Abstract Background: Currently available monoclonal antibodies (mAbs) for ovarian cancer research recognize the secreted protein CA125 and target extracellular epitopes on the cell surface. After the cleavage event and CA125 release occur, there have been few antibodies available to monitor the remaining MUC16 cleavage product(s). Recent studies (Guo, et. al. 2011) have shown that intracellular oncoproteins can be targeted with antibodies resulting in inhibition of tumor growth. The exact mechanism of antibody internalization is not well elucidated but potential mechanisms include pinocytosis or possibly internalization via conformational changes in surface lipids causing the antibody to flip into the cell. We reported novel monoclonal antibodies against the proximal (carboxy-terminal) portions of MUC16 (AIMM J, 2010) that recognize the external as well as the internal epitopes of MUC16. One of the monoclonal antibodies, 31A3, recognizes an internal epitope in the cytoplasmic portion of MUC16. Objectives: Our goals were: 1. To characterize the anti-tumor activity of our novel 31A3 antibody both in vivo (mice) and in vitro (various cell lines OVCAR3, 3T3, SKOV3); 2. To visualize and characterize the mechanism of 31A3 antibody internalization in situ. Methods/Results: FACS analysis revealed little, if any, specific surface binding of 31A3 antibody on SKOV3-phrGFP vector cell line or OVCAR3 cell line, incubated for 30 minutes on ice, followed by labeling for 30 min with a Goat anti-Mouse IgG1-PE secondary antibody. Alamar blue assay on in vitro starved OVCAR3 cell lines A and B (high CA125 and low CA125 producers, respectively) for 18 hrs did not show any difference in growth pattern when cultured with or without 31A3 mAb, however, clinical CA125 ELISA demonstrated statistically significant (P<0.0001) increase in CA125 units in the supernatants of cells treated with 31A3 antibody in low protein conditions. Morphologically, OVCAR3 cells become rounded and appear to agglutinate or clump, releasing CA125 into the supernatant. Additionally, decreased tumor growth in vivo was noted in mice flank implanted with stable 3T3-CDcGFP cell line, compared to 3T3-phrGFP vector controls when immunized IP with 31A3 mAb (at 50 or 200 ug/mouse) once/week for 4 weeks. During time lapse imaging we observed that 31A3 antibody, conjugated with Alexa-647 fluorochrome was internalized in OVCAR3 at approximately 4-10 hours in a sequence of events linked to focal cell surface binding and then gradual internalization. Conclusions: These results lead us to identify 31A3 as a potentially interesting anti-MUC16 antibody targeting an internal epitope. We are investigating the interactions of our new monoclonal antibodies, including 31A3, with potential molecular partners both in cancer cells and in the local environment. This will allow us to better understand MUC16 biology and may lead to a novel therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 513. doi:1538-7445.AM2012-513