Abstract POSTER-THER-1407: Cytotoxic natural human MAb against ovarian carcinoma

Abstract

Abstract MAb216 is a human IgM mAb encoded in germline configuration by the immunoglobulin heavy chain VH4-34 gene. This antibody and many others derived from the VH4-34 heavy chain gene bind to long chain poly n-acetyl lactosamine (P-NAL) on the cell surface, referred to as the i/I antigen and found on normal fetal red blood cells and mature B cells. We have shown that VH4-34 IgM mAbs that are strong B cell binders also exhibit cytotoxicity to normal and malignant B cells. The mechanism of cytoxicity employed in the absence of complement involves crosslinking of the carbohydrate ligand leading to membrane disruption and loss of cell integrity accompanied by formation of unusual giant pore-like structures on the cell membranes as demonstrated by scanning electron and confocal microscopy. The IgM isotype also activates complement mediated cytotoxicity. Interestingly mAb cytoxicity is greater in vitro at 4 degrees without complement than at room temperature perhaps due to lack of membrane repair. MAb216 has been studied in a phase I trial of acute B cell leukemia, showing some efficacy and no grade 3 toxicity at the doses tested. Recently we found that mAb216 and several similar VH4-34 encoded IgM mAbs bound to the surface of a subpopulation of serous ovarian carcinoma (EOC) cells from individual tumors, ascites and cell lines. Reactivity was assayed using immunofluorescence of live cells or flow cytometry analysis or both. mAb216 was labeled with biotin or directly labeled with Alexa488. The punctate pattern was similar to that seen on B lymphocytes. Of 33 tumor or ascites samples assayed, mAb216 bound to a cell population on 29 specimens (87%). Reactivity ranged from 5-80% of the tumor cells depending on the individual patient. Endometroid (n=3) and mucinous (n=2) ovarian cancer were negative for staining. Three EOC cell lines, OVCAR3, OVCAR5, and Kuramochi show a percent of cells that expressed the P-NAL antigen. OVCAR4, OVCAR8 and SKOV3 did not bind mAb216. In vitro cytotoxicity experiments with ascites and OVCAR3 cell line showed that mAb alone could kill the percent of cells expressing antigen, and the cytotoxicity effects dramatically increased with human complement. Cell death was detected using the CCK-sk assay or flow cytometry counting with propidium iodide to detect dead cells. Like mAb216 cytotoxicity on B lymphocytes, 4 degree temperature enhanced cytotoxicity of OVCAR3 cells. Further, endo-beta-galactosidase treatment removed mAb216 binding and scanning electron microscopy of OVCAR3 showed the same membrane “pores” seen on B lymphocytes after mAb216 treatment. Fluorescence cell sorting of the bright positive population and negative population after mAb216 staining showed that the negative mAb216 population re-expressed the P-NAL antigen by 72 hours indicating that epitope expression is in someway cell cycle or proliferation related. Human VH4-34 IgM mAbs could therefore be used as a new therapy and a new target antigen in treatment of EOC. Preclinical studies are planned. Citation Format: Yi Chen, Marcia Bieber, Neelima Bhat, Nelson N.H. Teng. Cytotoxic natural human MAb against ovarian carcinoma [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1407.