Antigen-binding proteins that bind to B7-H3

Abstract

The present disclosure provides B7-H3 binding proteins, particularly anti-B7-H3 antibodies, or antigen-binding portions thereof, that specifically bind to B7-H3, or uses thereof. Various aspects of the anti-B7-H3 antibodies relate to antibody fragments, single chain antibodies, pharmaceutical compositions, nucleic acids, recombinant expression vectors, host cells, and methods for preparing and using such anti-B7-H3 antibodies. Methods for using anti-B7-H3 antibodies include in vitro and in vivo methods for binding to B7-H3 and for treating diseases associated with B7-H3 expression.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 306,938, filed February 4, 2022, which is incorporated by reference herein in its entirety for all purposes.

[0002] Throughout this application, various publications, patents, and / or patent applications are referenced. The disclosures of these publications, patents, and / or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0003] Technical Field The present disclosure provides antigen binding proteins that specifically bind to B7-H3, and nucleic acids encoding the antigen binding proteins, vectors containing the nucleic acids, host cells harboring the vectors, and methods of use thereof. [Background technology]

[0004] background B7 family checkpoint molecules have come to the forefront of cancer research due to the concept that tumor cells exploit them to evade immune surveillance. B7 family molecules can control and suppress immune responses of NK cells as well as T cells. The ever-growing B7 family now includes 10 members: CD80 (also known as B7.1), CD86 (also known as B7.2), B7-H1 (also known as PD-L1 or CD274), B7-DC (also known as PD-L2 or CD273), B7-H2 (also known as ICOSL), B7-H3 (also known as CD276), B7-H4 (also known as B7S1, B7x or Vtcn1), B7-H5 (also known as VISTA, GI24, Dies1 or PD-1H), B7-H6 (also known as NCR3LG1) and B7-H7 (also known as HHLA2). Compelling evidence indicates that B7 molecules provide not only vital positive signals to stimulate and support T cell action, but also negative signals that regulate and inhibit T cell responses.

[0005] B7 homology 3 protein (B7-H3) (also known as CD276 and B7RP-2, referred to herein as "B7-H3") is a type I transmembrane glycoprotein of the immunoglobulin superfamily. Human B7-H3 contains a signal peptide at the N-terminus, an extracellular immunoglobulin-like variable region (Ig-like V type 1) and a constant region (Ig-like C2 type 1), a transmembrane region, and a cytoplasmic tail region with 45 amino acids (Zhou YH et al., 2007, Tissue Antigens. 70 (2): 96-104). B7-H3 has three splice variants, B7-H3α and B7-H3β. The extracellular domain of B7-H3α consists of two immunoglobulin domains, Ig-like V-Ig-like C (also known as 2IgB7-H3), while the extracellular domain of B7-H3β consists of four immunoglobulin domains, Ig-like V-Ig-like C-Ig-like V-Ig-like C (also known as 4IgB7-H3). The predominant B7-H3 isoform in human tissues and cell lines is the 4IgB7-H3 isoform (Steinberger et al., 2004, J. Immunol. 172(4): 2352-9).

[0006] B7-H3 has been reported to have both costimulatory and co-inhibitory signaling functions (see, e.g., Chapoval et al., 2001, Nat. Immunol. 2: 269-74; Suh et al., 2003, Nat. Immunol. 4: 899-906; Prasad et al., 2004, J. Immunol. 173: 2500-6; and Wang et al., 2005, Eur. J. Immunol. 35: 428-38; Yang, S. et al., 2020, Int. J. Biol. Sci. 16(11):1767-1773). For example, in vitro studies have shown that B7-H3 induces proliferation of cytotoxic T lymphocytes (CTLs) and enhances interferon gamma (IFN-γ) production in the presence of stimulatory anti-CD3 antibodies to mimic T cell receptor signals (Chapoval et al., 2001, Nat. Immunol. 2: 269-74), indicating a costimulatory function for B7-H3. Furthermore, in vivo studies using cardiac allografts in B7-h3 knockout / deficient- / - mice showed reduced production of vital cytokines, chemokines, and chemokine receptor mRNA transcripts (e.g., IL-2, IFN-γ, monocyte chemoattractant protein (MCP-1), and IFN-inducible protein (IP)-10) compared to wild-type littermate controls (Wang et al., 2005, Eur. J. Immunol. 35: 428-38). On the other hand, B7-H3 coinhibitory function has been observed, for example, in mice, where B7-h3 protein inhibited T cell activation and effector cytokine production (Suh et al., 2003, Nat. Immunol. 4: 899-906). Although the ligand for human B7-H3 has not been identified, mouse B7-h3 has been found to bind to triggering receptor expressed on myeloid cells (TREM)-like transcript 2 (TLT-2), a modulator of adaptive and innate immune cell responses. CD8 +Binding of mouse B7-h3 to TLT-2 on T cells induces T cell effector functions, such as proliferation, cytotoxicity and cytokine production (Hashiguchi et al., 2008, Proc. Nat'l. Acad. Sci. USA 105(30): 10495-500).

[0007] Enoblituzumab (MGA271), a humanized mAb targeting B7-H3, mediates potent antibody-dependent cellular cytotoxicity (ADCC) by recruiting NK cells against a wide range of tumor types. For example, it has been investigated in the treatment of refractory B7-H3-expressing tumors such as melanoma, as well as B7-H3-expressing neoplasms including osteosarcoma and Ewing's sarcoma. Furthermore, MGA271 has demonstrated potent antitumor activity in xenograft models of B7-H3-expressing renal cell and bladder cancer. Also, no significant safety findings were found in toxicity studies in cynomolgus monkeys (Loo, D. et al., 2012, Clin. Cancer Res. 18:3834-45).

[0008] B7-H3 protein is not or poorly expressed in normal tissues and cells, but is highly expressed in various tumors (not only solid tumors but also hematological malignancies) and is closely correlated with tumor progression, patient survival and disease prognosis. B7-H3 has been clinically reported to be overexpressed in many types of cancer, including prostate cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), ovarian cancer, colorectal cancer, colon cancer, renal cancer, hepatocellular carcinoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, breast cancer, urothelial carcinoma and urothelial cell carcinoma. Although the role of B7-H3 in cancer cells is unclear, its expression may orchestrate signaling events (and / or mediate cell-cell engagement) that may protect cancer cells from innate and adaptive immune responses. For example, B7-H3 is overexpressed in high-grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma, and high expression levels of B7-H3 in these cancerous cells are associated with an increased risk of cancer progression after surgery (Roth et al., 2007, Cancer Res. 67(16): 7893-900). Furthermore, tumor B7-H3 expression in NSCLC was inversely correlated with the number of tumor-infiltrating lymphocytes and significantly correlated with lymph node metastasis (Sun et al., 2006, Lung Cancer 53(2): 143-51). Levels of circulating soluble B7-H3 (sB7-H3) polypeptide in NSCLC patients have also been associated with more advanced tumor stage, increased / increasing tumor size, lymph node metastasis, and distant metastasis indicative of aggressive disease progression (Yamato et al., 2009, Br. J. Cancer 101(10):1709-16).

[0009] B7-H3 has co-inhibitory and co-stimulatory functions on T cells, but its expression on either tumor cells or diffuse tumor vasculature is significantly associated with increased risk of death and fatal outcomes. Targeted B7-H3 not only enhances antitumor immunity but also inhibits tumor angiogenesis, possibly through engagement with B7-H3 expressed by vasculature-associated macrophages, which are known to favor tumor angiogenesis by cytokine secretion.

[0010] There are reports that B7-H3 molecules are related to autoimmune diseases. In rheumatism and other autoimmune diseases, it has been reported that B7-H3 is important for the interaction between fibroblast-like synoviocytes and activated T cells (Yih-Wen, C. et al., 2008, Current Cancer Drug Targets 8: 404 - 413), and that B7-H3-activated macrophages are co-stimulatory factors in the release of cytokines from bacteremia and are involved in the development of sepsis (Harlow, E. and Lane, D.: "Antibodies: A Laboratory Manual", 1988, Cold Spring Harbor Laboratory). In addition, administration of anti-B7-H3 antibodies during the induction phase of a mouse asthma model suppressed cytokine production induced by Th2 cells in the draining lymph nodes and improved asthma. (Leitner, J. et al., 2009, European Journal of Immunology 39: 1754 - 1764). Thus, B7-H3 is an attractive antigen for targeting with antibodies. The present disclosure provides B7-H3 binding proteins, particularly anti-B7-H3 antibodies or antigen-binding portions thereof, that specifically bind to B7-H3, and uses thereof. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Zhou YH et al., 2007, Tissue Antigens. 70 (2): 96-104

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[0012] Abstract In one aspect, provided herein is an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region 1 (CDR1), a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 12, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 13, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 14, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 15, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 16, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 17. In embodiments, the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10, and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0013] In one aspect, provided herein is an antigen binding protein or fully human anti-B7-H3 antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11.

[0014] In one aspect, provided herein is an antigen-binding protein or fully human anti-B7-H3 antibody (e.g., referred to herein as VA), or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 10 and 11, respectively.

[0015] In embodiments, the antigen-binding fragment comprises a Fab fragment. In embodiments, the antigen-binding fragment comprises a single chain antibody in which the heavy chain variable domain and the light chain variable domain are linked together by a peptide linker. In embodiments, any one of the disclosed antigen-binding proteins, antibodies, or antigen-binding fragments thereof comprises an IgG antibody, e.g., an IgG1, IgG2, IgG3, or IgG4 class antibody. In embodiments, the IgG antibody is an IgG1 or IgG4 class antibody. In embodiments, the IgG1 antibody comprises a mutant Fc region. In embodiments, the mutant Fc region comprises one or more mutations selected from L234A and L235A. In embodiments, the mutant Fc region comprises mutations L234A and L235A (LALA). In embodiments, any one of the disclosed antigen-binding proteins, antibodies, or antigen-binding fragments thereof comprises 10 -7 M or less K D Binds to human anti-B7-H3 protein at 100 ng / mL.

[0016] In certain aspects, provided herein is a pharmaceutical composition comprising any one of the disclosed antigen binding proteins, antibodies, or antigen binding fragments and a pharma- ceutically acceptable excipient.

[0017] In certain aspects, provided herein is a kit comprising any one of the disclosed antigen binding proteins, antibodies, or antigen binding fragments and a pharma- ceutically acceptable excipient.

[0018] In certain aspects, provided herein is a nucleic acid encoding a polypeptide comprising the heavy chain variable region of any one of the disclosed antigen binding proteins, antibodies, or antigen binding fragments.

[0019] In certain aspects, provided herein is a nucleic acid encoding a polypeptide comprising the light chain variable region of any one of the disclosed antigen binding proteins, antibodies, or antigen binding fragments.

[0020] In certain aspects, provided herein is a nucleic acid encoding (i) a first polypeptide comprising a heavy chain variable region of any one of the disclosed antigen binding proteins, antibodies, or antigen binding fragments, and (ii) a second polypeptide comprising a light chain variable region of any one of the disclosed antigen binding proteins, antibodies, or antigen binding fragments.

[0021] In certain aspects, provided herein is a vector comprising any one of the disclosed nucleic acids.

[0022] In certain aspects, provided herein is a host cell harboring any of the disclosed vectors. In embodiments, the disclosed vectors comprise an expression vector and the host cell expresses a polypeptide comprising a heavy chain variable region. In embodiments, the disclosed vectors comprise an expression vector and the host cell expresses a polypeptide comprising a light chain variable region.

[0023] In one aspect, provided herein is a host cell harboring a first vector and a second vector. In embodiments, the first vector comprises a first expression vector and the second vector comprises a second expression vector, and the host cell expresses a first and a second polypeptide comprising heavy and light chain variable regions, respectively.

[0024] In certain aspects, provided herein is a method for preparing a polypeptide comprising a heavy chain variable region of an antigen binding protein, antibody, or antigen binding fragment, comprising culturing a population of host cells under conditions suitable for expressing the polypeptide comprising the heavy chain variable region of the antigen binding protein, antibody, or antigen binding fragment. In embodiments, the method further comprises recovering the expressed polypeptide comprising the heavy chain variable region of the antigen binding protein, antibody, or antigen binding fragment from the host cells.

[0025] In certain aspects, provided herein is a method for preparing a polypeptide comprising a light chain variable region of an antigen binding protein, antibody, or antigen binding fragment, comprising culturing a population of host cells under conditions suitable for expressing the polypeptide comprising the light chain variable region of the antigen binding protein, antibody, or antigen binding fragment. In embodiments, the method further comprises recovering the expressed polypeptide comprising the light chain variable region of the antigen binding protein, antibody, or antigen binding fragment from the host cells.

[0026] In certain aspects, provided herein is a method for preparing (i) a first polypeptide comprising a heavy chain variable region of an antigen binding protein, antibody, or antigen binding fragment, and (ii) a second polypeptide comprising a light chain variable region of the antigen binding protein, antibody, or antigen binding fragment, comprising culturing a population of host cells under conditions suitable for expression of (i) the first polypeptide and (ii) the second polypeptide. In embodiments, the method further comprises recovering (i) the expressed first polypeptide and (ii) the expressed second polypeptide from the host cells.

[0027] In certain aspects, provided herein are methods for inhibiting the growth or proliferation of B7-H3 expressing cells, comprising contacting a population of effector cells with a population of target cells expressing B7-H3 in the presence of any one of the disclosed antigen binding proteins, antibodies, or antigen binding fragments under conditions suitable for inhibiting the growth or proliferation of B7-H3 expressing cells. In embodiments, the population of effector cells comprises PBMCs or NK cells. In embodiments, the population of target cells comprises B7-H3 expressing human cancer cells, or transgenic cells expressing B7-H3. In embodiments, the ratio of effector cells to target cells is 1-5:1, e.g., 1:1, 2:1, 3:1, 4:1, or 5:1. In embodiments, the ratio of effector cells to target cells is 5-10:1, 10-20:1, or 20-30:1. In some embodiments, the method is an in vitro method. In some embodiments, the target cells are present in a subject and the antigen-binding protein, antibody, or antigen-binding fragment is administered to the subject. Effector cells can also be present in the subject.

[0028] In certain aspects, provided herein are methods for killing B7-H3 expressing cells, comprising contacting a population of effector cells with a population of target cells expressing B7-H3 in the presence of any one of the disclosed antigen binding proteins, antibodies, or antigen binding fragments under conditions suitable for killing the B7-H3 expressing cells. In embodiments, the population of effector cells comprises PBMCs or NK cells. In embodiments, the population of target cells comprises B7-H3 expressing human cancer cells, or transgenic cells expressing B7-H3. In embodiments, the ratio of effector cells to target cells is 1-5:1, e.g., 1:1, 2:1, 3:1, 4:1, or 5:1. In embodiments, the ratio of effector cells to target cells is 5-10:1, 10-20:1, or 20-30:1. In embodiments, provided herein are methods for restoring function of a B7-H3 expressing immune cell subset. In some embodiments, the method is an in vitro method. In some embodiments, the target cells are present in a subject and the antigen-binding protein, antibody, or antigen-binding fragment is administered to the subject. Effector cells can also be present in the subject.

[0029] In one aspect, provided herein is a method for treating a subject having a disease associated with B7-H3 expression, comprising administering to the subject an effective amount of a pharmaceutical composition comprising any one of the disclosed antigen binding proteins, antibodies, or antigen binding fragments. In an embodiment, the disease associated with B7-H3 expression is cancer. In an embodiment, the cancer is a carcinoma of the ovary, colon, prostate, skin, pancreas, kidney, urothelium, or lung cancer. In embodiments, the cancer is acute myeloid lymphoma (AML), non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), small cell lung cancer, urothelial cell carcinoma, esophageal cancer, hepatocellular carcinoma, glioma, neuroblastoma, glioblastoma multiforme, blastoma, sarcoma, leukemia, lymphoid malignancies, pancreatic cancer, head and neck cancer, ovarian cancer, oral cancer, breast cancer, triple negative breast cancer (TNBC), lymphoma, renal cell carcinoma, clear cell renal cell carcinoma, colon cancer, colorectal cancer, melanoma, gastric cancer, lung cancer, liver cancer, bladder cancer, prostate cancer, anal cancer, endometrial cancer, vulvar cancer, squamous cell tumor, hypopharyngeal squamous cell carcinoma, and squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer). In embodiments, the cancer, e.g., any one of the cancers listed above, is metastatic, refractory, or recurrent. [Brief description of the drawings]

[0030] [Figure 1A] FIG. 1A shows an SPR sensorgram of the binding kinetics of B7-H3-VA (anti-B7-H3 antibody clone VA).

[0031] [Figure 1B] FIG. 1B shows the SPR sensorgram of the binding kinetics of enoblituzumab, a commercially available anti-B7-H3 antibody developed by MacroGenics.

[0032] [Diagram 2]Figures 2A-B show the results of binding of anti-B7-H3 antibodies with human proteins B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, B7.1 and B7.2 assessed by ELISA assay. Figure 2A shows the results of binding of anti-B7-H3-VA antibodies. Figure 2B shows the results of binding of anti-B7-H3-VA LALA antibodies.

[0033] [Figure 3-1] FIG. 3 shows the results of binding of anti-B7-H3 antibody (clone VA) and enoblituzumab (humanized B7-H3 antibody, MacroGenics) with human, mouse, cynomolgus monkey, canine, rat and rabbit B7-H3 proteins assessed by ELISA assay. [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0034] [Figure 4] FIG. 4 shows the thermal stability of anti-B7-H3 antibody (clone VA).

[0035] [Diagram 5] Figure 5 shows immunostained tissue biopsies analyzed by confocal laser scanning microscopy that show increased binding of B7-H3 (clone VA) in malignant patient tissue but no binding in normal tissue or tissue adjacent to the cancer tissue.

[0036] [Figure 6A] Figure 6A shows the results of direct cell binding using flow cytometry comparing anti-B7-H3 antibody (VA clone) binding to enoblituzumab (anti-B7-H3 antibody, MacroGenics) binding to B7-H3-overexpressing osteosarcoma cells (U2OS).Anti-RSV antibody was used as a negative control.

[0037] [Figure 6B]Figure 6B shows the results of direct cell binding using flow cytometry comparing anti-B7-H3 antibody (VA clone) binding to enoblituzumab (anti-B7-H3 antibody, MacroGenics) binding to B7-H3-overexpressing neuroblastoma cells (SK-N-BE(2)). Anti-RSV antibody was used as a negative control.

[0038] [Figure 7A-B] 7A-C show the results of an antibody-dependent cell-mediated cytotoxicity (ADCC) assay of anti-B7-H3 antibody (VA clone) and enobrituzumab (anti-B7-H3 antibody, MacroGenics) in human osteosarcoma (U2OS) cells. FIG. 7A shows the results of an ADCC predictive assay of anti-B7-H3 antibody (VA clone) and enobrituzumab (anti-B7-H3 antibody, MacroGenics) where the effector cells were engineered Jurkat cells. FIG. 7B shows the results of an ADCC assay of anti-B7-H3 antibody (VA clone) and enobrituzumab (anti-B7-H3 antibody, MacroGenics) where the effector cells were NK cells. FIG. 7C shows the results of an ADCC assay of anti-B7-H3 antibody (VA clone) and anti-RSV antibody where the effector cells were NK cells. [Figure 7C] Same as above.

[0039] [Figure 8A] 8A-B show the results of an antibody-dependent cell-mediated cytotoxicity (ADCC) assay of anti-B7-H3 antibody (VA clone) and enoblituzumab (anti-B7-H3 antibody, MacroGenics) in human lung cancer cells A549 wild type (WT) or B7-H3 knockout (KO) (target cells), where the effector cells were engineered Jurkat cells. FIG. 8A shows the results of an ADCC assay of anti-B7-H3 antibody (VA clone) where the target cells were A549 WT. FIG. 8B shows the results of an ADCC assay of anti-B7-H3 antibody (VA clone) where the target cells were A549 KO. [Figure 8B] Same as above.

[0040] [Figure 9] Figure 9 shows B7-H3 surface expression by A549 and A549 B7-H3 KO cells assessed by flow cytometry analysis. Gating was focused on B7-H3 expressing cells and expressed as % of the population. ISO is an anti-RSV antibody used as an isotype control.

[0041] [Figure 10] Figure 10 shows the effect of anti-B7-H3 antibody (VA clone), anti-RSV antibody (isotype control), and HBSS buffer (vehicle control) on the SK-N-BE(2) human neuroblastoma tumor model when administered at high dosing frequency. **P<0.01; ***P<0.001 was obtained between the vehicle and anti-B7-H3 antibody (VA clone) groups.

[0042] [Figure 11] Figure 11 shows the effect of anti-B7-H3 antibody (VA clone), anti-RSV antibody (isotype control), and HBSS buffer (vehicle control) on the SK-N-BE(2) human neuroblastoma tumor model when administered at low dosing frequency. **P<0.01; ***P<0.001 was obtained between the vehicle and anti-B7-H3 antibody (VA clone) groups.

