Abstract IA25: Strategies for glycosylation-based immunotargeting of MUC16

The pathobiology of MUC16 / CA125 has been of great interest for three decades. Most recently, my laboratory has been focused on the biology of MUC16, the glycoprotein encoding the CA125 antigen. In particular, we have been interested in role of the carboxyterminal elements of this tethered mucin. Our work has required the development of new reagents including multiple antibodies specific for the most proximal, non-shed sequences adjacent to the cell surface. In our studies, we have shown that few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the EGFr, SRC AKT, and ERK pathways. MUC16 transformation was associated with upregulation of a number of metastases and invasion gene transcripts, including IL-1beta, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular “ectodomain” of MUC16 and did not require the cytoplasmic domain of MUC16 for transformation. In TP53 +/- knockout mice, the most proximal 114 aa of MUC16 is sufficient to generate spontaneous tumors (primarily sarcomas) in transgenic mice (Rao TD et al. PLoS One 2015;10(5):e0126633). Subsequent experiments have shown that MUC16 oncogenic effects are mediated through tetra-antennary N-glycosylation of asparagine sites within the 58-amino-acid domain between the putative cleavage site and the cell membrane. We have also established that oncogenic signaling from the C-terminal portion of MUC16 requires the presence of Galectin-3 and growth factor receptors colocalized on lipid rafts. These effects are blocked upon loss of either Galectin-3 expression or activity of the glycosylation enzyme MGAT5. With sufficient N-glycosylation and Galectin-3 MUC16 stabilizes pro-growth receptors on the cancer cell surface and enhances signaling through decreased receptor turnover. This inhibition is dependent on Galectin-3, MGAT5-dependent glycosylation, and MUC16 interactions. Using synthetic MUC16 glycopeptides, we have developed novel N-glycosylation site directed monoclonal antibodies that block Galectin-3-mediated MUC16 interactions with cell surface signaling molecules. These antibodies inhibit invasion of ovarian cancer cells, directly blocking the in vivo growth of MUC16-bearing ovarian cancer xenografts, elucidating new therapeutic modalities (Rao TD et al. ACS Chem Biol 2017 epub). We have also examined the use of MUC16 targeted CAR-T cells. These CAR T cells, specifically directed at the most proximal portions of MUC16, are in clinical testing and early results will be discussed. Enhancements in the design of MUC16-directed T-cell vectors and therapeutic directions will be addressed as well. Citation Format: David Spriggs, Roisin O’Cearbhaill, Oladapo Yeku, Marina Stasenko, Thapi D. Rao, Renier Brentjens. Strategies for glycosylation-based immunotargeting of MUC16. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA25.