Abstract C53: Potent CDC, ADCC, and antitumor activity of human monoclonal antibodies to Sialyl Lewisa.

Abstract

The carbohydrate antigen sialyl-Lewis A (sLea), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract, on breast cancer cells, and also on small cell lung cancer cells. sLea serves as a ligand for epithelial leukocyte adhesion molecules and over-expression of sLea appears to be a key event in invasion and metastasis of many tumor cells. Since tumor cells expressing sLea are highly susceptible to antibody mediated lysis mechanisms, sLea presents an attractive molecular target for tumor therapy in a minimal disease setting. Hybridomas were generated with human B cells isolated from individuals vaccinated with MSKCC's cancer vaccine. Antibody producing cells were identified by ELISA using sLea-HSA and synthetic sLea -PAA-biotin conjugates. Binding to the native antigen expressed on the cell surface was tested by FACS analysis on sLea positive DMS-79 cells and others and the biological activity was measured in CDC and ADCC assays. The binding affinity was determined by Surface Plasmon Resonance and the specificity of the carbohydrate binding was evaluated by ELISA and by glycan array analysis (CFG, Array 4.1). Recombinant antibodies were generated in CHO cells and purified on Protein A (IgG) or hydroxyapatite (IgM), respectively. We report the discovery and initial characterization of fully human antibodies that were generated from blood lymphocytes from individuals immunized with sLea -KLH vaccine. Two antibodies were selected for further studies based on the apparent high affinity, which was estimated by BiaCore at 0.14 nM for 5B1 (IgG/λ) and 0.04 nM for 7E3 (IgM/κ). Both antibodies were highly specific for Neu5Acα2–3Galβ1–3 (Fucα1–4) GlcNAcβ and did not bind to sialyl-Lewisx, Lewisa, and other related carbohydrates. Both antibodies have been expressed as fully functional human recombinant antibodies in CHO cells. Complement dependent cytotoxicity (CDC) against DMS-79 cells was approximately 60% and 70–90% for r5B1 and r7E3, respectively. Moreover, r5B1 antibodies showed approximately 50% ADCC of DMS-79 cells with human NK cells (at 5:1 ratio) and 80–90% ADCC with human peripheral blood mononuclear cells with two different blood donors (at 100:1 E/T ratio). These antibodies were tested in two xenograft models: 1) Treatment of animals with 5B1 on the day of engraftment with DMS-79 cells in a subcutaneous model completely prevented tumor growth. 2) Delayed treatment with various doses of 5B1 showed dose dependent protection up to complete cure in SCID mice engrafted (IV) with Colo205 cells. 7E3 antibodies did not show higher protection despite increased apparent affinity. The specificity and potency of 5B1 antibodies in CDC and ADCC assays translates into good activity in xenograft models. Further studies are warranted to explore the activity against additional cancer specific cell lines in animal models and to pursue preclinical development of 5B1 antibodies. Funding support by NCI #R42CA128362. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C53.