[0043] [Figure 12A-B]Figure 12A shows a schematic of a hemagglutination reaction. Figures 12B-C show images of a hemagglutination assay comparing the activity of the anti-B7-H3 antibodies STI-B7-H3-VA and MG-B7-H3 (enobritus from Macrogenics) and positive and negative controls (anti-CD47 antibody Hu5F9 and isotype, respectively). Figure 12B shows images of a hemagglutination assay comparing the activity of the anti-B7-H3 antibodies STI-B7-H3-VA and MG-B7-H3 (enobritus from Macrogenics) and positive and negative controls (anti-CD47 antibody Hu5F9 and isotype, respectively). FIG. 12C shows images of a hemagglutination assay comparing the activity of anti-B7-H3 antibodies STI-B7-H3-VA LALA and MG-B7-H3 (enoblitutuzumab from Macrogenics) and positive and negative controls (anti-CD47 antibodies Hu5F9 and isotype, respectively). [Figure 12C] Same as above.

[0044] [Figure 13] FIG. 13 shows binding of anti-B7-H3 antibodies B7-H3-VA and Macrogenics (MG-B7-H3, enoblituzumab) to human melanoma A375 cells, human lung carcinoma A549 cells, and human pancreatic carcinoma Panc-1 cells as a function of antibody concentration.

[0045] [Figure 14-1] Figure 14 provides a graph comparing the receptor engagement of anti-B7-H3 antibody B7-H3-VA with that of Macrogenics (enoblituumab, MG-B7-H3) in human melanoma A375 cells, human lung cancer A549 cells, and human pancreatic cancer Panc-1 cells as a function of antibody concentration. Additionally, a table of binding parameters is provided. [Figure 14-2] Same as above.

[0046] [Figure 15-1]Figure 15 provides a graph comparing the receptor engagement of the anti-B7-H3 antibody Macrogenics (enoblituzumab, MG-B7-H3) with that of B7-H3-VA (with LALA mutation in the Fc region) in human melanoma A375 cells, human lung cancer A549 cells, and human pancreatic cancer Panc-1 cells as a function of antibody concentration. Additionally, a table of binding parameters is provided. [Figure 15-2] Same as above.

[0047] [Figure 16-1] Figure 16 shows CFSE fluorescence of CD4+ T cells at day 0 and 72 hours (panels 1 and 2, respectively). Panel 3 shows CFSE fluorescence of CD4+ T cells after addition of anti-CD3 antibody. Panel 4 shows CFSE fluorescence of CD4+ T cells after addition of sB7-H3 polypeptide (after addition of anti-CD3 antibody). Panel 5 shows CD4+ T cells after addition of anti-B7-H3-VA antibody (after addition of sB7-H3). Panel 6 shows CD4+ T cells after addition of anti-RSV antibody (after addition of sB7-H3). Additionally, tables of division, proliferation, expansion and replication indices are provided. [Figure 16-2] Same as above.

[0048] [Figure 17] Figure 17 shows the results of antibody-dependent cell-mediated cytotoxicity (ADCC) assay of anti-B7-H3 antibodies B7-H3-VA, B7-H3-VA LALA (having LALA mutation in Fc region), and Macrogenetics (enoblitutumab, MG-B7-H3) in human lung cancer cells A549. R1 and R2 are two independent trials.

[0049] [Figure 18]Figure 18 shows the binding of antibodies to human RBCs. The top panel shows the binding of B7-H3-VA, MG-B7-H3 (enoblituzumab), anti-RSV antibody (isotype control), and anti-CD47 antibody Hu5F9 to human RBCs. The bottom panel shows the binding of B7-H3-VA LALA, MG-B7-H3, anti-RSV antibody (isotype control), and anti-CD47 antibody Hu5F9 to human RBCs.

[0050] [Figure 19-1] FIG. 19 shows SPR sensorgrams of the binding kinetics of B7-H3 antibodies (B3-A6, B3-4A1, B3-4F7, B3-D1, B3-2D1, B3-2H1, B3-2E3, and B3-5A6). [Figure 19-2] Same as above.

[0051] [Figure 20] FIG. 20 shows the results of a cell binding assay using flow cytometry comparing anti-B7-H3 antibody (B3-5A6, B3-2E3, B3-2D1, B3-D1, B3-2G11, and B3-4A1) binding to dendritic cells with MSKCC-B7-H3 antibody (anti-B7-H3 antibody, Memorial Sloan Kettering Cancer Center). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] explanation

[0053] Definition:

[0054] Unless otherwise defined, technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art unless otherwise defined.Generally, the terminology of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization techniques described herein are well known and commonly used in the art.The methods and techniques provided herein are generally performed according to conventional procedures well known in the art and as described in various general and more specific references cited and discussed herein, unless otherwise indicated.See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992).Several basic textbooks describe the standard antibody production process, including Borrebaeck (ed) Antibody Engineering, 2nd Edition Freeman and Company, NY, 1995;McCafferty et al. Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England, 1996;and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ, 1995; Paul (ed.), Fundamental Immunology, Raven Press, NY, 1993;Coligan (1991) Current Protocols in Immunology Wiley / Greene, NY;Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein;Coding Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New York, NY, 1986, and Kohler and Milstein Nature 256: 495-497, 1975. All references cited herein are incorporated by reference in their entirety. Enzymatic reactions and concentration / purification techniques are also well known and are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein.The terminology used in connection with, and the laboratory methods and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0055] The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole. In the event that any content incorporated by reference conflicts with specific content of the disclosure, the specific content shall control.

[0056] As used herein, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The singular forms "a," "an," and "the," as well as use of the singular form of any word, include plural referents unless expressly and unambiguously limited to one referent.

[0057] The use of the alternative (eg, "or") herein is to be construed as meaning either one or both of the alternatives, or any combination thereof.

[0058] The term "and / or" as used herein should be interpreted as meaning a specific disclosure of each of the specified features or components with or without the others. For example, when the term "and / or" is used herein in phrases such as "A and / or B", it is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, when the term "and / or" is used in phrases such as "A, B and / or C", it is intended to encompass each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0059] As used herein, the terms "comprising," "including," "having," and "containing," as well as grammatical variations thereof, are intended to be non-limiting, such that one or more items in a list do not exclude other items that may be substituted for or in addition to the listed items. Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0060] As used herein, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, where this acceptable error range will depend in part on how the value or composition is measured or determined, i.e., on the constraints of the measurement system. For example, "about" or "essentially consisting of" may mean within 1 standard deviation or may mean more than 1 standard deviation, according to practice in the art. Alternatively, "about" or "essentially consisting of" may mean a range of 10% (i.e., ±10%) or less, or may mean a greater range, depending on the constraints of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or biological processes, these terms may mean an order of magnitude or less or 5-fold less than the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of "about" or "essentially consisting of" should be considered to be within an acceptable error range for the particular value or composition.

[0061] Numeric ranges include the endpoints of the range, for example "between 4.5 mg and 5.5 mg" includes 4.5 mg, 5.5 mg, and all values ​​greater than 4.5 mg and less than 5.5 mg.

[0062] As used herein, the terms "peptide", "polypeptide" and "protein" and other related terms are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides can include natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor and mature molecules. Precursor molecules include those that have not yet undergone cleavage, e.g., cleavage by a secretory signal peptide or non-enzymatic cleavage at certain amino acid residues. Polypeptides include mature molecules that have undergone cleavage. These terms encompass natural and artificial proteins, protein fragments, and polypeptide analogs of protein sequences (e.g., muteins, variants, chimeric proteins, and fusion proteins), as well as proteins that have been post-translationally modified or otherwise covalently or non-covalently modified. Two or more polypeptides (e.g., three polypeptide chains) may be associated with each other by covalent and / or non-covalent association to form a multimeric polypeptide complex (e.g., a multispecific antigen-binding protein complex). Association of polypeptide chains may also include peptide folding. Thus, the polypeptide complex can be a dimer, trimer, tetramer, or higher order complex depending on the number of polypeptide chains that form the complex. Described herein are polypeptides that contain the amino acid sequence of a binding protein that binds to B7-H3 (e.g., an anti-B7-H3 antibody or antigen-binding portion thereof) prepared using recombinant procedures.

[0063] As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" and other related terms are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In embodiments, the nucleic acid molecules of the present disclosure include a contiguous open reading frame that encodes an antibody or a fragment or scFv, derivative, mutein or variant thereof. In one embodiment, the nucleic acid comprises one type of polynucleotide or a mixture of two or more types of polynucleotides. Nucleic acids encoding anti-B7-H3 antibodies or antigen-binding portions thereof are described herein.

[0064] The terms "recover" or "recovery" or "recovering" and other related terms refer to obtaining a protein (e.g., an antibody or antigen-binding portion thereof) from a host cell culture medium, or from a host cell lysate, or from a host cell membrane. In one embodiment, the protein is expressed by the host cell as a recombinant protein fused with a secretory signal peptide sequence that mediates secretion of the expressed protein. The secreted protein can be recovered from the host cell medium. In an embodiment, the protein is expressed by the host cell as a recombinant protein lacking a secretory signal peptide sequence, and the recombinant protein can be recovered from a host cell lysate. In one embodiment, the protein is expressed by the host cell as a membrane-bound protein, and the membrane-bound protein can be recovered by using a detergent to release the expressed protein from the host cell membrane. In one embodiment, regardless of the method used to recover the protein, the protein can be subjected to a procedure to remove cellular debris from the recovered protein. For example, the recovered protein can be subjected to chromatography, gel electrophoresis, and / or dialysis. In embodiments, the chromatography comprises any one or any combination or two or more procedures including affinity chromatography, hydroxyapatite chromatography, ion exchange chromatography, reverse phase chromatography, and / or chromatography using silica. In embodiments, the affinity chromatography comprises proteins A and G (cell wall components from Staphylococcus aureus).

[0065] The term "isolated" refers to a protein (e.g., an antibody or antigen-binding portion thereof) or polynucleotide that is substantially free of other cellular material. A protein can be rendered substantially free of naturally associated components (or components associated with the cellular expression system or chemical synthesis method used to produce the antibody) by isolation using protein purification techniques well known in the art. The term isolated also refers in some embodiments to a protein or polynucleotide that is substantially free of other molecules of the same species, e.g., other proteins or polynucleotides having different amino acid or nucleotide sequences, respectively. The purity of homogeneity of the desired molecule can be assayed using techniques well known in the art, including low-resolution methods, e.g., gel electrophoresis, or high-resolution methods, e.g., HPLC or mass spectrometry. In one embodiment, the multispecific antigen-binding protein complex or antigen-binding portion thereof of the present disclosure is isolated. In one embodiment, either the anti-B7-H3 antibody or its antigen-binding protein is isolated.

[0066] As used herein, "antigen-binding protein" and related terms refer to a protein that includes a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. Examples of antigen-binding proteins include antibodies, antibody fragments (e.g., antigen-binding portions of antibodies), antibody derivatives, and antibody analogs. Antigen-binding proteins may include, for example, alternative protein scaffolds or artificial scaffolds onto which CDRs or CDR derivatives are grafted. Such scaffolds include, but are not limited to, antibody-derived scaffolds that include mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein, as well as fully synthetic scaffolds, including, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129;Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics ("PAMs") can be used, as can antibody mimetic-based scaffolds that utilize fibronectin components as scaffolds. Described herein are antigen binding proteins that bind to B7-H3.

[0067] An antigen-binding protein may have a structure, for example, that of an immunoglobulin. In one embodiment, "immunoglobulin" refers to a tetrameric molecule composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha or epsilon, defining the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. The variable and constant regions in the light and heavy chains are joined by a "J" region of about 12 or more amino acids, with the heavy chain also containing a "D" region of about 10 amino acids. See generally Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair form the antibody binding site, and thus an intact immunoglobulin has two antigen binding sites. In embodiments, the antigen binding protein can be a synthetic molecule that has a structure that is different from a tetrameric immunoglobulin molecule but still binds to a target antigen or binds to two or more target antigens. For example, the synthetic antigen binding protein can include an antibody fragment, one to six or more polypeptide chains, an asymmetric assembly of polypeptides, or other synthetic molecules. Described herein are antigen binding proteins with immunoglobulin-like properties that specifically bind to B7-H3.

[0068] The variable regions of immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) bounded by three hypervariable regions, also called complementarity determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0069] One or more CDRs can be incorporated into a molecule, either covalently or non-covalently, to make the molecule an antigen-binding protein. An antigen-binding protein can incorporate a CDR as part of a larger polypeptide chain, can covalently link the CDR to another polypeptide chain, or can incorporate the CDR non-covalently. The CDRs enable the antigen-binding protein to specifically bind to a particular antigen of interest.

[0070] The amino acid assignments for each domain are given in Sequences of Proteins of Immunological Interest, 5 th The definition is based on that of Kabat et al., Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (international ImMunoGeneTics information system; Lefranc et al, 2005, Dev. Comp. Immunol. 29:185-203;) and Aho (Honegger and Pluckthun, 2001, J. Mol. Biol. 309(3):657-670); Chothia (Al-Lazikani et al., 1997 Journal of Molecular Biology 273:927-948); and Contact (Maccallum et al., 1996 Journal of Molecular Biology 262:732-745).

[0071] As used herein, "antibody" and "antibodies" and related terms refer to an intact immunoglobulin or to an antigen-binding portion thereof (or antigen-binding fragment thereof) that specifically binds to an antigen. Antigen-binding portions (or antigen-binding fragments) may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions (or antigen-binding fragments) include, among others, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin, which portion is sufficient to confer specific antigen binding to the polypeptide.

[0072] Antibodies include recombinantly produced antibodies and antigen-binding portions. Antibodies include non-human, chimeric, humanized and fully human antibodies. Antibodies include monospecific, multispecific (e.g., bispecific, trispecific and higher specific). Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers. Antibodies include F(ab')2 fragments, Fab' fragments and Fab fragments. Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragments (scFv), camelid antibodies, affibodies, disulfide-linked Fvs (sdFv), anti-idiotypic antibodies (anti-Id), minibodies. Antibodies include monoclonal and polyclonal populations. Anti-B7-H3 antibodies are described herein.

[0073] As used herein, "antigen-binding domain", "antigen-binding region" or "antigen-binding site" and other related terms refer to a portion of an antigen-binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the specificity and affinity of the antigen-binding protein for the antigen. For an antibody that specifically binds to its antigen, this will include at least a portion of at least one of its CDR domains. The antigen-binding domain of an anti-B7-H3 antibody is described herein. In some embodiments, the antigen-binding domain is formed from a VH domain and a VL domain.

[0074] The terms "specific binding," "specifically binds," or "specifically binding," and other related terms, as used herein in the context of an antibody or antigen-binding protein (e.g., a multispecific antigen-binding protein complex) or antibody fragment, refer to preferential non-covalent or covalent binding to an antigen relative to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen relative to other available antigens). In embodiments, an antibody binds to a target antigen such that it binds to that antigen 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or a dissociation constant K D Described herein are anti-B7-H3 antibodies that specifically bind to B7-H3.

[0075] In one embodiment, a BIACORE surface plasmon resonance (SPR) assay is used to determine the dissociation constant (K D) can be measured. Surface plasmon resonance refers to an optical phenomenon that allows real-time interaction analysis by detecting changes in protein concentration within a biosensor matrix, for example, using a BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0076] "Epitope" and related terms, as used herein, refer to a portion of an antigen to which an antigen-binding protein binds (e.g., by an antibody or antigen-binding portion thereof). An epitope may include portions of two or more antigens to which an antigen-binding protein binds. An epitope may include non-contiguous portions of an antigen (e.g., amino acid residues that are not contiguous in the primary sequence of the antigen, but are close enough to each other in the context of the antigen's tertiary and quaternary structure to allow binding by an antigen-binding protein) of the antigen. Generally, the variable regions of an antibody, particularly the CDRs, interact with the epitope. Described herein are anti-B7-H3 antibodies and antigen-binding proteins thereof that bind to epitopes of a B7-H3 polypeptide.

[0077] With respect to antibodies, the terms "antagonist" and "antagonistic" refer to a blocking antibody that binds to its cognate target antigen and inhibits or reduces the biological activity of the bound antigen. The terms "agonist" or "agonistic" refer to an antibody that binds to its cognate target antigen in a manner that mimics the binding of a physiological ligand, resulting in antibody-mediated downstream signaling.

[0078] As used herein, "antibody fragment", "antibody portion", "antigen-binding fragment of an antibody" or "antigen-binding portion of an antibody" and other related terms refer to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAbs; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and polypeptides that contain at least a portion of an antibody, the portion being sufficient to confer specific antigen binding to the polypeptide. Antigen-binding portions of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, among others, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen-binding properties to the antibody fragment. Antigen-binding fragments of anti-B7-H3 antibodies are described herein.

[0079] The terms "Fab", "Fab fragment" and other related terms refer to the variable light chain region (V L ), the constant light chain region (C L ), variable heavy chain region (V H ) and the first constant region (C H1 ) is a monovalent fragment that contains two Fab fragments linked by disulfide bridges at the hinge region. F(Ab')2 has antigen-binding ability. Fd fragments are monovalent fragments that contain two Fab fragments linked by disulfide bridges at the hinge region. F(Ab')2 has antigen-binding ability. H and C H1 The Fv fragment contains the V L and V H The Fv is capable of binding to an antigen. The dAb fragment comprises a V H Domain, V L Domain, or V HOr has an antigen-binding fragment of the VL domain (U.S. Patent Nos. 6,846,634 and 6,696,245; U.S. Patent Application Publication Nos. 2002 / 02512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958; and Ward et al., 1989, Nature 341:544-546). Fab fragments containing antigen-binding portions from anti-B7-H3 antibodies are described herein.

[0080] Single chain antibodies (scFv) are L Area and V H The antibody is one in which the domains are linked together via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain.Preferably, the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).A single-chain antibody comprising antigen-binding portion from anti-B7-H3 antibody is described herein.

[0081] A diabody is a bivalent antibody comprising two polypeptide chains, each of which has a V H and V LA bivalent antibody is an antibody that contains two domains, and the linker is too short to allow pairing between the two domains on the same chain, and therefore each domain will pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a diabody are identical, the resulting diabody will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to create a diabody with two different antigen-binding sites. Similarly, triabodies and tetrabodies are antibodies that contain three and four polypeptide chains, which can be the same or different, respectively, and form three and four antigen-binding sites, respectively. Diabody, triabody and tetrabody constructs can be prepared using antigen-binding portions from any of the anti-B7-H3 antibodies described herein.

[0082] The term "human antibody" refers to an antibody having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody). These antibodies can be prepared in a variety of ways, examples of which are described below, including by recombinant methodology or by immunization with the antigen of interest of mice that have been genetically modified to express antibodies derived from genes encoding human heavy and / or light chains. A fully human anti-B7-H3 antibody and its antigen-binding protein are described herein.

[0083] A "humanized" antibody refers to an antibody having a sequence that differs from that of an antibody derived from a non-human species in terms of one or more amino acid substitutions, deletions and / or additions, such that the humanized antibody is less likely to induce an immune response and / or induces a less vigorous immune response when it is administered to a human subject, compared to the non-human species antibody. In one embodiment, certain amino acids in the framework and constant domains of the heavy and / or light chains of the non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain from a human antibody is fused with a variable domain of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of the non-human antibody are altered to reduce the possible immunogenicity of the non-human antibody when it is administered to a human subject, the altered amino acid residues being non-critical for the immunospecific binding of the antibody to its antigen, or the amino acid sequence changes made are conservative changes such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies can be found in US Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.

[0084] The term "chimeric antibody" and related terms as used herein refers to an antibody that contains one or more regions from a first antibody and one or more regions from one or more other antibodies. In an embodiment, one or more of the CDRs are derived from a human antibody. In another embodiment, all of the CDRs are derived from a human antibody. In another embodiment, a chimeric antibody mixes and matches CDRs from more than one human antibody. For example, a chimeric antibody may contain CDR1 from a light chain of a first human antibody, CDR2 and CDR3 from a light chain of a second human antibody, and CDRs from a heavy chain from a third antibody. In another example, the CDRs originate from different species, e.g., human and mouse, or human and rabbit, or human and goat. It will be understood by those skilled in the art that other combinations are possible.

[0085] Furthermore, the framework regions may be from one of the same antibodies, from one or more different antibodies, such as human antibodies, or from humanized antibodies. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical to, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to, homologous to, or derived from an antibody from another species or belonging to another antibody class or subclass. Fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind to a target antigen) are also included. Chimeric antibodies can be prepared from portions of any of the anti-B7-H3 antibodies described herein.

