Purpose: The carbohydrate antigen sialyl-Lewis a (sLe a ), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract and breast and on small-cell lung cancers. Since overexpression of sLe a appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis, sLe a is an attractive molecular target for tumor therapy. Experimental Design: We generated and characterized fully human monoclonal antibodies (mAb) from blood lymphocytes from individuals immunized with a sLe a -KLH vaccine. Results: Several mAbs were selected based on ELISA and FACS including two mAbs with high affinity for sLe a (5B1 and 7E3, binding affinities 0.14 and 0.04 nmol/L, respectively) and further characterized. Both antibodies were specific for Neu5Aca2-3Galb1-3(Fuca1-4)GlcNAcb as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (EC 50 0.1 mg/mL vs. 1.7 mg/mL) for r7E3 (IgM) than for r5B1 (IgG1). In addition, r5B1 antibodies showed high level of antibodydependent cell-mediated cytotoxicity activity on DMS-79 cells with human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efficacy, the antibodies were tested in a xenograft model with Colo205 tumor cells engrafted into SCID (severe combined immunodeficient mice) mice. Treatment during the first 21 days with four doses of r5B1 (100 mg per dose) doubled the median survival time to 207 days, and three of five animals survived with six doses. Conclusion: On the basis of the potential of sLe a as a target for immune attack and their affinity, specificity, and effector functions, 5B1and 7E3 may have clinical utility.
The carbohydrate antigen sialyl-Lewis A (sLea) is widely expressed on epithelial tumors of the gastrointestinal tract, on breast cancer cells, and also on small cell lung cancer cells, but is expressed minimally or not at all on normal tissues. sLea serves as a ligand for epithelial leukocyte adhesion molecules and higher expression of sLea was observed in patients with greater node involvement. Since over-expression of sLea appears to be a key event in invasion and metastasis of many tumor cells and 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. Here we report the discovery and initial characterization of fully human antibodies that were generated from blood lymphocytes from individuals immunized with sLea vaccine at MSKCC. Antibodies were identified by ELISA using sLea-HSA conjugates and binding to the native antigen was verified by FACS analysis on DMS-79 cells. 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). These antibodies did not bind to sialyl-Lewis X, Lewis A 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 ratio). These results are very encouraging and we believe that further studies to scale up and test these antibodies in various tumor challenge models are warranted. Since sLea is widely expressed on human cancers, such antibodies could eventually find utility in approximately half of the new cancer cases occurring each year. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B122.
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.
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