MABC1731-25UL Sigma-AldrichAnti-B7-H3 (CD276) Antibody, clone 376.96

The high rate of relapse in patients with advanced ovarian cancer likely reflects the chemoresistance of cancer initiating cells (CICs). We evaluated the anti-tumor activity of monoclonal antibody (mAb) 376.96, which recognizes a B7-H3 epitope expressed on ovarian carcinoma cells (OCCs), in combination with the tyrosine kinase inhibitor Sunitinib and chemotherapy on chemosensitive and chemoresistant cells and CICs.Eight ovarian cancer cell lines including platinum- and taxane-resistant cell lines were analyzed by flow cytometry to establish expression of the mAb 376.96-defined-B7-H3-epitope on differentiated ovarian cancer cells and CICs. Samples from 10 ovarian cancer patients were analyzed via immunohistochemistry for mAb 376.96-defined-B7-H3-epitope expression. In vitro studies assessed mAb 376.96 alone and in combination with Sunitinib on the growth of chemosensitive and chemoresistant cell lines and on the content of CICs.The mAb-376.96-defined-B7-H3 epitope is expressed on both differentiated cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines and 10 patient derived ovarian cancer tumors. In vitro treatment of chemoresistant cell lines with mAb 376.96 resulted in decreased cell viability. mAb 376.96 enhanced the cytotoxicity of Sunitinib and reduced the content of CICs.The mAb-376.96-defined-B7-H3-epitope was found to be expressed on both differentiated ovarian cancer cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines. mAb 376.96 inhibited the in vitro growth of chemosensitive and chemoresistant OCCs and reduced the content of CICs when used with Sunitinib. Further studies examining B7-H3 as a potential target of mAb-based immunotherapy for this type of malignancy are warranted.

The monoclonal antibody (mAb) 376.96 has been used for detection of micrometastatic tumor cells due to its high binding specificity for a wide range of tumor cells, but the identity and function of its target antigen have not been known. Here, using immunoprecipitation and siRNA technology, we demonstrate that the antigen is the human 4Ig-B7H3 (4Ig-hB7H3) protein, previously known as an immunoregulatory protein in immune cells. Immunoblots of whole cell lysates, subcellular fractionation and tunicamycin treatment of human tumor cells indicated that 4Ig-hB7H3 is a approximately 100-kDa N-linked glycosylated membrane protein. The tumor promoter phorbol 12-myristate 13-acetate (PMA) enhanced the expression of 4Ig-hB7H3 in FEMX-I (melanoma), MA11 (breast cancer), and OHS (osteosarcoma) cells, suggesting that 4Ig-hB7H3 may be implicated in tumorigenesis. Most importantly, siRNA-downregulation of hB7H3 reduced cell adhesion to fibronectin of melanoma and breast cancer cells by up to 50 %, and migration and matrigel-invasion by more than 70 %, but surprisingly had no apparent impact on cell proliferation. In conclusion, our data present 4Ig-hB7H3 as a tumor-associated antigen and suggests a novel biological role of 4Ig-hB7H3 in tumor progression and metastasis.