[0086] As used herein, the terms "variant" polypeptide and "variant" of a polypeptide refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues are inserted, deleted, and / or substituted in the amino acid sequence compared to a reference polypeptide sequence. Polypeptide variants include fusion proteins. Similarly, variant polynucleotides include nucleotide sequences in which one or more nucleotides are inserted, deleted, and / or substituted in the nucleotide sequence compared to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.

[0087] As used herein, the term "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, by conjugation to another chemical moiety, such as polyethylene glycol, albumin (e.g., human serum albumin), etc., phosphorylation, and glycosylation. Unless otherwise indicated, the term "antibody" includes antibodies comprising two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below.

[0088] The term "hinge" refers to an amino acid segment typically found between two domains of a protein that may allow for general constructional freedom and freedom of movement of one or both of the domains relative to one another. Structurally, the hinge region comprises about 10 to about 100 amino acids, e.g., about 15 to about 75 amino acids, about 20 to about 50 amino acids, or about 30 to about 60 amino acids. In embodiments, the hinge region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. The hinge region may be derived from a naturally occurring protein hinge region, such as a CD8 hinge region or fragment thereof, a CD8α hinge region or fragment thereof, a hinge region of an antibody (e.g., an IgG, IgA, IgM, IgE or IgD antibody), or a hinge region that joins the constant domains CH1 and CH2 of an antibody. The hinge region may be derived from an antibody and may or may not include one or more constant regions of an antibody, or the hinge region includes the hinge region of an antibody and the CH3 constant region of an antibody, or the hinge region includes the hinge region of an antibody and the CH2 and CH3 constant regions of an antibody, or the hinge region is a non-naturally occurring peptide, or the hinge region is disposed between the C-terminus of the scFv and the N-terminus of the transmembrane domain. In one embodiment, the hinge region comprises any one or any combination of two or more regions comprising upper, middle or lower hinge sequences from an IgG1, IgG2, IgG3 or IgG4 immunoglobulin molecule. In an embodiment, the hinge region comprises the IgG1 upper hinge sequence EPKSCDKTHT (SEQ ID NO: 108). In an embodiment, the hinge region comprises the IgG1 middle hinge sequence CP X CP (in this sequence, Xis P, R or S) (SEQ ID NO: 109). In one embodiment, the hinge region comprises the lower hinge sequence APELLGGP (SEQ ID NO: 110). In an embodiment, the hinge is associated with an Fc region (CH2) having the amino acid sequence SVFLFPPKPKDT (SEQ ID NO: 111). In one embodiment, the hinge region comprises the amino acid sequence of the upper, middle and lower hinge and comprises EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 112). In an embodiment, the hinge region comprises one, two, three or more cysteines capable of forming at least one, two, three or more interchain disulfide bonds.

[0089] The term "Fc" or "Fc region" as used herein refers to a portion of an antibody heavy chain constant region beginning in or after the hinge region and ending at the C-terminus of the heavy chain. The Fc region includes at least a portion of the CH2 and CH3 regions, and may or may not include a portion of the hinge region. Two polypeptide chains, each carrying half of an Fc region, can dimerize to form a complete Fc region. In some embodiments, the Fc region can bind to an Fc cell surface receptor and to a protein that is part of the immune complement system. The Fc region can bind to complement component C1q. The Fc domain can exhibit effector functions including any one or any combination of two or more activities including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization, and / or cell binding. The Fc domain can bind to an Fc receptor, including FcγRI (e.g., CD64), FcγRII (e.g., CD32), and / or FcγRIII (e.g., CD16a). In embodiments, the Fc region can contain mutations that increase or decrease any one or any combination of these functions (e.g., an effector-negative Fc region).

[0090] The term "labeled antibody" or related terms, as used herein, refers to antibodies and their antigen-binding portions that are labeled for detection or conjugated with a detectable label or moiety, such as radioactive, colorimetric, antigenic, enzymatic, detectable beads (e.g., magnetic beads or electron-dense (e.g., gold) beads), biotin, streptavidin, or protein A. A variety of labels can be utilized, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens). Any of the anti-B7-H3 antibodies, antigen-binding proteins, and antibody fragments described herein can be unlabeled or conjugated with a detectable label or moiety.

[0091] "Percent identity" or "percent homology" as used herein and related terms refer to the quantitative measure of similarity between two polypeptides or two polynucleotide sequences.The percent identity between two polypeptide sequences is a function of the number of identical amino acids shared between two polypeptide sequences at aligned positions, taking into account the number of gaps and the length of each gap that may need to be introduced to optimize the alignment of two polypeptide sequences.Similarly, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides shared between two polynucleotide sequences at aligned positions, taking into account the number of gaps and the length of each gap that may need to be introduced to optimize the alignment of two polynucleotide sequences.Comparing sequences between two polypeptide sequences or two polynucleotide sequences and determining percent identity can be achieved using mathematical algorithms. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences can be determined using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) by comparing the sequences using its default parameters. A phrase such as "comprises a sequence having at least X% identity to Y" with respect to a test sequence means that the test sequence, when aligned with sequence Y as described above, contains residues that are identical to at least X% of the residues of Y.

[0092] In embodiments, the amino acid sequence of the test antibody may be similar, but not necessarily identical, to any of the amino acid sequences of any of the anti-B7-H3 antibodies described herein or any of the polypeptides constituting the antigen binding protein thereof. The similarity between the test antibody and the polypeptide may be at least 95%, or 96% or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the anti-B7-H3 antibodies described herein or any of the polypeptides constituting the antigen binding protein thereof. In one embodiment, the similar polypeptide may contain amino acid substitutions in the heavy and / or light chain. In one embodiment, the amino acid substitutions include one or more conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other in terms of conservative substitutions, the percent sequence identity or similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. For example, see Pearson (1994) Methods Mol. Biol. 24: 307-331, the entirety of which is incorporated herein by reference. Examples of groups of amino acids with side chains that have similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) basic side chains: lysine, arginine and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine.

[0093] Antibodies can be obtained from sources such as serum or plasma that contain immunoglobulins with diverse antigen specificities. Such antibodies can be subjected to affinity purification to enrich them for a particular antigen specificity. Such enriched preparations of antibodies are usually made with less than about 10% of the antibodies having specific binding activity for a particular antigen. These preparations can be subjected to several rounds of affinity purification to increase the percentage of antibodies having specific binding activity for the antigen. Antibodies prepared in this manner are often referred to as "monospecific". Monospecific antibody preparations can be composed of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% of antibodies having specific binding activity for a particular antigen. Antibodies can be produced using recombinant nucleic acid techniques described below.

[0094] As used herein, "vector" and related terms refer to a nucleic acid molecule (e.g., DNA or RNA) that can be operably linked to foreign genetic material (e.g., a nucleic acid transgene). A vector can be used as a vehicle to introduce foreign genetic material into a cell (e.g., a host cell). A vector can contain at least one restriction endonuclease recognition sequence for the insertion of a transgene into the vector. A vector can contain at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in the selection of host cells that harbor the vector-transgene construct. A vector can be a single-stranded or double-stranded nucleic acid molecule. A vector can be a linear or circular nucleic acid molecule. A donor nucleic acid used in gene editing methods that utilize zinc finger nucleases, TALENs or CRISPR / Cas can be a type of vector. One type of vector is a "plasmid," which refers to a linear or circular double-stranded extrachromosomal DNA molecule that can be linked to a transgene, can replicate in a host cell, and can transcribe and / or translate the transgene. Viral vectors typically contain viral RNA or DNA backbone sequences that can be linked to a transgene. The viral backbone sequences can be modified to render them incapable of infection, but to retain the insertion of the viral backbone and co-linked transgene into the host cell genome. Examples of viral vectors include retrovirus, lentivirus, adenovirus, adeno-associated, baculovirus, papovavirus, vaccinia virus, herpes simplex virus, and Epstein-Barr virus vectors. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors that contain a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome.

[0095] An "expression vector" is a type of vector that can contain one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers. An expression vector can include a ribosome binding site and / or a polyadenylation site. The regulatory sequence directs the transcription, or the transcription and translation, of a transgene linked to the expression vector that is transduced into a host cell. The regulatory sequence can control the level, timing and / or location of expression of the transgene. The regulatory sequence can, for example, exert its effect directly on the transgene or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). The regulatory sequence can be part of a vector. Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606. An expression vector can contain a nucleic acid encoding at least a portion of any of the anti-B7-H3 antibodies described herein.

[0096] A transgene is "operably linked" to a vector if there is a linkage between the transgene and the vector that allows for the function or expression of the transgene sequence contained in the vector. In one embodiment, a transgene is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.

[0097] The terms "transfected" or "transformed" or "transduced" or other related terms as used herein refer to the process by which an exogenous nucleic acid (e.g., a transgene) is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" host cell is one that has been transfected, transformed or transduced with an exogenous nucleic acid (transgene). A host cell includes a primary subject cell and its progeny. An exogenous nucleic acid encoding at least a portion of any of the anti-B7-H3 antibodies described herein can be introduced into a host cell. An expression vector comprising at least a portion of any of the anti-B7-H3 antibodies described herein can be introduced into a host cell, and the host cell can express a polypeptide comprising at least a portion of the anti-B7-H3 antibody.

[0098] The term "host cell" or "or population of host cells" or related terms, as used herein, refers to a cell (or population) into which a foreign (exogenous or transgene) nucleic acid has been introduced. The foreign nucleic acid can include an expression vector operably linked to a transgene, and the host cell can be used to express the nucleic acid and / or polypeptide encoded by the foreign nucleic acid (transgene). The host cell (or population) can be a cultured cell or can be extracted from a subject. The host cell (or population) includes the primary subject cell and its progeny regardless of the number of passages. The progeny cells may or may not harbor identical genetic material as compared to the parent cell. A host cell encompasses progeny cells. In embodiments, a host cell refers to any cell (including its progeny) that has been modified, transfected, transduced, transformed and / or manipulated in any way to express an antibody as disclosed herein. In one example, the host cell (or population) can be introduced with an expression vector operably linked to a nucleic acid encoding the desired antibody or antigen-binding portion thereof as described herein. Host cells and populations thereof can harbor expression vectors that are stably integrated into the host genome, or can harbor extrachromosomal expression vectors. In embodiments, host cells and populations thereof can harbor extrachromosomal vectors that are present after a few cell divisions, or that are transiently present and lost after a few cell divisions.

[0099] Transgenic host cells can be prepared using non-viral methods, including well-known designer nucleases, including zinc finger nucleases, TALENs, or CRISPR / Cas. Genome editing techniques, such as zinc finger nucleases, can be used to introduce transgenes into the genome of host cells. Zinc finger nucleases comprise a pair of chimeric proteins, each containing a non-specific endonuclease domain of a restriction endonuclease (e.g., FokI) fused with a DNA binding domain from an engineered zinc finger motif. The DNA binding domain can be engineered to bind to a specific sequence in the host genome, and the endonuclease domain creates a double-strand break. The donor DNA carries a transgene, for example, any of the nucleic acids encoding a CAR or DAR construct described herein, and flanking sequences that are homologous to regions on either side of the intended insertion site in the genome of the host cell. The DNA repair mechanism of the host cell allows for precise insertion of the transgene by homologous DNA repair. Transgenic mammalian host cells have been prepared using zinc finger nucleases (US Pat. Nos. 9,597,357, 9,616,090, 9,816,074 and 8,945,868). Transgenic host cells can be prepared using TALENs (transcription activator-like effector nucleases), which are similar to zinc finger nucleases in that they contain a non-specific endonuclease domain fused with a DNA binding domain that can result in precise transgene insertion. Like zinc finger nucleases, TALENs also introduce double-strand breaks into the host's DNA. Transgenic host cells can be prepared using CRISPRs (clustered regularly interspaced short palindromic repeats). CRISPRs utilize a Cas endonuclease coupled with a guide RNA for target-specific donor DNA integration.The guide RNA contains a conserved multinucleotide containing a protospacer adjacent motif (PAM) sequence upstream of the gRNA binding region in the target DNA, and hybridizes with the host cell target site where the Cas endonuclease cleaves the double-stranded target DNA. The guide RNA can be designed to hybridize to a specific target site. Similar to zinc finger nucleases and TALENs, the CRISPR / Cas system can be used to introduce site-specific insertion of donor DNA with adjacent sequences with homology to the insertion site. Examples of CRISPR / Cas systems used to modify genomes are described, for example, in U.S. Patent Nos. 8,697,359, 10,000,772, 9,790,490, and U.S. Patent Application Publication No. 2018 / 0346927. In one embodiment, the transgenic host cell can be prepared using zinc finger nucleases, TALENs, or CRISPR / Cas systems, and the host target site can be the TRAC gene (T cell receptor alpha constant). The donor DNA can include, for example, any of the nucleic acids encoding the CAR or DAR constructs described herein. Electroporation, nucleofection or lipofection can be used to co-deliver the donor DNA and zinc finger nucleases, TALEN or CRISPR / Cas systems into the host cell.

[0100] The host cell may be a prokaryote, e.g., E. coli, or it may be a eukaryote, e.g., a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), a mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. In one embodiment, an expression vector operably linked to a nucleic acid encoding a desired antibody may be introduced into the host cell, thereby generating a transfected / transformed host cell, the transfected / transformed host cell may be cultured under conditions suitable for expression of the antibody by the transfected / transformed host cell, and, if desired, the antibody may be recovered from the transfected / transformed host cell (e.g., recovered from a host cell lysate) or from the culture medium. In one embodiment, the host cell comprises a non-human cell, including CHO, BHK, NS0, SP2 / 0, and YB2 / 0. In embodiments, host cells include human cells, including HEK293, HT-1080, Huh-7 and PER.C6.Exemplary host cells include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or derivatives thereof, such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31), or the CHO line DX-B 11, which is deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, the BHK (ATCC CRL10) cell line, the CV1 / EBNA cell line (ATCC CCL 70) derived from the African green monkey kidney cell line CV1 (McMahan et al., 1991, EMBO J. 10:2821), human fetal kidney cells, e.g., 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo 205 cells, other transformed primate cell lines, normal diploid cells, cell lines obtained from in vitro culture of primary tissues, primary explants, HL-60, U937, HaK or Jurkat cells. In one embodiment, the host cell comprises a lymphoid cell, e.g., Y0, NS0 or Sp20. In an embodiment, the host cell is a mammalian host cell, but is not a human host cell. Typically, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, such that the nucleic acid can be expressed in the host cell. The phrase "transgenic host cell" or "recombinant host cell" can be used to mean a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that the regulatory sequence is operably linked to the nucleic acid. The term host cell is understood to refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell.Because certain modifications may occur in subsequent generations, due to, for example, mutations or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term as used herein.

[0101] The polypeptides (e.g., antibodies and antigen-binding proteins) of the present disclosure can be produced using any method known in the art. In one example, the polypeptides are produced by recombinant nucleic acid methods, in which a nucleic acid sequence (e.g., DNA) encoding the polypeptide is inserted into a recombinant expression vector, which is introduced into a host cell and expressed by the host cell under conditions promoting expression.

[0102] General techniques for recombinant nucleic acid manipulation are described, for example, in Sambrook et al., in Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., in Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and periodic revisions, and these references are incorporated herein by reference in their entirety. The nucleic acid (e.g., DNA) encoding the polypeptide is operably linked to an expression vector carrying one or more suitable transcriptional or translational regulatory elements derived from mammalian, viral or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that controls the termination of transcription and translation. The expression vector may contain an origin of replication that confers the ability to replicate in a host cell. The expression vector may contain a gene that provides for selection to facilitate recognition of transgenic host cells (e.g., transformants).

[0103] Recombinant DNA may also code for any type of protein tag sequence that may be useful for protein purification.Examples of protein tags include, but are not limited to, histidine tag, FLAG tag, myc tag, HA tag, or GST tag.Appropriate cloning and expression vectors for use with bacteria, fungi, yeast and mammalian cell hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, NY, 1985).

[0104] Expression vector construct can be introduced into host cell by using the method suitable for host cell.Various methods for introducing nucleic acid into host cell are known in the art, including but not limited to electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; viral transfection; non-viral transfection; particle bombardment; lipofection; and infection (for example, when vector is an infectious agent).Suitable host cell includes prokaryotic, yeast, mammalian cell, or bacterial cell.

[0105] Suitable bacteria include gram-negative or gram-positive bacteria, such as E. coli or Bacillus spp. Yeast, preferably from the Saccharomyces species, such as S. cerevisiae, can also be used to produce polypeptides. Various mammalian or insect cell culture systems can also be utilized to express recombinant proteins. Baculovirus systems for the production of heterologous proteins in insect cells are reviewed by Luckow and Summers, (Bio / Technology, 6:47, 1988). Examples of suitable mammalian host cell systems include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines. Purified polypeptides are prepared by culturing a suitable host / vector system to express the recombinant protein. For many applications, the small size of many of the polypeptides disclosed herein will make expression in E. coli the preferred expression method. The protein is then purified from the culture medium or cell extracts. Any of the anti-B7-H3 antibodies, antigen binding proteins, or antigen-binding portions thereof, can be expressed by a transgenic host cell.

[0106] The antibodies and antigen-binding proteins disclosed herein can also be produced using a cell translation system. For such purposes, the nucleic acid encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA, and to allow cell-free translation of the mRNA in the particular cell-free system used (eukaryotic, e.g., mammalian or yeast cell-free translation system, or prokaryotic, e.g., bacterial cell-free translation system).

[0107] Nucleic acids encoding any of the various polypeptides disclosed herein can be chemically synthesized. Codon usage can be selected to improve expression in cells. Such codon usage will depend on the cell type selected. Specialized codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See, e.g., Mayfield et al., 2003, Proc. Natl. Acad. Sci. USA. 100(2):438-42; Sinclair et al., 2002, Protein Expr. Purif. (1):96-105; Connell N D., 2001, Curr. Opin. Biotechnol. 12(5):446-9; Makrides et al., 1996, Microbiol. Rev. 60(3):512-38; and Sharp et al., 1991, Yeast. 7(7):657-78.

[0108] The antibodies and antigen binding proteins described herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to the proteins can also be made by chemical synthesis.

[0109] The antibody and antigen-binding protein described herein can be purified by protein isolation / purification methods generally known in the field of protein chemistry.Non-limiting examples include extraction, recrystallization, salting out (e.g., using ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination thereof.After purification, the polypeptide can be exchanged into different buffers and / or concentrated by any of a variety of methods known in the art, including but not limited to filtration and dialysis.

[0110] The purified antibodies and antigen-binding proteins described herein are preferably at least 65% pure, at least 75% pure, at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact numerical value of the purity, the polypeptide is sufficiently pure for use as a pharmaceutical product. Any of the anti-B7-H3 antibodies or antigen-binding proteins described herein can be expressed by a transgenic host cell and then purified to about 65-98% purity or a high level of purity using any method known in the art.

[0111] In certain embodiments, the antibodies and antigen-binding proteins herein may further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation or addition of polypeptide side chains or hydrophobic groups. As a result, modified polypeptides may contain non-amino acid elements, such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation, which conjugates one or more sialic acid moieties to the polypeptide. Sialic acid moieties improve solubility and serum half-life, but also reduce the potential immunogenicity of the protein. See Raju et al., 2001, Biochemistry. 31; 40(30):8868-76.

[0112] In embodiments, the antibodies and antigen binding proteins described herein can be modified to increase solubility, for example, by linking the antibodies and antigen binding proteins to a nonproteinaceous polymer. In one embodiment, the nonproteinaceous polymer comprises polyethylene glycol ("PEG"), polypropylene glycol, or polyoxyalkylenes in the manner set forth in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0113] PEG is a water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of size or modification at the termini of the PEG, and has the formula: XO(CH2CHO). n-CH2CH2OH(1) (wherein n is 20 to 2300 and X is H or a terminal modification, such as C 1~4 In embodiments, PEG is terminated at one end with hydroxy or methoxy, i.e., X is H or CH3 ("methoxy PEG"). PEG may contain additional chemical groups that are essential for the conjugation reaction, that result from the chemical synthesis of the molecule, or that are spacers for optimal distance of the parts of the molecule. In addition, such PEG may consist of one or more PEG side chains linked together. PEGs with more than one PEG chain are called multi-armed or branched PEGs. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, branched PEGs with four arms can be prepared from pentaerythritol and ethylene oxide. Branched PEGs are described, for example, in EP-A 0 473 084 and U.S. Pat. No. 5,932,462. One form of PEG contains two PEG side chains (PEG2) linked through the primary amino groups of lysine (Monfardini et al., 1995, Bioconjugate Chem. 6: 62-69).

[0114] The serum clearance rate of the PEG-modified polypeptide can be modulated (e.g., increased or decreased) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90% compared to the clearance rate of unmodified antibodies and antigen binding proteins that bind to the polypeptide. PEG-modified antibodies and antigen binding proteins have a half-life (t 1 / 2). The half-life of PEG-modified polypeptides may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, or 500%, or even 1000%, as compared to the half-life of unmodified antibodies and antigen-binding proteins. In some embodiments, the protein half-life is determined in vitro, e.g., in a buffered saline solution or in serum. In other embodiments, the protein half-life is the in vivo half-life, e.g., the half-life of the protein in serum or other bodily fluids of an animal.

