when grouping was performed, and the average body weight was 19.8-20.9 when the study was terminated (day 17 after grouping in the first experiment and day 20 after grouping in the second experiment), so the body weight change was between 103.2% and 111.1%. The results showed that the tested anti-human B7-H3 antibodies were well tolerated by the mice, i.e., all the antibodies were non-toxic to the mice.

[0178] Figure 12 shows the change in tumor size in mice from each group. Compared with the control group, it can be seen that 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 anti-tumor effects than the positive control enoblituzumab.

[0179] The tumor size on the day of grouping, and on the 7th or 14th day after grouping, the tumor volume at the end of the experiment, the mouse survival rate, and the tumor (volume) inhibition rate (TGI TV The main data and analysis results, including the mean (%) and statistically significant differences (P values) between the treatment and control groups, are listed 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 in vivo anti-cancer activity of the fully human anti-human B7-H3 antibody 10F5-Mut [Table 6] Table 6 Summary of in vivo anti-cancer activity of fully human anti-human B7-H3 antibodies 6B5-Mut and 15F11-Mut [Table 7] Example 10 Cross-reactive staining of normal human tissues with fully human anti-human B7-H3 antibody

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

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

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

[0183] From the human tissue staining results in Table 8, it can be seen that the tissue staining specificity of the fully human anti-B7-H3 antibody 6B5-AF is consistent with that of enobritusumab. That is, among 35 normal human tissues tested, 24 normal human tissues obtained negative staining results, and only 11 normal tissues obtained weak staining. Meanwhile, as the difference of B7-H3 antigen epitopes between 6B5-AF and enobritusumab, the staining intensity is slightly different in some tissues with positive staining. As shown in Table 8, compared with enobritusumab, 6B5-AF has relatively strong staining in pituitary gland, but slightly weak staining in thymus, cervix and endometrium.

[0184] FIG. 13 shows representative tissue staining results, where 1-1, 2-1, 3-1, and 4-1 show enoblituzumab staining in the spleen, skeletal muscle, adrenal gland, and bladder, and 1-2, 2-2, 3-2, and 4-2 show 6B5-AF staining 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 percentage of positive cells in cells of the same type: Positive cell staining intensity: 0 = no cell staining; 1 = weak or equivocal staining; 2 = weak positive staining; 3 = moderate positive staining; 4 = strong positive staining; M = tissue loss or not applicable; percentage of positive cells in cells of the same type: 0 = no cell staining; 1 = positive cells account for less than 25% of cells of the same type; 2 = positive cells account for 25%-50% of cells of the same type; 3 = positive cells account for 50%-75% of cells of the same type; 4 = positive cells account for more than 75% of cells of the same type; M = tissue loss or not applicable.

[0185] The sequences referred to in this application are summarized in Table 9. Table 9. Sequences [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0186] While the present invention has been described with respect to one or more embodiments, it is understood that the invention is not limited to those embodiments, and that the above description is intended to include all other alternatives, modifications, and equivalents falling within the spirit and scope of the appended claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. 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, the VH CDR2 region, and the VH CDR3 region comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) 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; 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, the VL CDR2 region, and the VL CDR3 region comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) 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) SEQ ID NOs: 26, 27, and 28, respectively.

2. An isolated monoclonal antibody, or antigen-binding portion thereof, that binds to B7-H3, comprising:

2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 29, 31, 33, 35 or 37.

3. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 30, 32, 34, 36 or 38.

4. The isolated monoclonal antibody or antigen-binding portion thereof of claim 2, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to: (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.

5. i) a heavy chain variable region, wherein said heavy chain variable region comprises a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein said VH CDR1 region, said VH CDR2 region, and said VH CDR3 region have identity to the VH CDR1 region, VH CDR2 region, and VH CDR3 region, respectively, of a specified VH sequence; and / or ii) a light chain variable region, wherein said light chain variable region comprises a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein said VL CDR1 region, the VL CDR2 region, and the VL CDR3 region have identity to the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region, respectively, of a designated VL sequence; wherein the designated VH sequence and the designated VL sequence are (1) the designated VH sequence and the designated VL sequence, as set forth in SEQ ID NOs: 29 and 30, respectively; (2) the designated VH sequence and the designated VL sequence, as set forth in SEQ ID NOs: 31 and 32, respectively; (3) the designated VH sequence and the designated VL sequence, as set forth in SEQ ID NOs: 33 and 34, respectively; (4) the designated VH sequence and the designated VL sequence, as set forth in SEQ ID NOs: 35 and 36, respectively; (5) The designated VH sequence and the designated VL sequence are set forth in SEQ ID NOs: 37 and 38, respectively. selected from the group consisting of 2. An isolated monoclonal antibody, or antigen-binding portion thereof, that binds to B7-H3, comprising:

6. 6. The isolated monoclonal antibody or antigen-binding portion thereof of any one of claims 1 to 5, 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 kappa constant region.

7. 7. The isolated monoclonal antibody or antigen-binding portion thereof of claim 6, 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.

8. 8. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1 , which is an afucosylated monoclonal antibody or antigen-binding portion thereof.

9. (a) binds to human B7-H3, (b) binds to monkey B7-H3, (c) binds to mouse B7-H3, and (d) binds to B7-H3. ＋ (e) binds to cells, (f) is capable of activating immune cells, and (g) binds to B7-H3 ＋ 9. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, which is capable of inducing ADCC against a cell, and / or (g) has an in vivo anti-cancer effect.

10. 10. A bispecific molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the isolated monoclonal antibody or an antigen-binding portion thereof of any one of claims 1 to 9.

11. A nucleic acid molecule encoding the isolated monoclonal antibody or antigen-binding portion thereof of any one of claims 1 to 9.

12. 10. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding portion thereof of any one of claims 1 to 9 and a pharma- ceutically acceptable carrier.

13. 12. Use of the isolated monoclonal antibody or antigen-binding portion thereof of any one of claims 1 to 9, or the pharmaceutical composition of claim 11, in the preparation of a medicament for treating a B7-H3-related disease.

14. The use according to claim 13, wherein the B7-H3-related disease is a tumor.

15. 15. The use according to claim 14, 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, central nervous system neuroblastoma, glioblastoma, desmoplastic small cell tumor, diffuse pontine glioma, medulloblastoma, pancreatic cancer, liver cancer, colorectal cancer, non-Hodgkin's lymphoma, esophageal cancer, ovarian cancer, bladder cancer, small cell carcinoma, endometrial cancer, renal cancer, gastric cancer, acute myeloid leukemia, hepatocellular carcinoma, hypopharyngeal squamous cell carcinoma, urothelial cell carcinoma.

16. 12. Use of an isolated monoclonal antibody or an antigen-binding portion thereof according to any one of claims 1 to 9, or a pharmaceutical composition according to claim 11, in the preparation of a medicament for enhancing an immune response.

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