Abstract 1776: High-affinity anti-galectin-3 antibodies targeting oncogenic properties in serous ovarian cancer

Abstract

Abstract Objective: Galectin-3 (LGALS3) regulates the interaction of surface proteins with the extracellular membrane domain and mediates a signal cascade leading to invasion, oncogene activation and growth. We targeted LGALS3 cancer promotion by developing high-affinity anti-galectin-3 antibodies directed either at the carbohydrate recognition domain (CRD) of the galectin-3 carboxyl-terminus (to block sugar binding) or at the N-terminus (NT) (to inhibit N-terminal mediated oligomerization of galectin-3) in order to prevent formation of the cell surface galectin lattice and suppress the oncogenic effects of the glycoprotein. Methods: Two distinct types of murine monoclonal antibodies (MAb) were generated by creating hybridomas from mice immunized either with a fusion protein derived from the 128-amino acid LGALS3 carboxyl-terminus fused to a human IgG1-Fc backbone followed by immunization with a recombinant human LGALS3 protein, or the mice were further immunized with a 25-amino acid peptide derived from the LGALS3-N-terminus conjugated to KLH. The resulting antibodies were confirmed to recognize either the LGALS3 CRD or NT by ELISA. Functional assays (Matrigel invasion, inhibition of laminin binding, and oncogene expression) and cell surface expression by FACS analysis were utilized to confirm MAb binding and activity. Mice bearing A2780 and SKOV3 ovarian cancer cell lines were treated with purified MAb to determine the effects of MAb on tumor growth in vivo. Data were analyzed for statistical significance using Student's t-test. Results: Initial screening yielded two candidate antibodies, of which one (named 14D11.2D2) had superior binding to galectin-3 by ELISA and Surface Plasmon Resonance (SPR). The antibody dissociation constant was 14.6 nM by SPR. At a concentration as low as 150 nM, the 14D11.2D2 MAb decreased galectin-3 binding to laminin by 36.6% compared to untreated control. In a Matrigel invasion assay, the anti-CRD MAb significantly inhibited invasion by MUC16 expressing SKOV3 cells and by wild-type MUC16 positive OVCAR3 cells. MUC16 expressing A2780 cells showed a statistically nonsignificant decrease in invasion. In vivo experiments with MUC16 expressing A2780 or SKOV3 tumors showed a significant inhibition of tumor growth in mice treated with anti-CRD MAb. Six candidate antibodies directed against the LGALS3-N-terminus have been identified. Characterization of two NT antibodies (1F5.D4 and 7D4.A1) is under way via in in vitro and in vivo experiments. Conclusion: High-affinity CRD and NT anti-galectin-3 antibodies inhibit MUC16-induced cellular invasion and growth. Anti-LGALS3 MAbs hold therapeutic potential for inhibition of glycosylation-dependent invasion. Citation Format: Dharmarao Thapi, Marina Stasenko, Thomas White, Sven J. Walderich, Noah Feit, Frances Weis-Garcia, David R. Spriggs. High-affinity anti-galectin-3 antibodies targeting oncogenic properties in serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1776.