[0115] The present disclosure provides pharmaceutical compositions comprising any of the anti-B7-H3 antibodies or antigen-binding proteins thereof described herein in admixture with pharma- ceutically acceptable excipients. Excipients include carriers, stabilizers and excipients. Excipients or pharma-ceutically acceptable excipients include, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricants, glidants, and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Additional examples include buffers, stabilizers, preservatives, non-ionic surfactants, antioxidants, and isotonicity agents.

[0116] Pharmaceutical compositions and methods for their preparation are well known in the art and can be found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. AR Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.). Pharmaceutical compositions that can be formulated for parenteral administration may or may not contain, for example, excipients, sterile water, saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the antibodies (or antigen-binding proteins thereof) described herein. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the antibodies (or antigen-binding proteins thereof). Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the antibody (or antigen-binding protein thereof) in the formulation will vary depending on several factors, including the dosage of the drug to be administered, and the route of administration.

[0117] Any of the anti-B7-H3 antibodies (or antigen-binding portions thereof) can be administered as a pharma- ceutically acceptable salt, such as non-toxic acid addition salts or metal complexes commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids, such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid; polymeric acids, such as tannic acid, carboxymethylcellulose; and inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like. Metal complexes include zinc, iron, and the like. In one example, the antibody (or antigen-binding portion thereof) is formulated in the presence of sodium acetate to increase thermal stability.

[0118] Any of the anti-B7-H3 antibodies (or antigen-binding portions thereof) can be administered as a pharma- ceutically acceptable salt, such as non-toxic acid addition salts or metal complexes commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids, such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid; polymeric acids, such as tannic acid, carboxymethylcellulose; and inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like. Metal complexes include zinc, iron, and the like. In one example, the antibody (or antigen-binding portion thereof) is formulated in the presence of sodium acetate to increase thermal stability.

[0119] Any of the anti-B7-H3 antibodies (or antigen-binding portions thereof) can be formulated for oral use, including tablets containing the active ingredient in a mixture with non-toxic pharma- ceutically acceptable excipients.Formulations for oral use can also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium.

[0120] The term "subject" as used herein refers to humans and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, the subject may be a human. In other embodiments, the subject may be a non-human primate, monkey, ape, murine (e.g., mouse and rat), bovine, porcine, equine, canine, feline, caprine, wolf, frog or fish subject.

[0121] The terms "administering", "administered" and grammatical variations refer to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" as used herein means a method of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered by a route other than a parenteral route, for example, orally. Other non-parenteral routes include topical, epidermal or mucosal administration routes, such as intranasally, vaginally, rectally, sublingually or topically. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods. Any of the anti-B7-H3 antibodies (or antigen-binding proteins thereof) described herein can be administered to a subject using methods and delivery routes known in the art.

[0122] The terms "effective amount," "therapeutically effective amount," or "effective dose," or related terms, may be used interchangeably and refer to an amount of an antibody or antigen binding protein (e.g., any of the anti-B7-H3 antibodies described herein or antigen binding proteins thereof) that is sufficient when administered to a subject to result in a measurable amelioration or prevention of a disease or disorder associated with tumor or cancer antigen expression. Therapeutically effective amounts of antibodies provided herein, when used alone or in combination, will vary depending on the relative activities of the antibodies and combinations (e.g., in inhibiting cell growth), as well as on the subject and condition to be treated, the subject's weight and age and sex, the severity of the condition in the subject, the mode of administration, etc., and can be readily determined by one of skill in the art.

[0123] In embodiments, the therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated, and one of skill in the art can ascertain the therapeutically effective amount using known techniques. In general, the polypeptide is administered at about 0.01 g / kg to about 50 mg / kg per day, preferably 0.01 mg / kg to about 30 mg / kg per day, and most preferably 0.1 mg / kg to about 20 mg / kg per day. The polypeptide can be administered daily (e.g., once, twice, three or four times daily) or preferably less frequently (e.g., once a week, once every two weeks, once every three weeks, once a month, or four times a year). In addition, as known in the art, adjustments for age, as well as weight, overall health, sex, diet, administration frequency, drug interactions and disease severity may be necessary.

[0124] The term "B7-H3 expressing tumor" as used herein refers to a tumor that expresses B7-H3 protein. In embodiments, B7-H3 expression in a tumor is determined using immunohistochemical staining of tumor cell membranes, and any immunohistochemical staining above background levels in a tumor sample indicates that the tumor is a B7-H3 expressing tumor. Methods for detecting expression of B7-H3 in tumors are known in the art and include immunohistochemical assays. In any of the disclosed embodiments relating to the medical use of the disclosed antibodies or antigen binding proteins for the treatment of tumor-related conditions, the tumor may be a B7-H3 expressing tumor.

[0125] The terms "overexpress", "overexpression", or "overexpressed" refer synonymously to a gene that is detectably transcribed or translated, usually in cancer cells, as compared to normal cells. Overexpression therefore refers to both protein and RNA overexpression (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability, and altered proteolysis), as well as local overexpression due to altered protein trafficking patterns (increased nuclear localization) and increased functional activity, such as in the case of increased enzymatic hydrolysis of a substrate. Overexpression therefore refers to either the protein level or the RNA level. Overexpression can be 50%, 60%, 70%, 80%, 90% or more compared to normal or comparative cells. In certain embodiments, anti-B7-H3 antibodies are used to treat solid tumors that are likely to overexpress B7-H3.

[0126] The term "combination therapy" or "combination" as used herein in the context of a method of therapy (e.g., a method of treatment) refers to the administration of two or more therapeutic substances, e.g., an anti-B7-H3 antibody and an additional therapeutic agent. The additional therapeutic agent may be administered concomitantly with, prior to, or following administration of the anti-B7-H3 antibody.

[0127] The present disclosure provides methods for treating a subject having a disease associated with expression or overexpression of B7-H3. In embodiments, the disease is an autoimmune disease. In embodiments, the disease is HIV. In embodiments, the disease is an inflammatory disease. In embodiments, the disease is an infectious disease. In embodiments, the disease comprises a cancer or tumor cell expressing a tumor-associated antigen. In embodiments, the cancer or tumor comprises cancer of the breast, prostate, colon, liver, ovary, bladder, pancreas, lung (including non-small cell lung or small cell lung), esophagus, stomach, skin and kidney.

[0128] In embodiments, the cancer is non-small cell lung cancer, small cell lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer (Johnson, KA et al, 2007, Analytical Biochemistry, 360:75 - 83; Winter, G. et al., 1994, Annu. Rev. Immunol., 12:433 - 455; Ashraf, SQ et al., 2009, British Journal Of Cancer 101(10):1758 - 1768; Barbara, BM and Stanley, MS, 1980, "Selected Methods in Cellular Immunology"; WH Freeman and company; Yamato, I. et al., 2009, British Journal Of Cancer 101(10):1709 - 1716; Sun, J. et al., 2010, Cancer Immunology, Immunotherapy 59: 1163 - 1171;Roth, TJ et al., 2007, Cancer Research 67: 7893 - 7900;Carmen, S. et al., 2002, Briefings In Functional Genomics and Proteomics 1(2):189 - 203). In prostate cancer, B7-H3 expression intensity is reported to be positively correlated with clinicopathological grade, such as tumor volume, extraprostatic invasion, and Grimson score, as well as with cancer progression. (Yamato, I. et al., 2009, British Journal of Cancer 101(10):1709 - 1716). Similarly, B7-H3 expression is negatively correlated with recurrence-free survival in glioblastoma multiforme (Sun, J. et al., 2010, Cancer Immunology, Immunotherapy 59: 1163 - 1171). In pancreatic cancer, B7-H3 expression correlates with lymph node metastasis and disease progression.(Carmen, S. et al., 2002, Briefings in Functional Genomics and Proteomics 1(2):189 - 203). In ovarian cancer, B7-H3 expression correlates with lymph node metastasis and disease progression.

[0129] In embodiments, the cancer includes, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. In embodiments, the cancer includes glioblastoma, glioma, neuroblastoma, melanoma, hepatocellular carcinoma, clear cell renal cell carcinoma, acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), colorectal cancer, oral cancer, head and neck cancer, breast cancer (e.g., triple-negative breast cancer), squamous cell tumor, hypopharyngeal squamous cell carcinoma, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), urothelial cell carcinoma, gastric cancer, anal cancer, endometrial cancer, and vulvar cancer. In embodiments, the cancer is metastatic cancer, refractory cancer, or recurrent cancer.

[0130] The present disclosure provides a B7-H3 binding protein, particularly an anti-B7-H3 antibody, or an antigen-binding portion thereof, that specifically binds to B7-H3, and uses thereof. In an embodiment, the B7-H3 binding protein binds to an epitope of B7-H3. B7-H3 is a type I transmembrane protein (e.g., UniProt Q5ZPR3-1) that is encoded by chromosome 9 in mice and chromosome 15 in humans. The extracellular domain is composed of a single pair of immunoglobulin variable domain and immunoglobulin constant domain in mice (2IgB7-H3 isoforms) and two identical pairs in humans due to exon duplication (4IgB7-H3 isoforms). The intracellular tail of B7-H3 is short, and its signaling motif is unknown.

[0131] Various aspects of the anti-B7-H3 antibodies relate to antibody fragments, single chain antibodies, pharmaceutical compositions, nucleic acids, recombinant expression vectors, host cells, and methods for preparing and using such anti-B7-H3 antibodies. Methods for using the anti-B7-H3 antibodies include in vitro and in vivo methods for binding to B7-H3, detecting B7-H3, and treating diseases associated with B7-H3 expression.

[0132] The present disclosure provides antigen binding proteins that specifically bind to B7-H3 polypeptides (e.g., antigen targets) or to fragments of B7-H3 polypeptides. In embodiments, the B7-H3 target antigen comprises a naturally occurring polypeptide (e.g., UniProt Accession No. Q5ZPR3-1) having a wild-type or polymorphic or mutant amino acid sequence. The B7-H3 target antigen can be prepared by recombinant methods or chemically synthesized. The B7-H3 target antigen can be in soluble or membrane-bound form (e.g., expressed by cells or phage).

[0133] In an embodiment, the B7-H3 target antigen is expressed by a cell, for example, a cancer or non-cancer cell line that naturally expresses B7-H3 or is engineered to express B7-H3, such as A549, U-2197, ASC TERT1, CACO-2 or HHSteC. Cell lines that do not express B7-H3, such as Jurkat, Daudi or K562 cell lines or B7-H3 KO cell lines, are not expected to bind to anti-B7-H3 antibodies. The B7-H3 target antigen can be a fusion protein with a detectable moiety, such as a fluorophore, or can be conjugated to a detectable moiety, such as a fluorophore. The B7-H3 target antigen can be a fusion protein with an affinity tag, such as a His tag, or can be conjugated to an affinity tag, such as a His tag. In embodiments, the human B7-H3 target antigen comprises the amino acid sequence of SEQ ID NO:1 (eg, UniProt Accession No. Q5ZPR3-1) or SEQ ID NO:2 (eg, recombinant his-tagged human B7-H3 from Sino Biologicals catalog no. 11188-H08H-50).

[0134] In embodiments, the antigen-binding protein is an IgG, IgA, IgD, IgE or IgM antibody with one or more mutations in the Fc region, for example, one or more mutations that attenuate antibody-dependent enhancement (ADE) and / or one or more mutations that extend antibody half-life. Mutations that reduce or eliminate the interaction of the Fc region of the antibody with its receptor (e.g., FcγR) on such cells can reduce or eliminate ADE. For example, the antigen-binding protein as provided herein can be an IgG1 or IgG4 antibody with a mutation in the Fc region that reduces ADE, for example, a LALA mutation, or can be a single chain antibody (ScFv) that optionally includes an Fc region that can optionally include a mutation that reduces ADE, for example, a LALA mutation. In further embodiments, the antigen-binding protein as provided herein can be a Fab, Fab', or F(ab')2 antibody fragment.

[0135] The present disclosure provides a fully human antibody of the IgG class that binds to a B7-H3 polypeptide. In embodiments, the anti-B7-H3 antibody comprises a heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100, or a combination thereof; and / or the anti-B7-H3 antibody comprises a light chain variable region having 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101, or a combination thereof. In an embodiment, the anti-B7-H3 antibody comprises an IgG1, IgG2, IgG3 or IgG4 class antibody. In an embodiment, the anti-B7-H3 antibody comprises an IgG1 or IgG4 class antibody. In an embodiment, the anti-B7-H3 antibody comprises an IgG1 class antibody. In an embodiment, the anti-B7-H3 antibody comprises an IgG4 class antibody. In an embodiment, the IgG1 class antibody may have a mutation in the Fc region selected from L234A or L235A. In an embodiment, the mutation in the Fc region may include mutations L234A and L235A (LALA). In an embodiment, the mutation in the Fc region is an LA mutation or two LALA mutations. In an embodiment, the LALA mutation in the Fc region reduces the effector function of the anti-TIGIT antibody compared to an anti-TIGIT antibody that does not have a LALA mutation in the Fc region.

[0136] In embodiments, the anti-B7-H3 antibody or fragment thereof has a binding affinity to an epitope of the B7-H3 target antigen (polypeptide). -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9M or less, or 10 -10 Binding affinity (K D ) (see FIG. 1A and Table 2). In embodiments, the anti-B7-H3 antibodies or fragments thereof described herein comprise antigen-binding portions that bind to an epitope of the B7-H3 target antigen with similar binding affinity, regardless of whether the antibody comprises the mutations L234A and L235A in the Fc region (see FIGS. 14 and 15). In embodiments, the binding affinity of the anti-B7-H3 antibodies or fragments thereof described herein to the B7-H3 target antigen is essentially the same for the wild-type antibody and the mutant antibody in which the mutations are L234A and L235A in the Fc region.

[0137] In embodiments, the B7-H3 antigen comprises cell surface B7-H3 antigen or soluble B7-H3 antigen. In embodiments, the B7-H3 antigen comprises the extracellular portion of cell surface B7-H3 antigen. In embodiments, the B7-H3 antigen comprises human or non-human B7-H3 antigen. In embodiments, the B7-H3 antigen is expressed by human or non-human cells. In embodiments, the anti-B7-H3 antibody binds to human B7-H3 expressed by antigen presenting cells (APCs), macrophages, or expressed by human tumor cells (e.g., clear cell renal carcinoma and colon carcinoma). In embodiments, the binding between the anti-B7-H3 antibody or fragment thereof can be detected and measured using surface plasmon resonance, flow cytometry, and / or ELISA.

[0138] The term "cross-reacting" as used herein refers to the ability of the antibodies described herein to bind to B7-H3 from different species. The present disclosure provides anti-B7-H3 antibodies that can bind to an epitope of B7-H3 from humans or bind to an epitope of B7-H3 (e.g., homologous antigens) from any one or any combination of non-human animals, such as mice, rats, goats, rabbits, hamsters, and / or monkeys (e.g., cynomolgus monkeys).

[0139] In embodiments, the anti-B7-H3 antibody or antigen-binding fragment is a 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 Binding affinity K below M D In embodiments, the anti-B7-H3 antibody or antigen-binding fragment binds to cynomolgus B7-H3 at 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 Binding affinity K below M D Combine with.

[0140] In an embodiment, cynomolgus B7-H3 is commercially available from Sino Biologicals (catalog number 90806-C08H-20). In an embodiment, mouse B7-H3 is commercially available from Sino Biologicals (catalog number 50973-M08H-50). In an embodiment, human B7-H3 is commercially available from Sino Biologicals (catalog number 11188-H08H-50).

[0141] In embodiments, the anti-B7-H3 antibody has enhanced thermal stability. In embodiments, the anti-B7-H3 antibody has a T of at least 30° C., or at least 35° C., or at least 40° C., or at least 45° C., or at least 50° C., or at least 55° C., or at least 60° C., or at least 65° C., or at least 70° C., or at least 75° C., or at least 80° C., or at least 85° C., or at least 90° C. m (See, for example, FIG. 4). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0142] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (referred to herein as VA), or an antigen-binding fragment thereof, comprising a heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 14, and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 17.

[0143] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (referred to herein as B3-A6), or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:22, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:23, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:24, and a light chain CDR1 having the amino acid sequence of SEQ ID NO:25, a light chain CDR2 having the amino acid sequence of SEQ ID NO:26, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:27.

[0144] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (herein referred to as B3-D1), or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 33, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 34, and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 36, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 37.

[0145] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (herein referred to as B3-2D1), or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 44, and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 45, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 46, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 47.

[0146] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (referred to herein as B3-2E3), or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:52, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:53, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:54, and a light chain CDR1 having the amino acid sequence of SEQ ID NO:55, a light chain CDR2 having the amino acid sequence of SEQ ID NO:56, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:57.

[0147] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (herein referred to as B3-2G11), or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:62, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:63, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:64, and a light chain CDR1 having the amino acid sequence of SEQ ID NO:65, a light chain CDR2 having the amino acid sequence of SEQ ID NO:66, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:67.

[0148] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (referred to herein as B3-2H1), or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 74, and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 75, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 76, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 77.

[0149] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (herein referred to as B3-4A1), or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 82, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 83, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 84, and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 85, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 86, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 87.

[0150] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (referred to herein as B3-4F7), or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:92, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:93, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94, and a light chain CDR1 having the amino acid sequence of SEQ ID NO:95, a light chain CDR2 having the amino acid sequence of SEQ ID NO:96, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:97.

[0151] The present disclosure provides an anti-B7-H3 antigen binding protein or fully human anti-B7-H3 antibody (referred to herein as B3-5A6), or an antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104, and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 105, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 106, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 107.

[0152] The present disclosure relates to fully human antibodies that bind to B7-H3, comprising both heavy and light chains, wherein the heavy / light chain variable region amino acid sequences are selected from the following set of amino acid sequences: SEQ ID NOs: 10 and 11 (referred to herein as VA), SEQ ID NOs: 20 and 21 (referred to herein as B3-A6), SEQ ID NOs: 30 and 31 (referred to herein as B3-D1), SEQ ID NOs: 40 and 41 (referred to herein as B3-2D1), SEQ ID NOs: 50 and 51 (referred to herein as B3-2E3), SEQ ID NOs: 60 and 61 (referred to herein as B3-2G11), and SEQ ID NOs: 70 and 72 (referred to herein as B3-2G21). In one embodiment, the fully human antibody has at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to any of SEQ ID NOs: 70 and 71 (referred to herein as B3-2H1), 80 and 81 (referred to herein as B3-4A1), 90 and 91 (referred to herein as B3-4F7), or SEQ ID NOs: 100 and 101 (referred to herein as B3-5A6). In an embodiment, the VA antibody comprises mutations L234A and L235A in the Fc region (VA-LALA). In an embodiment, the B3-A6 antibody comprises mutations L234A and L235A in the Fc region (B3-A6-LALA). In an embodiment, the B3-D1 antibody comprises mutations L234A and L235A in the Fc region (B3-D1-LALA). In an embodiment, the B3-2D1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2D1-LALA). In an embodiment, the B3-2E3 antibody comprises the mutations L234A and L235A in the Fc region (B3-2E3-LALA). In an embodiment, the B3-2G11 antibody comprises the mutations L234A and L235A in the Fc region (B3-2G11-LALA). In an embodiment, the B3-2H1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2H1-LALA). In an embodiment, the B3-4A1 antibody comprises the mutations L234A and L235A in the Fc region (B3-4A1-LALA). In embodiments, the B3-4F7 antibody comprises the mutations L234A and L235A in the Fc region (B3-4F7-LALA).In embodiments, the B3-5A6 antibody contains the mutations L234A and L235A in the Fc region (B3-5A6-LALA).

[0153] The present disclosure provides a Fab fully human antibody fragment comprising a heavy chain variable region from a heavy chain and a variable region from a light chain, wherein the sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, SEQ ID NO: 90 or SEQ ID NO: 100, or a combination thereof. The sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, SEQ ID NO: 81, SEQ ID NO: 91 or SEQ ID NO: 101, or a combination thereof.

[0154] The present disclosure relates to a Fab fully human antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy / light chain variable region amino acid sequences are selected from the following set of amino acid sequences: SEQ ID NOs: 10 and 11 (referred to herein as VA), SEQ ID NOs: 20 and 21 (referred to herein as B3-A6), SEQ ID NOs: 30 and 31 (referred to herein as B3-D1), SEQ ID NOs: 40 and 41 (referred to herein as B3-2D1), SEQ ID NOs: 50 and 51 (referred to herein as B3-2E3), SEQ ID NOs: 60 and 61 (referred to herein as B3-2E4), SEQ ID NOs: 62 and 63 (referred to herein as B3-2E5), SEQ ID NOs: 63 and 64 (referred to herein as B3-2E6), SEQ ID NOs: 64 and 65 (referred to herein as B3-2E7), SEQ ID NOs: 65 and 66 (referred to herein as B3-2E8), SEQ ID NOs: 66 and 67 (referred to herein as B3-2E9), SEQ ID NOs: 67 and 68 (referred to herein as B3-2E1), SEQ ID NOs: 68 and 69 (referred to herein as B3-2E2), SEQ ID NOs: 69 and 70 (referred to herein as B3-2E3), SEQ ID NOs: 71 and 72 (referred to herein as B3-2E4), SEQ ID NOs: 73 and 74 (referred to herein as B3-2E5), SEQ ID NOs: 74 and 75 (referred to herein as B3-2E6), SEQ ID NOs: 75 and B3-2G11), SEQ ID NOs: 70 and 71 (referred to herein as B3-2H1), SEQ ID NOs: 80 and 81 (referred to herein as B3-4A1), SEQ ID NOs: 90 and 91 (referred to herein as B3-4F7), or SEQ ID NOs: 100 and 101 (referred to herein as B3-5A6). In an embodiment, the VA antibody comprises mutations L234A and L235A in the Fc region (VA-LALA). In an embodiment, the B3-A6 antibody comprises mutations L234A and L235A in the Fc region (B3-A6-LALA). In an embodiment, the B3-D1 antibody comprises mutations L234A and L235A in the Fc region (B3-D1-LALA). In an embodiment, the B3-2D1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2D1-LALA). In an embodiment, the B3-2E3 antibody comprises the mutations L234A and L235A in the Fc region (B3-2E3-LALA). In an embodiment, the B3-2G11 antibody comprises the mutations L234A and L235A in the Fc region (B3-2G11-LALA). In an embodiment, the B3-2H1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2H1-LALA). In an embodiment, the B3-4A1 antibody comprises the mutations L234A and L235A in the Fc region (B3-4A1-LALA). In embodiments, the B3-4F7 antibody comprises the mutations L234A and L235A in the Fc region (B3-4F7-LALA).In embodiments, the B3-5A6 antibody contains the mutations L234A and L235A in the Fc region (B3-5A6-LALA).

[0155] The disclosure provides a single chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain and optionally a linker joining the variable heavy and variable light chain regions, wherein the variable heavy chain region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90 or SEQ ID NO:100 or a combination thereof. The variable light chain region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91 or SEQ ID NO:101, or a combination thereof.

[0156] The present disclosure relates to single chain fully human antibodies comprising a polypeptide chain having a heavy chain variable region and a light chain variable region, wherein the heavy / light chain variable region amino acid sequence set is selected from the following amino acid sequence sets: SEQ ID NOs: 10 and 11 (referred to herein as VA), SEQ ID NOs: 20 and 21 (referred to herein as B3-A6), SEQ ID NOs: 30 and 31 (referred to herein as B3-D1), SEQ ID NOs: 40 and 41 (referred to herein as B3-2D1), SEQ ID NOs: 50 and 51 (referred to herein as B3-2E3), SEQ ID NOs: 60 and 61 (referred to herein as B3-2E4), SEQ ID NOs: 62 and 63 (referred to herein as B3-2E5), SEQ ID NOs: 63 and 64 (referred to herein as B3-2E6), SEQ ID NOs: 64 and 65 (referred to herein as B3-2E7), SEQ ID NOs: 65 and 66 (referred to herein as B3-2E8), SEQ ID NOs: 66 and 67 (referred to herein as B3-2E9), SEQ ID NOs: 67 and 68 (referred to herein as B3-2E1), SEQ ID NOs: 68 and 69 (referred to herein as B3-2E2), SEQ ID NOs: 69 and 70 (referred to herein as B3-2E3), SEQ ID NOs: 71 and 72 (referred to herein as B3-2E4), SEQ ID NOs: 73 and 74 (referred to herein as B3-2E5), SEQ ID NOs: 74 and 75 (referred to herein as B3-2E6), SEQ ID NOs: 75 In one embodiment, the VA antibody comprises mutations L234A and L235A in the Fc region (VA-LALA). In another embodiment, the B3-A6 antibody comprises mutations L234A and L235A in the Fc region (B3-A6-LALA). In another embodiment, the B3-D1 antibody comprises mutations L234A and L235A in the Fc region (B3-D1-LALA). In an embodiment, the B3-2D1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2D1-LALA). In an embodiment, the B3-2E3 antibody comprises the mutations L234A and L235A in the Fc region (B3-2E3-LALA). In an embodiment, the B3-2G11 antibody comprises the mutations L234A and L235A in the Fc region (B3-2G11-LALA). In an embodiment, the B3-2H1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2H1-LALA). In an embodiment, the B3-4A1 antibody comprises the mutations L234A and L235A in the Fc region (B3-4A1-LALA). In embodiments, the B3-4F7 antibody comprises the mutations L234A and L235A in the Fc region (B3-4F7-LALA).In embodiments, the B3-5A6 antibody contains the mutations L234A and L235A in the Fc region (B3-5A6-LALA).

[0157] The present disclosure provides a pharmaceutical composition comprising any of the anti-B7-H3 antibodies or antigen-binding proteins thereof described herein in admixture with a pharma- ceutically acceptable excipient. Excipients include carriers and stabilizers. In one embodiment, the pharmaceutical composition comprises an anti-B7-H3 antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy / light chain variable region amino acid sequences are selected from the following set of amino acid sequences: SEQ ID NOs: 10 and 11 (referred to herein as VA), SEQ ID NOs: 20 and 21 (referred to herein as B3-A6), SEQ ID NOs: 30 and 31 (referred to herein as B3-D1), SEQ ID NOs: 40 and 41 (referred to herein as B3-2D1), SEQ ID NOs: 50 and 51 (referred to herein as B3-2E3), SEQ ID NOs: 52 and 53 (referred to herein as B3-2E4), SEQ ID NOs: 54 and 55 (referred to herein as B3-2E5), SEQ ID NOs: 56 and 57 (referred to herein as B3-2E6), SEQ ID NOs: 59 and 60 (referred to herein as B3-2E7), SEQ ID NOs: 61 and 62 (referred to herein as B3-2E8), SEQ ID NOs: 63 and 64 (referred to herein as B3-2E9), SEQ ID NOs: 65 and 66 (referred to herein as B3-2E1), SEQ ID NOs: 67 and 68 (referred to herein as B3-2E2), SEQ ID NOs: 68 and 69 (referred to herein as B3-2E3), SEQ ID NOs: 69 and 70 (referred to herein as B3-2 At least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of sequence numbers 60 and 61 (referred to herein as B3-2G11), SEQ ID NOs: 70 and 71 (referred to herein as B3-2H1), SEQ ID NOs: 80 and 81 (referred to herein as B3-4A1), SEQ ID NOs: 90 and 91 (referred to herein as B3-4F7), or SEQ ID NOs: 100 and 101 (referred to herein as B3-5A6). In an embodiment, the VA antibody comprises mutations L234A and L235A in the Fc region (VA-LALA). In an embodiment, the B3-A6 antibody comprises mutations L234A and L235A in the Fc region (B3-A6-LALA). In an embodiment, the B3-D1 antibody comprises mutations L234A and L235A in the Fc region (B3-D1-LALA). In an embodiment, the B3-2D1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2D1-LALA). In an embodiment, the B3-2E3 antibody comprises the mutations L234A and L235A in the Fc region (B3-2E3-LALA). In an embodiment, the B3-2G11 antibody comprises the mutations L234A and L235A in the Fc region (B3-2G11-LALA). In an embodiment, the B3-2H1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2H1-LALA). In an embodiment, the B3-4A1 antibody comprises the mutations L234A and L235A in the Fc region (B3-4A1-LALA).In an embodiment, the B3-4F7 antibody comprises the mutations L234A and L235A in the Fc region (B3-4F7-LALA). In an embodiment, the B3-5A6 antibody comprises the mutations L234A and L235A in the Fc region (B3-5A6-LALA).

[0158] The present disclosure provides kits comprising any one or any combination of two or more of the anti-B7-H3 antibodies or antigen-binding fragments thereof described herein. In embodiments, the kits comprise any one or any combination of two or more anti-B7-H3 antibodies or antigen-binding fragments thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy / light chain variable region amino acid sequences are selected from the following sets of amino acid sequences: SEQ ID NOs: 10 and 11 (referred to herein as VA), SEQ ID NOs: 20 and 21 (referred to herein as B3-A6), SEQ ID NOs: 30 and 31 (referred to herein as B3-D1), SEQ ID NOs: 40 and 41 (referred to herein as B3-2D1), SEQ ID NOs: 50 and 51 (referred to herein as B3-2D1), SEQ ID NOs: 52 and 53 (referred to herein as B3-2D2), SEQ ID NOs: 54 and 54 (referred to herein as B3-2D3), SEQ ID NOs: 55 and 56 (referred to herein as B3-2D4), SEQ ID NOs: 57 and 58 (referred to herein as B3-2D5), SEQ ID NOs: 59 and 60 (referred to herein as B3-2D6), SEQ ID NOs: 61 and 62 (referred to herein as B3-2D7), SEQ ID NOs: 63 and 64 (referred to herein as B3-2D8), SEQ ID NOs: 64 and 65 (referred to herein as B3-2D9), SEQ ID NOs: 65 and 66 (referred to herein as B3-2D1), SEQ ID NOs: 66 and 67 (referred to herein as B3-2D1), SEQ ID NOs: 67 and In an embodiment, the VA antibody comprises mutations L234A and L235A in the Fc region (VA-LALA). In an embodiment, the B3-A6 antibody comprises mutations L234A and L235A in the Fc region (B3-A6-LALA). In an embodiment, the B3-D1 antibody comprises the mutations L234A and L235A in the Fc region (B3-D1-LALA). In an embodiment, the B3-2D1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2D1-LALA). In an embodiment, the B3-2E3 antibody comprises the mutations L234A and L235A in the Fc region (B3-2E3-LALA). In an embodiment, the B3-2G11 antibody comprises the mutations L234A and L235A in the Fc region (B3-2G11-LALA). In an embodiment, the B3-2H1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2H1-LALA). In embodiments, the B3-4A1 antibody contains the mutations L234A and L235A in the Fc region (B3-4A1-LALA).In an embodiment, the B3-4F7 antibody comprises the mutations L234A and L235A in the Fc region (B3-4F7-LALA). In an embodiment, the B3-5A6 antibody comprises the mutations L234A and L235A in the Fc region (B3-5A6-LALA).

[0159] The kit can be used to detect the presence or absence of B7-H3 antigen, for example, in biological samples.The kit can be used to carry out in vitro reactions, such as antigen binding assays in the form of ELISA, flow cytometry or surface plasmon resonance; in vitro cell activation assays; luciferase-reporter assays; Western blotting and detection; and other such in vitro assays.The kit can be used to treat subjects with B7-H3-related diseases or conditions, for example, sarcoma, osteosarcoma, pancreatic adenocarcinoma, head and neck cancer, or neuroblastoma.

[0160] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100, or a combination thereof.

[0161] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., VA) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 14.

[0162] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-A6) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:22, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO:23, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO:24.

[0163] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-D1) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 33, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 34.

[0164] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2D1) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 42, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 43, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 44.

[0165] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2E3) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:52, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO:53, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO:54.

[0166] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2G11) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 63, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 64.

[0167] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2H1) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 73, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 74.

[0168] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-4A1) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 82, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 83, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 84.

[0169] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-4F7) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 92, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 93, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 94.

[0170] The present disclosure provides a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-5A6) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 103, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 104.

[0171] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100. In one embodiment, the first vector comprises an expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0172] The present disclosure provides a first vector (e.g., a first expression vector) operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody heavy chain variable region having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100. In one embodiment, the first vector comprises an expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0173] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., VA) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 14. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0174] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-A6) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 23, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 24. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0175] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-D1) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 33, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 34. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0176] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2D1) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 42, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 43, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 44. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0177] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2E3) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 52, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 53, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 54. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0178] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2G11) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 63, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 64. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0179] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2H1) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 73, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 74. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0180] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-4A1) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 82, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 83, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 84. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0181] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-4F7) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 92, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 93, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 94. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0182] The present disclosure provides a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody (e.g., B3-5A6) heavy chain variable region having a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO: 103, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 104. In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first vector comprises at least one promoter operably linked to the first nucleic acid.

[0183] The disclosure provides a first host cell harboring a first vector operably linked to a first nucleic acid encoding a first polypeptide comprising an anti-B7-H3 antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100.

[0184] In one embodiment, the first vector comprises a first expression vector. In one embodiment, the first host cell expresses a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100.

[0185] The present disclosure provides a method for preparing a first polypeptide comprising an antibody heavy chain variable region, the method comprising culturing a population of first host cells (e.g., a plurality of first host cells) harboring a first expression vector under conditions suitable for expressing a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100.

[0186] In one embodiment, the method further comprises recovering from the first population of host cells an expressed first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90 or SEQ ID NO:100.

[0187] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101, or a combination thereof.

[0188] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., VA) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 17.

[0189] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-A6) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:25, a light chain CDR2 region having the amino acid sequence of SEQ ID NO:26, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO:27.

[0190] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-D1) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 35, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 36, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 37.

[0191] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2D1) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 45, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 46, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 47.

[0192] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2E3) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:55, a light chain CDR2 region having the amino acid sequence of SEQ ID NO:56, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO:57.

[0193] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2G11) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:65, a light chain CDR2 region having the amino acid sequence of SEQ ID NO:66, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO:67.

[0194] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2H1) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 75, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 76, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 77.

[0195] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-4A1) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 85, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 86, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 87.

[0196] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-4F7) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:95, a light chain CDR2 region having the amino acid sequence of SEQ ID NO:96, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO:97.

[0197] The disclosure provides a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-5A6) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 106, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 107.

[0198] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0199] The present disclosure provides a second vector (e.g., a second expression vector) operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody light chain variable region having at least 95% sequence identity to SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101. In one embodiment, the second vector comprises an expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0200] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., VA) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 17. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0201] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-A6) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 25, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 27. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0202] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-D1) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 35, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 36, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 37. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0203] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2D1) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 45, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 46, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 47. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0204] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2E3) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 55, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 56, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 57. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0205] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2G11) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 65, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 66, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 67. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0206] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-2H1) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 75, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 76, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 77. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0207] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-4A1) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 85, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 86, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 87. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0208] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-4F7) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 95, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 96, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 97. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0209] The present disclosure provides a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody (e.g., B3-5A6) light chain variable region having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 106, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 107. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second vector comprises at least one promoter operably linked to the second nucleic acid.

[0210] The disclosure provides a second host cell harboring a second vector operably linked to a second nucleic acid encoding a second polypeptide comprising an anti-B7-H3 antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101. In one embodiment, the second vector comprises a second expression vector. In one embodiment, the second host cell expresses a second polypeptide comprising an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101.

[0211] The present disclosure provides a method for preparing a second polypeptide comprising an antibody light chain variable region, the method comprising culturing a population of second host cells (e.g., a plurality of second host cells) harboring a second expression vector under conditions suitable for expressing a second polypeptide comprising an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO: 101. In one embodiment, the method further comprises recovering the expressed second polypeptide comprising an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101 from the population of second host cells.

[0212] The disclosure provides first and second nucleic acids, where (a) the first nucleic acid encodes a first polypeptide comprising an anti-B7-H3 antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100, and (b) a second polypeptide comprising an anti-B7-H3 antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101.

[0213] The disclosure provides a vector operably linked to a first and a second nucleic acid, where (a) the first nucleic acid encodes a first polypeptide comprising an anti-B7-H3 antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100, and (b) a second polypeptide comprising an anti-B7H3 antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101. In one embodiment, the vector comprises an expression vector. In one embodiment, the vector comprises at least a first promoter operably linked to a first nucleic acid. In one embodiment, the vector comprises at least a second promoter operably linked to a second nucleic acid.

[0214] The disclosure provides a host cell harboring a vector operably linked to a first and a second nucleic acid, wherein (a) the first nucleic acid is an anti-B7-H3 antibody heavy chain having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100. (b) a second nucleic acid encodes a first polypeptide comprising a variable region, and (b) a second nucleic acid encodes a second polypeptide comprising an anti-B7-H3 antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, SEQ ID NO: 81, SEQ ID NO: 91, or SEQ ID NO: 101. In one embodiment, the vector comprises an expression vector. In one embodiment, the host cell expresses (a) a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100, and (b) a second polypeptide comprising an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101.

[0215] The present disclosure provides a method for preparing a first polypeptide having an antibody heavy chain variable region and a second polypeptide having an antibody light chain variable region, the method comprising culturing a population of host cells (e.g., a plurality of host cells) harboring an expression vector operably linked to a first and second nucleic acid encoding the first and second polypeptides, respectively. In one embodiment, the culturing is performed under conditions suitable for expressing (a) a first polypeptide having an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90, or SEQ ID NO:100, and (b) a second polypeptide having an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91, or SEQ ID NO:101. In one embodiment, the method further comprises recovering from the population of host cells an expressed first polypeptide having an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:90 or SEQ ID NO:100, and an expressed second polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:81, SEQ ID NO:91 or SEQ ID NO:101.

[0216] In an embodiment, the host cell, or population of host cells, harbors one or more expression vectors capable of directing the transient introduction of a transgene into the host cell or the stable insertion of a transgene into the genome of the host cell, the transgene comprising a nucleic acid encoding any of the first and / or second polypeptides described herein. The expression vector can direct the transcription and / or translation of the transgene in the host cell. The expression vector can include one or more regulatory sequences, e.g., inducible and / or constitutive promoters and enhancers. The expression vector can include a ribosome binding site and / or a polyadenylation site. In one embodiment, the expression vector operably linked to the nucleic acid encoding the first and / or second polypeptide can direct the production of the first and / or second polypeptide that can be displayed on the surface of the transgenic host cell, or the first and / or second polypeptide can be secreted into the cell culture medium.

[0217] The present disclosure provides a method for inhibiting growth or proliferation of target cells or for killing target cells, comprising contacting a population of effector cells with a population of target cells (e.g., target cells expressing B7-H3) in the presence of an anti-B7-H3 antibody (or antibody fragment thereof) under conditions suitable for inhibiting growth or proliferation of target cells or for killing target cells. In embodiments, the population of effector cells comprises peripheral blood mononuclear cells (PBMCs) or natural killer (NK) cells. PBMCs may comprise lymphocytes, including T cells, B cells and / or NK cells. In embodiments, the population of target cells comprises cells that naturally express B7-H3, including T cells; NK cells; antigen-presenting cells (APCs), including dendritic cells and macrophages. B7-H3 is found on fibroblasts, fibroblast-like synoviocytes, and epithelial cells, and may play diverse roles in regulating the growth and differentiation of non-hematopoietic tissues. In embodiments, the population of target cells includes cells that naturally express B7-H3, including, for example, prostate cancer cells, non-small cell lung cancer cells, gastric cancer cells, and ovarian cancer cells from a subject having a disease associated with B7-H3 expression. In embodiments, the population of target cells is any type of transgenic cell that has been engineered to express B7-H3. In embodiments, the ratio of effector cells to target cells can be about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 5-10:1, or about 10-20:1, or about 20-30:1.

[0218] The present disclosure provides in vitro methods for blocking the interaction (eg, binding) of B7-H3 with its receptor.

[0219] In embodiments, a method for blocking the interaction of a B7-H3 polypeptide with its receptor comprises contacting any of the anti-B7-H3 antibodies (or antibody fragments thereof) described herein (e.g., VA, B3-A6, B3-D1, B3-2D1, B3-2E3, B3-2G11, B3-2H1, B3-4A1, B3-4F7, or B3-5A6) with a B7-H3 polypeptide and a B7-H3 receptor under conditions suitable for binding of the anti-B7-H3 antibody (or antibody fragment thereof) to the B7-H3 receptor and for blocking between the B7-H3 polypeptide and the B7-H3 receptor. In embodiments, the anti-B7-H3 antibody (or antibody fragment thereof) can be contacted with the B7-H3 polypeptide and the B7-H3 receptor at the same time (essentially simultaneously) or sequentially in any order. In embodiments, the blocking method can be performed in vitro or in vivo.

[0220] In embodiments, a method for blocking the interaction between a B7-H3 expressing cell and a cell expressing a receptor for B7-H3 comprises contacting a B7-H3 expressing cell and a cell expressing a receptor for B7-H3 with any of the anti-B7-H3 antibodies (or antibody fragments thereof) described herein (e.g., VA, B3-A6, B3-D1, B3-2D1, B3-2E3, B3-2G11, B3-2H1, B3-4A1, B3-4F7, or B3-5A6) under conditions suitable for binding between the anti-B7-H3 antibody (or antibody fragment thereof) and the cell expressing the B7-H3 receptor, and for blocking between the B7-H3 expressing cell and the cell expressing the B7-H3 receptor. In embodiments, the anti-B7-H3 antibody (or antibody fragment thereof) can be contacted with the B7-H3 expressing cell and the cell expressing the B7-H3 receptor at the same time (essentially simultaneously) or sequentially in any order. In embodiments, the blocking method can be performed in vitro or in vivo. In embodiments, the B7-H3 expressing cell comprises a T cell. In embodiments, the B7-H3 expressing cell comprises a NK cell. In embodiments, the B7-H3 expressing cell comprises a tumor cell. In embodiments, the cell expressing the B7-H3 receptor comprises a NK cell. In embodiments, the cell expressing the B7-H3 receptor comprises a T cell. In embodiments, the VA antibody comprises mutations L234A and L235A in the Fc region (VA-LALA). In embodiments, the B3-A6 antibody comprises mutations L234A and L235A in the Fc region (B3-A6-LALA). In an embodiment, the B3-D1 antibody comprises the mutations L234A and L235A in the Fc region (B3-D1-LALA). In an embodiment, the B3-2D1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2D1-LALA). In an embodiment, the B3-2E3 antibody comprises the mutations L234A and L235A in the Fc region (B3-2E3-LALA). In an embodiment, the B3-2G11 antibody comprises the mutations L234A and L235A in the Fc region (B3-2G11-LALA). In an embodiment, the B3-2H1 antibody comprises the mutations L234A and L235A in the Fc region (B3-2H1-LALA). In embodiments, the B3-4A1 antibody contains the mutations L234A and L235A in the Fc region (B3-4A1-LALA).In an embodiment, the B3-4F7 antibody comprises the mutations L234A and L235A in the Fc region (B3-4F7-LALA). In an embodiment, the B3-5A6 antibody comprises the mutations L234A and L235A in the Fc region (B3-5A6-LALA).

[0221] The present disclosure provides a method for treating a subject having a disease associated with B7-H3 expression, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an anti-B7-H3 antibody or antigen-binding fragment thereof selected from the group consisting of any of the fully human anti-B7-H3 antibodies described herein, any of the Fab fully human anti-B7-H3 antibodies described herein, and any of the single chain human anti-B7-H3 antibodies described herein. The present disclosure provides a method for treating a subject having a disease associated with B7-H3 expression or overexpression. In an embodiment, the disease associated with B7-H3 expression is an autoimmune disease. In an embodiment, the disease associated with B7-H3 expression is HIV. In an embodiment, the disease associated with B7-H3 expression is an inflammatory disease. In an embodiment, the disease associated with B7-H3 expression is an infectious disease. In an embodiment, the disease associated with B7-H3 expression comprises carcinoma of ovarian, colon, prostate, skin, pancreatic, renal, urothelial, or lung cancer. In embodiments, diseases associated with B7-H3 expression include cancer of the breast, prostate, colon, liver, ovary, bladder, pancreas, lung (eg, non-small cell lung or small cell lung), esophagus, stomach, skin, or kidney. In embodiments, the disease associated with B7-H3 expression is selected from the group consisting of acute myeloid lymphoma (AML), non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), small cell lung cancer, urothelial cell carcinoma, esophageal cancer, hepatocellular carcinoma, glioma, neuroblastoma, glioblastoma multiforme, blastoma, sarcoma, leukemia, lymphoid malignancies, pancreatic cancer, head and neck cancer, ovarian cancer, oral cancer, breast cancer, triple-negative breast cancer (TNBC), lymphoma, renal cell carcinoma, clear cell renal cell carcinoma, colon cancer, colorectal cancer, melanoma, gastric cancer, lung cancer, liver cancer, bladder cancer, prostate cancer, anal cancer, endometrial cancer, vulvar cancer, squamous cell tumors, hypopharyngeal squamous cell carcinoma, and squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer).

[0222] In embodiments, the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer.

[0223] Anti-B7-H3 antibodies can be used alone to inhibit the growth of cancerous tumors. In embodiments, anti-B7-H3 antibodies can be used in combination with another agent for the treatment of diseases associated with B7-H3 expression (or increased B7-H3 expression), such as other immunogenic agents, standard cancer treatments, or other antibodies.

[0224] In an embodiment, the disease associated with B7-H3 expression is cancer. In an embodiment, a method for treating a subject with a cancer expressing B7-H3, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an anti-B7-H3 antibody or an antigen-binding fragment thereof selected from the group consisting of any of the fully human anti-B7-H3 antibodies described herein, any of the Fab fully human anti-B7-H3 antibodies described herein, and any of the single chain human anti-B7-H3 antibodies described herein. The method further comprises co-administration of a cytotoxic agent, a cystostatic agent, or an anti-angiogenic agent suitable for treating the cancer.

[0225] In embodiments, treatment of a subject having a disease associated with B7-H3 expression (e.g., cancer) with a B7-H3 antibody as provided herein (e.g., a VA clone), or with an antibody having a heavy chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10 and a light chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 11, can result in reduced toxicity to the subject, including, but not limited to, less anemia, hemagglutination of peripheral blood cells, or reduced immune cells, than treatment of the patient with the same tumor-targeting antibody and a different anti-B7-H3 antibody.

[0226] In embodiments, B7-H3 (VA clone) exhibits reduced hemagglutination when contacted with human red blood cells compared to the Macrogenics anti-B7-H3 antibody (MG-B7-H3).

[0227] Array Listing: Human B7-H3 protein (UniProt Q5ZPR3-1) SEQ ID NO:1: [ka]

[0228] Recombinant truncated human his-tagged B7-H3 protein (amino acids 1-461 of SEQ ID NO:1 with a carboxy-terminal polyhistidine tag) SEQ ID NO:2: [ka]

[0229] Enoblituzumab (B7-H3 Macrogenics; MGA271; MG-B7-H3) SEQ ID NO:3: Heavy Chain: [ka] SEQ ID NO:4: Light chain: [ka]

[0230] B7-H3 Memorial Sloan Kettering Cancer Center (MKSCC B7-H3, Hu8H9) SEQ ID NO:5: Heavy Chain: [ka] SEQ ID NO:6: Light chain: [ka] [ka]

[0231] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] EXAMPLES

[0232] Working Example

[0233] The following examples are intended to be illustrative and can be used to further understand embodiments of the present disclosure and should not be construed as limiting the scope of the present teachings in any way.

[0234] Example 1 Measurement of binding affinity using surface plasmon resonance.

[0235] The binding kinetics of anti-B7-H3 antibodies and his-tagged B7-H3 protein was measured using surface plasmon resonance (SPR). Anti-B7-H3 antibodies tested included an anti-B7-H3 antibody according to the present disclosure (VA clone, heavy and light chain variable regions comprising the amino acid sequences of SEQ ID NOs: 10 and 11, respectively) and the commercially available B7-H3 antibody Enobrituzumab (MacroGenics). Anti-human fragment crystallizable region (Fc region) antibodies diluted in pH 5.0 acetate buffer were immobilized on a CM5 sensor chip to approximately 8,000 RU using standard N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (NHS / EDC) coupling methodology automated by a Biacore model T200 (described in the Biacore Concentration Analysis Handbook).

[0236] Anti-B7-H3 antibody (VA clone) or enoblituzumab (2 μg / mL) was captured for 60 seconds at a flow rate of 10 μL / min. Recombinant human his-tagged B7-H3 protein comprised amino acid 1 to amino acid 461 of SEQ ID NO:1 (i.e., SEQ ID NO:2) (Sino Biologicals; Cat. No. 11188-H08H-50). The polypeptide was serially diluted 1:1 in running buffer HBS-EP+ (Cytiva; Cat. No. BR100669) starting at 100 nM. All measurements were performed in HBS-EP+ buffer at a flow rate of 30 μL / min, automated by a Biacore model T200. The surface was regenerated with 3 M MgCl2 for 60 seconds. Data were fitted using a 1:1 (Langmuir) binding model. All BIACORE assays were performed at room temperature using a Biacore model T200 (GE Healthcare).

[0237] The SPR sensorgrams of anti-B7-H3 antibodies B7-H3 (VA clone) and enoblituzumab are shown in Figures 1A-1B, respectively, and their corresponding binding kinetics are listed below in Table 2. Both B7-H3 antibodies B7-H3 (VA clone) and enoblituzumab showed affinity for their cognate antigens in the nM range.

[0238] [Table 2]

[0239] Example 2 Measurement of antigen-binding specificity by ELISA.

[0240] Anti-B7-H3 antibodies (VA clone and VA LALA clone) were analyzed for antigen binding specificity by ELISA assay focusing on human antigens B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, B7.1, and B7.2 (Sino Biologicals). Each antigen protein was dissolved in PBS buffer (50 ng / well) and coated on the plate at 50 μl per well overnight at 4°C. The plate was then washed three times with 150 μL / well of KPL buffer (Sera Care; Cat. No. 5150-0008). The plate was then blocked with 50 μL / well of Superblock buffer (Scytek; Cat. No. AAA500) for 1 h at room temperature with gentle shaking. After blocking, the plate was washed three times with 150 μL / well of KPL buffer. Anti-B7-H3 antibodies (VA and VA LALA clones) were serially diluted in Superblock buffer at 50 μL / well (triplicates noted) in a separate plate. The diluted antibodies were added (50 μL / well) to the coated plate and incubated for 1 hour at room temperature with gentle shaking. The plate was washed three times with 150 μL / well KPL buffer. 50 μL / well HRP-conjugated detection antibody (Jackson Immuno Research; Cat. No. 109-036-097) diluted 1:10,000 in KPL buffer was added and incubated for 1 hour at room temperature with gentle shaking. The plate was then washed three times with 150 μL / well KPL buffer and binding was revealed by applying 50 μL / well KPL SureBlue substrate (Sera Care; Cat. No. 5120-0075). Signal development was stopped at the desired saturation point by using a 1N HCl stop solution and read at 450 nm using a plate reader.

[0241] Figure 2A shows that anti-B7-H3 antibody (VA clone) specifically binds to human B7-H3 antigen and does not engage with human B7-H1, B7-H2, B7-H4, B7-H5, B7-H6, B7-H7, B7.1, or B7.2. Figure 2B shows that anti-B7-H3 antibody (VA LALA clone) specifically binds to human B7-H3 antigen and does not engage with human B7-H1, B7-H2, B7-H4, B7-H5, B7-H6, B7-H7, B7.1, or B7.2.

[0242] Example 3 ELISA cross-reactivity.

[0243] The cross-reactivity of anti-human B7-H3 antibodies (B7-H3 VA and MG-B7-H3) with his-tagged B7-H3 recombinant proteins from human, mouse, cynomolgus monkey, canine, rat and rabbit was analyzed using an ELISA assay. Mouse antigen protein (Sino Biologicals; Catalog No. 50973-M08H-50), cynomolgus monkey antigen protein (Sino Biologicals; Catalog No. 90806-C08H-20), rat antigen protein (Sino Biologicals; Catalog No. 80380-R02H-20), rabbit antigen protein, canine antigen protein, or human antigen protein (Sino Biologicals; Catalog No. 11188-H08H-50) was dissolved in PBS buffer (50 ng / well) and coated on plates at 50 μl per well overnight at 4°C. The plate was then washed three times with 150 μL / well KPL buffer (Sera Care; Cat. No. 5150-0008). The plate was then blocked with 50 μL / well Superblock buffer (Scytek; Cat. No. AAA500) for 1 hour at room temperature with gentle shaking. After blocking, the plate was washed three times with 150 μL / well KPL buffer. Anti-B7-H3 (VA clone) or Enoblituzumab (MacroGenics MG-B7-H3) was serially diluted in Superblock buffer at 50 μL / well (triplicates noted) in a separate plate. The diluted antibodies were added (50 μL / well) to the coated plate and incubated for 1 hour at room temperature with gentle shaking. The plate was washed three times with 150 μL / well KPL buffer. 50 μL / well of HRP-conjugated detection antibody (Jackson Immuno Research; Catalog No. 109-036-097) diluted 1:10,000 in KPL buffer was added and incubated for 1 hour at room temperature with gentle shaking. Plates were then washed three times with 150 μL / well of KPL buffer and binding was revealed by applying 50 μL / well of KPL SureBlue substrate (Sera Care; Catalog No. 5120-0075).The signal was stopped at the desired saturation point by using a 1N HCl stop solution and read at 450 nm using a plate reader.

[0244] FIG. 3 shows that anti-B7-H3 antibody (VA clone) binds to canine, rat, rabbit and cynomolgus monkey B7-H3 antigens to the same extent as Macrogenics' B7-H3.

[0245] Example 4 Thermal stability of anti-B7-H3 antibody (VA clone).

[0246] The thermal stability of IgG protein was evaluated by the melting temperature T m The antibody was evaluated by determining the melting temperature. The antibody was diluted to 1 mg / ml in PBS buffer, and 9 μL of the antibody solution was loaded onto the UNI sample cartridge. A heating range of 30 °C to 90 °C was set with a heating rate of 1 °C per minute. Fluorescence was detected in the range of 250 to 720 nm. Curve calculation and fitting by centroid mean fluorescence (BCM) was performed to evaluate the melting temperature of the antibody.

[0247] FIG. 4 shows improved antibody thermostability T proteins that contribute to antibody stability, promote extended serum half-life, and facilitate ease of manufacturing. m Indicates >70°C.

[0248] Example 5 Antigen recognition of anti-B7-H3 antibody (clone VA) in patient biopsies.

[0249] Antigen / target recognition ability of anti-B7-H3 antibody (clone VA) was tested in solid tumors, e.g., sarcoma, head and neck cancer, and pancreatic adenocarcinoma. To prevent tissue detachment during antigen retrieval and to facilitate deparaffinization, slides were baked at 60°C for 30 min, up to 120 min, using a heating block. Slides were then placed in a coupling jar (Fisher Scientific; 19-4) and further deparaffinized / rehydrated in xylene for 5 min at room temperature three times. Slides were then immersed in 100% ethanol for 10 min twice at room temperature, followed by immersing in 95% ethanol for 10 min twice at room temperature, immersing in 70% ethanol for 10 min twice at room temperature, immersing in 50% ethanol for 10 min twice at room temperature, and finally immersing in DI water for 5 min twice at room temperature. Slide racks were submerged in antigen retrieval solution (Vector Laboratories; Cat. No. H3300-250) and then autoclaved for 10 min in solid phase. Slides were then rinsed with cold tap water for 5 min. Pancreatic cancer tissue (Biomax.US; Cat. No. HPan-Ade060CS-01), sarcoma tissue (Biomax.US; Cat. No. SO809a), and head and neck cancer tissue (Biomax.US; Cat. No. T342) were fixed with 2% paraformaldehyde diluted in PBS for 15 min at room temperature. Slides were washed three times with PBS for 5 min at room temperature. Tissue slides were precipitated / permeabilized with ice-cold methanol for 15 min at room temperature with gentle shaking. Slides were then washed three times with PBS for 5 min at room temperature. Wax was applied to close a ring around the tissue biopsy using an ImmEdge Pen. Blocking buffer containing 1% mouse serum and 2.5% goat serum diluted in PBS was applied for 1 hour at room temperature, and the tissue biopsies were then washed with PBS to remove any residual fluid.

[0250] Antibody staining solution was prepared as follows: 1% mouse serum and 2.5% goat serum diluted in PBS, and 1 μg / ml anti-B7-H3 antibody (VA clone). Tissue biopsies were covered with the primary antibody staining solution, placed in a wet chamber, and incubated overnight at 4°C. Tissue biopsies were then washed with PBS and all residual liquid was removed. Antibody staining solution was prepared as follows: 1% mouse serum and 2.5% goat serum diluted in PBS, and fluorescently labeled secondary antibodies diluted in the range of 1:1000 to 1:25. Tissue biopsies were covered with the secondary antibody staining solution, placed in a wet chamber, and incubated for 4 hours at room temperature. Tissue biopsies were then washed with PBS and all residual liquid was removed. Up to 25 μL of Mowiol mounting solution (Sigma; Cat. No. 81381-250G) was applied (depending on the size of the tissue biopsy) and covered with a microscope cover slip. Mowiol-mounted biopsies were allowed to dry (protected from light) at room temperature for 24 hours. Fluorescence emission was analyzed using a confocal laser scanning microscope.

[0251] Figure 5 shows human cancer tissue arrays (sarcoma, head and neck cancer, and pancreatic adenocarcinoma) immunostained with anti-B7-H3 antibody (VA clone) to assess antigen recognition in malignant patient tissues (green-grey) compared to adjacent normal tissues (blue-grey). Anti-B7-H3 antibody (VA clone) shows increased binding to malignant patient tissue biopsies but no binding to adjacent normal tissues.

[0252] Example 6 Flow cytometric cell binding assay.

[0253] Flow cytometry was used to test the binding ability of anti-B7-H3 antibody (VA clone) to human osteosarcoma cells (U2OS) and human neuroblastoma cells (SK-N-BE(2)). Human U2OS (ATCC; Catalog No. HTB-96) and SK-N-BE(2) (ATCC; Catalog No. CRL-2271) cells were subcultured in McCoy's 5a medium (Thermo Fisher Scientific; Catalog No. 16600082) or EMEM:F12 1:1 medium (ATCC; Catalog No. 30-2003: Thermo Scientific; Catalog No. 31765035) supplemented with 1% antibiotic / antimycotic (Fisher Scientific; Catalog No. 15240062) and 10% FBS (Genesee Scientific; Catalog No. 25550). Cells were harvested using trypsin / EDTA (Thermo Fisher Scientific; Cat. No. 25300120), pelleted by centrifugation at 1500 rpm for 5 min, and washed once with PBS (CellGro; Cat. No. 21-031-CM). Cells were stained with anti-RSV (control IgG), enoblituzumab (Macrogenics) or anti-B7-H3 antibody (VA clone) diluted in staining buffer (PBS / 1% BSA) for 30 min at room temperature or left unstained. Cells were washed once with PBS followed by centrifugation at 100 rpm for 5 min. For cells stained with anti-RSV, enoblituzumab or anti-B7-H3 antibody (VA clone), secondary antibody staining was performed at room temperature for 30 min using anti-human IgG 488 (Invitrogen; Cat. No. A11036) diluted 1:200 in staining buffer. Cells were washed with PBS, spun down at 1500 rpm for 5 min, and resuspended in 300 μL staining buffer, followed by flow cytometry analysis using a BD Accuri C6 plus cytometer. Data were collected and analyzed using FlowJo software.

[0254] Figure 6A shows the results of immunostaining human osteosarcoma (U2OS) cells with anti-RSV (control IgG), enoblitzumab, or anti-B7-H3 antibody (VA clone), as well as cells left unstained (control). Figure 6A shows that anti-B7-H3 antibody (VA clone) shows stronger binding than enoblitzumab at all concentrations. It also shows that anti-B7-H3 antibody (VA clone) clearly binds to U2OS cells even at concentrations as low as 0.01 μg / ml.

[0255] Figure 6B shows the results of immunostaining of human neuroblastoma (SK-N-BE(2)) cells with anti-RSV (control IgG), enoblituzumab, or anti-B7-H3 antibody (VA clone), as well as cells left unstained (control). Figure 6B shows that anti-B7-H3 antibody (VA clone) exhibits stronger binding than enoblituzumab at all concentrations. It also shows that anti-B7-H3 antibody (VA clone) clearly binds to SK-N-BE(2) cells even at concentrations as low as 0.01 μg / ml.

[0256] Example 7 Antibody-dependent cell-mediated cytotoxicity (ADCC) prediction.

[0257] The potential of anti-B7-H3 antibody (VA clone) to recruit NK cells was predicted by artificial ADCC assay. The artificial ADCC assay uses an engineered T cell line carrying an inducible luciferase transgene. The resulting luminescence upon engagement of the IgG Fc portion indicates the antibody-cell interaction and helps predict the NK recruitment ability of anti-B7-H3 antibody (VA clone).

[0258] Human osteosarcoma (U2OS) cells were cultured in McCoy's 5a medium (Thermo Fisher Scientific; Catalog No. 16600082) supplemented with 1% antibiotic / antimycotic (Fisher Scientific; Catalog No. 15240062) and 10% FBS (Genesee Scientific; Catalog No. 25550). Cells were harvested using trypsin / EDTA (Thermo Fisher Scientific; Catalog No. 25300120), pelleted by centrifugation at 1500 rpm for 5 min, and washed once with PBS (CellGro; Catalog No. 21-031-CM). Cells were counted and plated at 3 × 10 in a 96-well plate. 3 Cells / well were plated. Antibody (anti-B7-H3 antibody (VA clone) or enoblituzumab) was added to each well at the indicated concentration. Effector cells (engineered Jurkat cells) (Promega, Core Kit; Cat# G7018) were added to the 96-well plate at an E:T ratio of 25:1. Target / effector cells were then incubated for 6 hours at 37°C, 5% CO2. Luciferase substrate (from the kit) was added to the wells, followed by incubation for 10 minutes at room temperature. Luminescence was read using a Spark microplate reader and data were analyzed.

[0259] Figure 7A shows the ADCC activity of anti-B7-H3 antibody (VA clone) and enoblituumab by luminescence acquisition. Figure 7A shows that the predicted ADCC ability of anti-B7-H3 antibody (VA clone) is superior to that of enoblituumab. In addition, it shows that anti-B7-H3 antibody (VA clone) has ADCC ability even at low concentration.

[0260] Example 8 Antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

[0261] Human osteosarcoma (U2OS) cells were cultured in McCoy's 5a medium (Thermo Fisher Scientific; Catalog No. 16600082) or EMEM:F12 1:1 medium (ATCC; Catalog No. 30-2003: Thermo Scientific; Catalog No. 31765035) supplemented with 1% antibiotic / antimycotic (Fisher Scientific; Catalog No. 15240062) and 10% FBS (Genesee Scientific; Catalog No. 25550). Cells were harvested using trypsin / EDTA (Thermo Fisher Scientific; Catalog No. 25300120), pelleted by centrifugation at 1500 rpm for 5 min, and washed once with PBS (CellGro; Catalog No. 21-031-CM). Cells were counted and plated at 3 × 10 in 96-well plates. 3 Cells / well were plated. Antibodies (anti-B7-H3 antibody (VA clone), enoblituzumab or anti-RSV antibody) were added to each well at the indicated concentrations. Effector cells (NK cells) were added to 96-well plates at an E:T ratio of 25:1. NK cells were purified from human PBMCs (ATCC; Cat. No. PCS800011) using the EasySep™ Human NK Cell Enrichment Kit according to the manufacturer's instructions (Stemcell technologies; Cat. No. 19055). Target / effector cells were then incubated for 6 hours at 37°C and 5% CO2. Recombinant luciferase and its substrate (CellTiter-Glo; Promega; Cat. No. G7572) were added to the wells, followed by incubation at room temperature for 10 minutes to allow ATP-dependent enzyme substrate turnover according to the manufacturer's instructions. Luminescence was read using a Spark microplate reader and data were analyzed.

[0262] Figure 7B shows the ADCC activity (by indirect killing) of anti-B7-H3 antibody (VA clone) compared to enoblitzumab. Figure 7B shows that anti-B7-H3 antibody (VA clone)-mediated target cell killing by NK cells is significantly superior to enoblitzumab-mediated target cell killing.

[0263] Figure 7C compares the ADCC activity (by indirect killing) of anti-B7-H3 antibody (VA clone) compared to anti-RSV antibody (isotype IgG control). Figure 7C shows that the anti-RSV antibody has no ADCC activity.

[0264] Example 9 Antigen-restricted target cell engagement ADCC assay.

[0265] To test whether anti-B7-H3 antibody (clone VA)-mediated target cell engagement was restricted to its antigen, loss-of-function B7-H3 knockout cells were generated and expressed against the parental B7-H3 in an artificial ADCC assay. + compared with cells.

[0266] Human lung cancer A549 cells (wild type WT, or knockout KO for B7-H3) were cultured in F-12K cell culture medium (ATCC; Catalog No. 30-2004) supplemented with 1% antibiotic / antimycotic (Fisher Scientific; Catalog No. 15240062) and 10% FBS (Genesee Scientific; Catalog No. 25550). Cells were harvested using trypsin / EDTA (Thermo Fisher Scientific; Catalog No. 25300120), pelleted by centrifugation at 1500 rpm for 5 min, and washed once with PBS (CellGro; Catalog No. 21-031-CM). Cells were counted and plated at 3 × 10 in 96-well plates. 3 Cells / well were plated. Antibody (anti-B7-H3 antibody (VA clone) or enoblituzumab) was added to each well at the indicated concentration. Effector cells (engineered Jurkat cells) (Promega; Core Kit; Cat# G7018) were added to the 96-well plate at an E:T ratio of 15:1. Target / effector cells were then incubated for 6 hours at 37°C, 5% CO2. Luciferase substrate (from the kit) was added to the wells, followed by incubation for 10 minutes at room temperature. Luminescence was read using a Spark microplate reader and data was analyzed.

[0267] FIG. 8A demonstrates the role of the B7-H3 antigen in anti-B7-H3 antibody (clone VA)-mediated cell engagement. + Mediates cell engagement with A549 lung cancer cells. Figure 8B shows that loss of B7-H3 cell expression (A549 B7-H3 KO cells) completely desensitizes anti-B7-H3 antibody (clone VA)-mediated cell engagement. Loss of B7-H3 expression is proportional to loss of luminescence.

[0268] Example 10 B7-H3 surface expression by A549 WT or A549 B7-H3 KO cells by flow cytometry.

[0269] The procedure for flow cytometry analysis was described above in Example 6, except that in this procedure, APC-labeled anti-human CD276 (B7-H3) antibody (Biolegend; 351006) was used (1:200) as the primary antibody for cell immunostaining (instead of B7-H3 VA clone). Alternatively, cells were unstained or stained with anti-RSV antibody for isotype control.

[0270] Figure 9 shows B7-H3 surface expression by A549 WT and A549 B7-H3 KO cells. Gating was focused on B7-H3 expressing cells and expressed as % of the population. Figure 9 demonstrates that B7-H3 is expressed only by A549 WT cells.

[0271] Example 11 In vivo efficacy study of anti-B7-H3 clone VA in a human neuroblastoma tumor model.

[0272] Human neuroblastoma tumor growth kinetics was evaluated after high or low dosing of anti-B7-H3 antibody (clone VA). Human neuroblastoma cells (SK-N-BE(2)) were cultured in EMEM:F12 1:1 cell culture medium (ATCC; Catalog No. 30-2003: Thermo Scientific; Catalog No. 31765035) supplemented with 1% antibiotic / antimycotic (Fisher Scientific; Catalog No. 15240062) and 10% FBS (Genesee Scientific; Catalog No. 25550). Cells were harvested using trypsin / EDTA (Thermo Fisher Scientific; Catalog No. 25300120), pelleted by centrifugation at 1500 rpm for 5 min, and resuspended in HBSS / Matrigel (Corning; Catalog Nos. 21-022-CM and 356237) mixed at a 1:1 ratio at 5 × 10 6 cells / 100 μL HBSS / Matrigel were obtained. SCID mice were subjected to a hair removal procedure using a hair removal cream (Nair) 24 hours prior to tumor cell inoculation. On day 0, 5×10 6 100 μL of HBSS / Matrigel containing cells was administered subcutaneously into the shaved flank of each mouse. Starting on day 4, tumors were grown to between 50 and 100 mm 3 Once tumor volume reached 100 μL, tumor-bearing mice were randomized (N=8 for all groups) and subjected to systemic treatment at the indicated doses and frequencies (see paragraph

[0161] below). Tumor-bearing mice were administered systemically via the retro-orbital sinus route with 200 μL of HBSS (buffer control), anti-RSV antibody (isotype control, 10 mg / kg), or various doses of anti-B7-H3 antibody (clone VA) diluted in HBSS. For dose administration, mice were anesthetized using 2-4% isoflurane / oxygen.

[0273] Mice were dosed at 1 mg / kg, 5 mg / kg or 10 mg / kg every other day, 3 days per week (for high-frequency dosing experiments) or 0.1 mg / kg, 1 mg / kg or 10 mg / kg once every 2 weeks (for low-frequency dosing experiments). Tumor dimensions were assessed using calipers. Tumor volume was calculated as volume = 1 / 2 x length x width. 2 It was calculated as:

[0274] Figure 10 shows the effect of 1 mg / kg, 5 mg / kg or 10 mg / kg of anti-B7-H3 antibody (clone VA) and 10 mg / kg of isotype control (anti-RSV) on tumor volume when administered every other day, three times a week, averaged over 8 animals and measured over 36 days. **P<0.01; ***P<0.001 between vehicle and anti-B7-H3 antibody (VA clone) groups is considered statistically significant.

[0275] Figure 11 shows the effect of 0.1 mg / kg, 1 mg / kg or 10 mg / kg of anti-B7-H3 antibody (clone VA) and 10 mg / kg of isotype control (anti-RSV) on tumor volume when administered once every two weeks, averaged over 8 animals and measured over 32 days. **P<0.01; ***P<0.001 between vehicle and anti-B7-H3 antibody (VA clone) groups is considered statistically significant.

[0276] Figures 10 and 11 show that systemically administered anti-B7-H3 antibody (VA clone) reduces tumor growth kinetics at doses as low as 1 mg / kg. Anti-B7-H3 antibody (VA clone) treatment reduces tumor growth kinetics in a dose-dependent manner.

[0277] Example 12 Hemagglutination assessment of anti-B7-H3 clone VA.

[0278] Hemagglutination of red blood cells (RBCs) was measured using anti-B7-H3 antibodies STI-B7-H3-VA (the name was used synonymously with the B7-H3 VA clone), B7-H3-VA LALA, and anti-CD47 Hu5F9 (HC [ka] [ka]

[0279] Red blood cells (RBCs) were prepared from human patient blood by mixing 1 ml of blood with 14 ml of PBS in a 15 ml Falcon tube and centrifuged at 800 rpm for 10 minutes at room temperature. The supernatant was discarded and another 15 ml of PBS was added to the cell pellet and the centrifugation was repeated. 9 ml of PBS was added to the cell pellet, thereby generating a 10% stock solution of RBCs. The RBC count was determined.

[0280] A 0.5% RBC working solution was prepared by diluting a 10% stock solution with assay buffer (2% FBS, 2 mM EDTA in DBPS).

[0281] 50 μl / well of working solution was added to a U-bottom low-attachment 96-well plate. The following antibodies were used in this assay: anti-CD47 Hu5F9, B7-H3-VA (anti-B7-H3), B7-H3-VA LALA (anti-B7-H3), MG-B7-H3 (anti-B7-H3, enoblituzumab from MacroGenics), and isotype control (anti-RSV antibody). Serial dilutions of antibodies were made in PBS (1:5 serial dilutions). Antibody dilutions were transferred to plates containing RBCs (100 μL / well, starting antibody concentration: 100 μg / mL). Plates were incubated for 24 hours at 37° C. and images were acquired. In a similar experiment, 1:2 serial dilutions were made starting at 200 μg / ml, and the B7-H3 VA antibody did not show any hemagglutination even at 200 μg / ml.

[0282] A negative result (no hemagglutination) will appear as a red dot in the center of the round bottom plate. A positive result (hemagglutination) will form a uniform reddish color across the well.

[0283] Figure 12A provides a schematic diagram and examples of positive and negative results. Figures 12B and 12C (plate images) show the results of the assay. In Figure 12B, the MG-B7-H3 antibody induces hemagglutination in wells corresponding to antibody concentrations starting from 100 μg / mL, and hemagglutination disappears at antibody concentrations of 50 μg / mL and below. In contrast, even at the highest antibody concentration of STI-B7-H3-VA (200 μg / mL), hemagglutination is not evident. The positive control antibody Hu5F9 induces hemagglutination in wells corresponding to antibody concentrations starting from 0.032 μg / mL. The negative control non-binding anti-RSV antibody (isotype control) did not induce hemagglutination at any concentration. In Figure 12C, MG-B7-H3, B7-H3-VA LALA, and isotype control (anti-RSV) antibodies did not induce hemagglutination up to the highest antibody concentration tested (100 μg / mL). The positive control antibody Hu5F9 induces hemagglutination in wells corresponding to antibody concentrations starting at 0.16 μg / mL.

[0284] Conclusions: Neither the STI-B7-H3-VA nor the B7-H3-VA LALA antibodies induce hemagglutination even at high concentrations up to 200 μg / mL.

[0285] Example 13 Cell-binding activity of B7-H3 clone VA measured by flow cytometry.

[0286] Flow cytometry was used to test the binding ability of anti-B7-H3 antibody (VA clone) to human melanoma cells (A375), human lung cancer cells (A549) and human pancreatic cancer cells (Panc-1).

[0287] Human cancer cell lines were subcultured using media conditions as recommended by the supplier. Briefly, human malignant melanoma A375 and human pancreatic cancer PANC-1 cells were cultured using DMEM medium, human lung cancer A549 cells were cultured using F-12K medium, and all cell culture media were supplemented with 10% FBS and antibiotic / antimycotic.

[0288] Cells were harvested with DPBS containing 10 mM EDTA and plated in 2 ml deep-bottom 96-well plates at 100,000 cells / well suspended in 50 μl staining buffer (1% BSA in DPBS). The following antibodies were used in this assay: B7-H3-VA (anti-B7-H3), MG-B7-H3 (anti-B7-H3, enoblituzumab from MacroGenics), and isotype control (anti-RSV antibody). Serial dilutions of the antibodies were made in staining buffer (containing 1% BSA in DPBS) (1:5 serial dilutions) starting at a final concentration of 100 μg / ml (prepared at 200 μg / ml).

[0289] Antibody dilutions were transferred to the plates containing the cells (50 μL / well of antibody was added to each well; starting antibody concentration: 100 μg / mL). Plates were incubated for 25 minutes at 37° C., then washed with 1 ml / well of staining buffer (1% BSA in DPBS), centrifuged at 1,500 rpm for 3 minutes at room temperature, and the supernatant was discarded.

[0290] 100 μl of fluorescently labeled secondary antibody diluted 1:500 in staining buffer (1% BSA in DPBS) was added to each well and the plate was incubated for 20 minutes at 37°C. The plate was washed with 1 ml of staining buffer (1% BSA in DPBS) and centrifuged at 1,500 rpm for 3 minutes at room temperature. 100 μl of cold fixation buffer (4% paraformaldehyde in PBS) was added to each well and the plate was incubated for 20 minutes at 37°C (protected from light). 100 μl of staining buffer was then added to each well and the cells were analyzed by flow cytometry using an AttuneNXT instrument. The collected data was analyzed by FlowJo v10 software, converting raw fluorescence to geometric mean fluorescence values.

[0291] Dose-dependent binding of the antibodies was performed in triplicate and acquired by flow cytometry. Figure 13 shows the results of immunostaining of human melanoma cells (A375), human lung cancer cells (A549) or human pancreatic cancer cells (Panc-1) with anti-RSV (isotype control IgG), Macrogenics (MG-B7-H3 antibody) or anti-B7-H3 antibody (VA clone). Figure 13 demonstrates that MG-B7-H3 has reduced binding compared to B7-H3-VA in all three cell types. B7-H3-VA shows 10-100 times stronger cell binding activity (cell type dependent) than MG-B7-H3. B7-H3-VA shows binding to human cancer cells A375, A549 and Panc-1 at very low concentrations (even 0.0001 μg / ml).

[0292] Example 14 Receptor occupancy of B7-H3 clone VA measured by flow cytometry.

[0293] Flow cytometry was used to measure receptor engagement of anti-B7-H3 antibody (VA clone) with human melanoma cells (A375), human lung cancer cells (A549), and human pancreatic cancer cells (Panc-1).

[0294] Human cancer cell lines were subcultured using media conditions as recommended by the supplier. Briefly, human malignant melanoma A375 and human pancreatic cancer PANC-1 cells were cultured using DMEM medium, human lung cancer A549 cells were cultured using F-12K medium, and all cell culture media were supplemented with 10% FBS and antibiotic / antimycotic.

[0295] Cells were detached from culture dishes using cell dissociation buffer according to the manufacturer's instructions and plated into 96-well U-bottom plates at 100,000 cells / well suspended in 50 μl assay buffer (4% FBS in RPMI 1640 medium) immediately prior to antibody application.

[0296] All antibodies were fluorescently labeled (in-house) with fluorophores excitable at 647 nm (near infrared, nIR).

[0297] The following antibodies were used in this assay: B7-H3-VA (anti-B7-H3), B7-H3 MG (anti-B7-H3, enoblituzumab from MacroGenics), and isotype control (anti-RSV antibody).

[0298] Serial dilutions (1:5 dilutions) of AF647 labeled antibody were made in assay buffer (4% FBS in RPMI1640) starting at a final concentration of 100 μg / ml (prepared at 200 μg / ml).

[0299] Cells were incubated on ice (4° C.) in the dark for 1 hour, then centrifuged at 300 g for 5 minutes to pellet the cells (supernatant was discarded). Cells were washed three times with wash buffer (2% FBS, 5 mM EDTA diluted in DPBS) and centrifuged at 300 g for 5 minutes to pellet the cells (supernatant was discarded). Plates were analyzed by flow cytometry using an AttuneNXT instrument and quantified using AF647 quantification beads from Bangslab.

[0300] MESF (molecular equivalent soluble fluorescent dye) was used to generate a standard curve. MESF standard microsphere solutions were prepared by dilution with assay buffer according to the manufacturer's instructions. Alexa Fluor 647 fluorescently labeled MESF beads were read by the cytometer and the values ​​were entered into the "QuickCal Template" according to the manufacturer's instructions to generate a regression curve.

[0301] Differences in fluorophore to antibody (F / P) ratios were adjusted by normalizing to the known F / P ratios for each antibody conjugate to obtain the geometric mean of total antibody bound. Collected data were analyzed by FlowJo v10 software, which converts raw fluorescence to geometric mean fluorescence values. Standard curves fitted using GraphPad software according to the manufacturer's instructions were used to calculate Kd [μg / ml] and B max was calculated.

[0302] Figure 14 shows that B7-H3-VA exhibits significantly increased engagement with surface antigens expressed by human cancer cells A375, A549 and Panc-1. B7-H3-VA exhibits higher receptor engagement activity compared to MG-B7-H3.

[0303] Example 15 Receptor occupancy of the B7-H3 clone VA (harboring the LALA mutation in the Fc region) measured by flow cytometry.

[0304] Flow cytometry was used to measure receptor engagement of anti-B7-H3-VA antibody (with the LALA mutation in the Fc region) with human melanoma cells (A375), human lung cancer cells (A549) and human pancreatic cancer cells (Panc-1).

[0305] The assay was performed according to the method described above in Example 14. The following antibodies were used in the assay: B7-H3-VA-LALA (anti-B7-H3), B7-H3 MG (anti-B7-H3, enoblituzumab from MacroGenics), and an isotype control (anti-RSV antibody).

[0306] Figure 15 shows that B7-H3-VA LALA exhibits significantly increased engagement with surface antigens expressed by human cancer cells A375, A549 and Panc-1. B7-H3-VA LALA exhibits receptor engagement activity comparable to the parent B7-H3-VA antibody and significantly higher than the MG-B7-H3 antibody (Macrogenics).

[0307] Example 16 Restoration of CD4+ T cell proliferation by B7-H3-VA.

[0308] The efficacy of B7-H3-VA antibody was tested based on its ability to restore CD4+ T cell proliferation. sB7-H3 (soluble B7-H3) polypeptide is a known inhibitor of CD4+ T cell activation and proliferation, which contributes to tumor cell immune escape. The restoration of CD4+ T cell proliferation was evaluated using B7-H3-VA antibody and isotype control (anti-RSV antibody).

[0309] CD4+ T cells were isolated by negative isolation from human blood according to the manufacturer's instructions. CD4+ T cells were subjected to staining with CFSE cell permeable dye in 5 ml of PBS at 37°C for 20 min according to the manufacturer's instructions. 10 ml of RPMI1640 supplemented with 10% FBS was added to the CD4+ T cells and the cells were incubated for 10 min at room temperature. The cells were centrifuged at 1,500 rpm for 3 min at room temperature and the supernatant was discarded. The cells were resuspended in RPMI1640 supplemented with 10% FBS and then plated in 48-well plates at 500 μl / well (500,000 cells).

[0310] 500 ng / ml of anti-CD3 antibody was added to each well (to induce CD4+ T cell proliferation) followed by addition of soluble B7-H3 polypeptide (sB7-H3, 0.5 μg / well).

[0311] Finally, 50 μg / well of anti-B7-H3-VA antibody or isotype control (anti-RSV antibody) (300 nM antibody) was added to the plate, which was incubated for 72 hours at 37°C.

[0312] Cells were fixed with 2% paraformaldehyde in PBS for 20 min at room temperature and CFSE fluorescence was acquired by flow cytometry. Analysis was gated to exclude cell debris and the total cell population and subjected to automated proliferation modeling provided by FlowJo software.

[0313] Panel 1 of FIG. 16 shows CFSE fluorescence of CD4+ T cells on day 0, and panel 2 shows CFSE fluorescence of CD4+ T cells after 72 hours. Panel 3 of FIG. 16 demonstrates that the addition of anti-CD3 antibodies enhances CD4+ T cell proliferation. Panel 4 of FIG. 16 demonstrates that the addition of sB7-H3 polypeptide blocks (and reverses) CD4+ T cell proliferation. Panel 5 of FIG. 16 demonstrates that the addition of anti-B7-H3-VA antibodies (after the addition of sB7-H3) restores CD4+ T cell proliferation (by blocking the B7-H3 receptor). Panel 5 of FIG. 16 demonstrates that the addition of anti-RSV (non-targeting) antibodies does not restore CD4+ T cell proliferation. CD4+ T cell proliferation was acquired by flow cytometry, whereby cell division was assessed by CFSE dilution of dividing T cells.

[0314] Conclusion: Figure 16 shows that B7-H3-VA restores CD4+ T cell proliferation and thus attenuates the T cell suppressive activity of soluble B7-H3 polypeptide, thus demonstrating the efficacy of the B7-H3-VA antibody as a blocking antibody.

[0315] (Example 17) Antibody-dependent cell-mediated cytotoxicity (ADCC) efficacy loss-of-function.

[0316] The potential for antibody-mediated recruitment of effector cells was assessed by a reporter assay. The following antibodies were compared in this assay: B7-H3-VA and B7-H3-VA LALA (anti-B7-H3), MG-B7-H3 (anti-B7-H3, enoblituzumab from MacroGenics), and an isotype control (anti-RSV antibody).

[0317] 5,000 human melanoma target cells / well (A375) were plated in 96-well plates (100 μl / well) and incubated overnight at 37°C.

[0318] Serial dilutions of antibodies were prepared in ADCC assay buffer according to the manufacturer's instructions. 95 μl / well cell solution was removed and 25 μl / well assay buffer was added, followed by 25 μl / well of serially diluted antibody solution. 25 μl / well effector cells (engineered Jurkat cells expressing NFAT luciferase; = 75,000 cells / well) were then added to achieve a target:effector cell ratio of 1:15, and the plate was incubated for 6 hours at 37°C. After incubation, 40 μl / well OneGlo™ luciferase substrate solution was added to the plate, and the plate was incubated for 5-10 minutes at room temperature. Induced bioluminescence was acquired using a plate reader.

[0319] Figure 17 displays the results of two independent experiments (replicates of the same experiment, R1 and R2). Both graphs in Figure 17 show that the LALA mutation in the Fc region of the anti-B7-H3-VA antibody results in a loss of function, i.e., the antibody does not induce cell engagement-dependent bioluminescence compared to the parental B7-H3-VA antibody or the MG-B7-H3 (Macrogenics) antibody.

[0320] (Example 18) Red blood cell (RBC) binding activity measured by flow cytometry.

[0321] Flow cytometry was used to test the binding ability of anti-B7-H3 antibodies (MG-B7-H3, VA clone and VA LALA clone), an isotype control antibody (anti-RSV), and an anti-CD47 Hu5F9 antibody to human red blood cells (RBCs).

[0322] RBCs from three donors were prepared as described in Example 12 above (paragraph

[0267] ).

[0323] Antibody immunostaining: The following antibodies were used in this assay: B7-H3-VA (anti-B7-H3), B7-H3-VA LALA (anti-B7-H3), MG-B7-H3 (anti-B7-H3, enoblituzumab from MacroGenics), Hu5F9 (anti-CD47 antibody), and isotype control (anti-RSV antibody). Serial dilutions of the antibodies were made in staining buffer (containing 1% BSA in DPBS) (1:5 serial dilutions) starting at a final concentration of 100 μg / ml (prepared at 200 μg / ml).

[0324] Antibody dilutions were transferred to plates containing 100 μl / well of RBCs (50 μL / well of antibody was added to each well; starting antibody concentration: 100 μg / mL) and mixed gently. Plates were incubated for 25 minutes at 37° C., then washed with 1 ml / well of staining buffer (1% BSA in DPBS), centrifuged at 1,500 rpm for 3 minutes at room temperature, and the supernatant was discarded.

[0325] 100 μl of fluorescently labeled secondary antibody diluted 1:500 in staining buffer (1% BSA in DPBS) was added to each well and the plate was incubated for 20 minutes at 37°C. The plate was washed twice with 200 μl / well of staining buffer (1% BSA in DPBS), centrifuged at 1,500 rpm for 3 minutes at room temperature and the supernatant was removed. 100 μl of staining buffer was then added to each well and the cells were analyzed by flow cytometry using an AttuneNXT instrument. The collected data was analyzed by FlowJo v10 software, which converts raw fluorescence to geometric mean fluorescence values.

[0326] Dose-dependent binding of antibodies was performed in triplicate and obtained by flow cytometry. Figure 18 shows the results of immunostaining of human RBCs with anti-RSV (isotype control IgG), Macrogenics (MG-B7-H3 antibody), anti-B7-H3 antibody (VA clone and VA LALA clone), and Hu5F9 (anti-CD47 antibody). Figure 18 shows that only Hu5F9 (anti-CD47 antibody) shows RBC binding, while MG-B7-H3 antibody, anti-B7-H3 antibody (VA clone and VA LALA clone), and anti-RSV (isotype control IgG) did not show any discernible RBC binding.

[0327] (Example 19) Measurement of binding affinity using surface plasmon resonance.

[0328] The binding kinetics of anti-B7-H3 antibodies to his-tagged B7-H3 protein was measured using surface plasmon resonance (SPR). Anti-B7-H3 antibodies tested included the tagged antibodies (B3-A6, B3-4A1, B3-4F7, B3-D1, B3-2D1, B3-2H1, B3-2E3, and B3-5A6). Anti-human Fc antibodies from the Human Antibody Capture Kit (Cytiva; catalog no. BR100839) were immobilized onto a CM5 sensor chip to approximately 5,000 RU using standard N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (NHS / EDC) coupling methodology.

[0329] Anti-B7-H3 antibody (approximately 5 μg / mL) was captured for 60 seconds at a flow rate of 10 μL / min. Recombinant human his-tagged B7-H3 protein comprised amino acid 1 to amino acid 461 of SEQ ID NO:1 (i.e., SEQ ID NO:2) (Sino Biologicals; Cat. No. 11188-H08H-50). The polypeptide was serially diluted 1:1 in running buffer HBS-EP (Cytiva; Cat. No. BR100188) starting at 100 nM. All measurements were performed in HBS-EP buffer at a flow rate of 30 μL / min automated by a BIAcore 3000. The surface was regenerated with 3 M MgCl2 for 60 seconds. Data were fitted using a 1:1 (Langmuir) binding model. All BIACORE assays were performed at room temperature using a BIAcore 3000 (GE Healthcare).

[0330] The SPR sensorgrams of the anti-B7-H3 antibodies are shown in Figure 19, and their corresponding binding kinetics are listed below in Table 3. All B7-H3 antibodies showed affinity for their cognate antigens in the nM range.

[0331] [Table 3]

[0332] (Example 20) Cell-binding activity of B7-H3 clones (B3-5A6, B3-2E3, B3-2D1, B3-D1, B3-2G11, B3-4A1) measured by flow cytometry.

[0333] Flow cytometry was used to test the binding ability of anti-B7-H3 antibodies (B3-5A6, B3-2E3, B3-2D1, B3-D1, B3-2G11, B3-4A1) to dendritic cells (DCs).

[0334] Dendritic cells were prepared by culturing purified monocytes with 1,000 U / mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 500 U / mL of interleukin 4 (IL-4) for 6 to 7 days.

[0335] Monocytes were obtained from peripheral blood mononuclear cells (PBMCs) by reactivity with biotin anti-human CD14 after addition of magnetic anti-biotin beads and separation by passage through a column attached to a magnet, typically resulting in a population of >90% pure CD14-positive monocytes.

[0336] A B7-H3 antibody from Memorial Sloan Kettering Cancer Center, MSKCC B7-H3, was expressed in-house and used as a positive control.

[0337] Test antibodies (B3-5A6, B3-2E3, B3-2D1, B3-D1, B3-2G11, B3-4A1, and positive control MSKCC B7-H3) were added to cells at a final concentration of 10 mg / ml, 1 mg / ml, or 0.1 mg / ml. After 20 min at 4° C., cells were washed and then resuspended in PBS+1% FCS containing APC-conjugated goat anti-human IgG at a 1:1,000 dilution. After 20 min at 4° C., cells were washed, resuspended in PBS+1% FCS, and analyzed by flow cytometry.

[0338] Figure 20 shows the results of B7-H3 antibody binding to dendritic cells. Figure 20 shows that some anti-B7-H3 antibodies (B3-5A6, B3-2D1, and B3-4A1) show stronger binding than the MKSCC B7-H3 control antibody at some, but not all, concentrations. It also shows that the anti-B7-H3 antibodies (B3-2D1 and B3-4A1) clearly bind to dendritic cells at concentrations as low as 0.1 μg / ml.

[0339] The detailed description given above is provided to aid those skilled in the art in practicing the present invention. However, the present invention described and claimed herein is not limited in scope by the specific embodiments disclosed herein, since these embodiments are intended to illustrate some aspects of the present invention. Any equivalent embodiments are considered to be within the scope of the present invention. Indeed, various modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the above description, which do not depart from the spirit or scope of the discovery of the present invention. Such modifications are also considered to be within the scope of the appended claims.

[0340] All publications, patents, patent applications, and other references cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention.

Claims

1. comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementarity determining region 1 CDR1, heavy chain CDR2 and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3; a. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 12, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 13, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 14, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 15, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 16, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 17; or b. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:22, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:23, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:24, the light chain CDR1 has the amino acid sequence of SEQ ID NO:25, the light chain CDR2 has the amino acid sequence of SEQ ID NO:26, and the light chain CDR3 has the amino acid sequence of SEQ ID NO:27; or c. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 32, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 33, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 34, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 35, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 36, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 37; or d. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 42, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 43, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 44, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 45, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 46, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 47; or e. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:52, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:53, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:54, the light chain CDR1 has the amino acid sequence of SEQ ID NO:55, the light chain CDR2 has the amino acid sequence of SEQ ID NO:56, and the light chain CDR3 has the amino acid sequence of SEQ ID NO:57; or f. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 62, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 63, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 64, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 65, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 66, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 67; or g. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 72, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 73, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 74, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 75, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 76, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 77; or h. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 82, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 83, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 84, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 85, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 86, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 87; or i. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 92, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 93, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 94, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 95, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 96, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 97; or j. the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 102, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 103, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 104, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 105, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 106, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 107; An anti-B7-H3 antigen binding protein or a fully human anti-B7-H3 antibody, or an antigen binding fragment thereof.

2. a. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11; or b. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:20 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:21; or c. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:30 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:31; or d. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:40 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:41; or e. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:50 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:51; or f. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:60 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:61; or g. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 70 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 71; or h. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 80 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 81; or i. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 90 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 91; or j. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 100, and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 101; An antigen-binding protein, antibody or antigen-binding fragment thereof according to claim 1.

3. comprising a heavy chain variable region and a light chain variable region, a. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11; or b. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:20 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:21; or c. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:30 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:31; or d. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:40 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:41; or e. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:50 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:51; or f. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:60 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:61; or g. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 70 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 71; or h. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 80 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 81; or i. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 90 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 91; or j. the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 100, and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 101; An antigen-binding protein or a fully human anti-B7-H3 antibody, or an antigen-binding fragment thereof.

4. a. a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 10 and 11, respectively; or b. a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 20 and 21, respectively; or c. a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 30 and 31, respectively; or d. a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 40 and 41, respectively; or e. a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 50 and 51, respectively; or f. a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 60 and 61, respectively; or g. a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 70 and 71, respectively; or h. a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 80 and 81, respectively; or i. a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 90 and 91, respectively; or j. A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 100 and 101, respectively; An antigen-binding protein or a fully human anti-B7-H3 antibody, or an antigen-binding fragment thereof.

5. The antigen-binding protein, antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 12, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 13, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 14, the light chain CDR1 has the amino acid sequence of SEQ ID NO: 15, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 16, and the light chain CDR3 has the amino acid sequence of SEQ ID NO:

17.

6. 5. The antigen-binding protein, antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the heavy chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10 and the light chain variable region has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

11.

7. 5. An antigen-binding protein, antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 10 and 11, respectively.

8. 8. An antigen-binding fragment according to any one of claims 1 to 7, comprising a Fab fragment.

9. 8. An antigen-binding fragment according to any one of claims 1 to 7, comprising a single-chain antibody in which the heavy chain variable domain and the light chain variable domain are linked together by a peptide linker.

10. 2. The antigen-binding protein, antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising an IgG antibody, optionally wherein the IgG antibody is an IgG1, IgG2, IgG3 or IgG4 class antibody.

11. The antigen-binding protein, antibody or antigen-binding fragment thereof of claim 10, wherein the IgG antibody is an IgG1 or IgG4 class antibody.

12. The antigen-binding protein, antibody or antigen-binding fragment thereof of claim 11 , wherein the IgG1 antibody comprises a variant Fc region.

13. The antigen-binding protein, antibody or antigen-binding fragment thereof of claim 12, wherein the variant Fc region comprises one or more mutations selected from L234A and L235A.

14. The antigen-binding protein, antibody or antigen-binding fragment thereof of claim 13, wherein the variant Fc region comprises the mutations L234A and L235A (LALA).

15. 10 －７ M or less K Ｄ 2. The antigen-binding protein, antibody or antigen-binding fragment thereof of any one of the preceding claims, which binds to human B7-H3 protein at

16. 20. A pharmaceutical composition comprising an antigen-binding protein, antibody or antigen-binding fragment according to any one of the preceding claims and a pharma- ceutical acceptable excipient.

17. 16. A kit comprising an antigen-binding protein, antibody or antigen-binding fragment of any one of claims 1 to 15 and a pharma- ceutically acceptable excipient.

18. A nucleic acid encoding a polypeptide comprising the heavy chain variable region of an antigen-binding protein, antibody or antigen-binding fragment of any one of claims 1 to 15.

19. A nucleic acid encoding a polypeptide comprising the light chain variable region of an antigen-binding protein, antibody or antigen-binding fragment of any one of claims 1 to 15.

20. 16. A nucleic acid encoding (i) a first polypeptide comprising the heavy chain variable region of an antigen-binding protein, antibody or antigen-binding fragment of any one of claims 1 to 15, and (ii) a second polypeptide comprising the light chain variable region of the antigen-binding protein, antibody or antigen-binding fragment.

21. A vector comprising the nucleic acid of claim 18.

22. A vector comprising the nucleic acid of claim 19.

23. A vector comprising the nucleic acid of claim 20.

24. A host cell harboring the vector of claim 21.

25. 25. The host cell of claim 24, wherein the vector is an expression vector and the host cell expresses the polypeptide comprising the heavy chain variable region.

26. A host cell harboring the vector of claim 22.

27. 27. The host cell of claim 26, wherein the vector is an expression vector and the host cell expresses the polypeptide comprising the light chain variable region.

28. A host cell harboring a first vector comprising the vector of claim 21 and a second vector comprising the vector of claim 22.

29. 29. The host cell of claim 28, wherein the first vector is a first expression vector and the second vector is a second expression vector, and the host cell expresses the polypeptide comprising the heavy chain variable region and the polypeptide comprising the light chain variable region.

30. A host cell harboring the vector of claim 23.

31. 31. The host cell of claim 30, wherein the vector is an expression vector and the host cell expresses the first and second polypeptides.

32. 26. A method for preparing a heavy chain variable region of an antigen binding protein, antibody or antigen binding fragment, comprising culturing a population of host cells of claim 25 under conditions suitable for expressing the heavy chain variable region of the antigen binding protein, antibody or antigen binding fragment.

33. 33. The method of claim 32, further comprising recovering the expressed heavy chain variable region of the antigen binding protein, antibody or antigen binding fragment from the host cell.

34. 30. A method for preparing a polypeptide comprising a light chain variable region of an antigen binding protein, antibody or antigen binding fragment, comprising culturing a population of host cells of claim 27 under conditions suitable for expressing the polypeptide comprising the light chain variable region of the antigen binding protein, antibody or antigen binding fragment.

35. 35. The method of claim 34, further comprising recovering the expressed light chain variable region of the antigen binding protein, antibody or antigen binding fragment from the host cell.

36. 30. A method for preparing (i) a first polypeptide comprising a heavy chain variable region of an antigen binding protein, antibody or antigen binding fragment, and (ii) a second polypeptide comprising a light chain variable region of an antigen binding protein, antibody or antigen binding fragment, comprising culturing a population of host cells according to claim 29 under conditions suitable for expression of (i) the first polypeptide and (ii) the second polypeptide.

37. 37. The method of claim 36, further comprising recovering (i) the expressed first polypeptide and (ii) the expressed second polypeptide from the host cell.

38. 32. A method for preparing (i) a first polypeptide comprising a heavy chain variable region of an antigen binding protein, antibody or antigen binding fragment, and (ii) a second polypeptide comprising a light chain variable region of an antigen binding protein, antibody or antigen binding fragment, comprising culturing a population of host cells according to claim 31 under conditions suitable for expression of (i) the first polypeptide and (ii) the second polypeptide.

39. 39. The method of claim 38, further comprising recovering (i) the expressed first polypeptide and (ii) the expressed second polypeptide from the host cell.

40. 16. A method for inhibiting the growth or proliferation of B7-H3 expressing cells, comprising the step of contacting (i) a population of effector cells with (ii) a population of target cells expressing B7-H3, in the presence of (iii) an antigen binding protein, antibody or antigen binding fragment thereof according to any one of claims 1 to 15, under conditions suitable for inhibiting the growth or proliferation of said B7-H3 expressing cells.

41. 41. The method of claim 40, wherein the population of effector cells comprises peripheral blood mononuclear cells (PBMCs) or natural killer (NK) cells.

42. The method of claim 40 or 41, wherein the population of target cells comprises B7-H3-expressing human cancer cells or transgenic cells expressing B7-H3.

43. 43. The method of any one of claims 40 to 42, wherein the ratio of effector cells to target cells is 1-5:1, optionally the ratio is 1:1, 2:1, 3:1, 4:1 or 5:

1.

44. 43. The method of any one of claims 40 to 42, wherein the ratio of effector cells to target cells is 5-10:1, 10-20:1, or 20-30:

1.

45. 16. A method for killing B7-H3 expressing cells, comprising the steps of: (i) contacting a population of effector cells with (ii) a population of target cells expressing B7-H3, (iii) in the presence of an antigen binding protein, antibody or antigen binding fragment thereof according to any one of claims 1 to 15, under conditions suitable for killing the B7-H3 expressing cells.

46. 46. ​​The method of claim 45, wherein the population of effector cells comprises PBMCs or NK cells.

47. The method of claim 45 or 46, wherein the population of target cells comprises B7-H3-expressing human cancer cells or transgenic cells expressing B7-H3.

48. 48. The method of any one of claims 45 to 47, wherein the ratio of effector cells to target cells is 1-5:1, optionally the ratio is 1:1, 2:1, 3:1, 4:1 or 5:

1.

49. 48. The method of any one of claims 45 to 47, wherein the ratio of effector cells to target cells is 5-10:1, 10-20:1, or 20-30:

1.

50. 50. The method of any one of claims 40-49, wherein the population of target cells is present in a subject and the antigen binding protein, antibody, or antigen binding fragment thereof is administered to the subject.

51. 50. The method of any one of claims 40 to 49, which is an in vitro method.

52. 16. A method for treating a subject having a disease associated with B7-H3 expression, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antigen-binding protein, antibody or antigen-binding fragment of any one of claims 1 to 15.

53. The method of claim 52, wherein the disease associated with B7-H3 expression is cancer.

54. 54. The method of claim 53, wherein the cancer is ovarian, colon, prostate, skin, pancreatic, renal, urothelial or lung carcinoma.

55. 54. The method of claim 53, wherein the cancer is acute myeloid lymphoma (AML), non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), small cell lung cancer, urothelial cell carcinoma, esophageal cancer, hepatocellular carcinoma, glioma, neuroblastoma, glioblastoma multiforme, blastoma, sarcoma, leukemia, lymphoid malignancy, pancreatic cancer, head and neck cancer, ovarian cancer, oral cancer, breast cancer, triple-negative breast cancer (TNBC), lymphoma, renal cell carcinoma, clear cell renal cell carcinoma, colon cancer, colorectal cancer, melanoma, gastric cancer, lung cancer, liver cancer, bladder cancer, prostate cancer, anal cancer, endometrial cancer, vulvar cancer, squamous cell tumor, hypopharyngeal squamous cell carcinoma, or squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer).

56. 56. The method of any one of claims 53 to 55, wherein the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer.

57. 57. An antigen-binding protein, antibody or antigen-binding fragment according to any one of claims 1 to 15 for use in a method according to any one of claims 40 to 56.

58. 57. Use of an antigen-binding protein, antibody or antigen-binding fragment according to any one of claims 1 to 15 in the manufacture of a medicament for use in a method according to any one of claims 40 to 56.

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