Construction, Production, and Characterization of Humanized Anti-Lewis Y Monoclonal Antibody 3S193 for Targeted Immunotherapy of Solid Tumors

The Lewis Y (Le y ) antigen is a blood group-related antigen that is expressed in a high proportion of epithelial cancers (including breast, colon, ovary, and lung cancer) and is an attractive target for monoclonal antibody-directed therapy. The murine monoclonal 3S193 (IgG3) was generated in BALB/c mice by immunization with Le y -expressing cells of the MCF-7 breast carcinoma cell-line. The murine 3S193 showed high specificity for Le y in ELISA tests with synthetic Le y and Le y -containing glycoproteins and glycolipids and also reacted strongly in rosetting assays and cytotoxic tests with Le y -expressing cells. We generated a humanized form of the murine 3S193 antibody by linking cDNA sequences encoding the variable region of murine 3S913 with frameworks of the human KOL heavy chain and REI k chain. The genes for the humanized 3S193 monoclonal antibody IgG1 were transfected into mouse myeloma NS0 cells and cloned for the establishment of high antibody-producing colonies. Humanized 3S193 antibody was subsequently produced through in vitro culture and under good manufacturing practice conditions using hollow-fiber bioreactors. The purified humanized 3S193 (hu3S193) was subsequently characterized and validated for use in preliminary immunotherapy investigations. hu3S193 reacted specifically with Le y antigen, with similar avidity to the murine form. hu3S193 demonstrated potent immune effector function, with higher antibody-dependent cell-mediated cytotoxicity than its murine counterpart and potent complement-dependent cytotoxicity (ED50, 1.0 mg/ml). The in vivo immunotherapeutic potential of hu3S193 was assessed in a human breast xenograft model using MCF-7, Le y -positive cells. Six i.v. doses of up to 1 mg of hu3S193 were administered to animals bearing established tumors (120 ‐130 mm 3 ) with no significant effect on tumor growth. In contrast, in an MCF-7 xenograft preventive model, a 1-mg hu3S193 dosage schedule was able to significantly slow tumor growth compared with placebo and isotype-matched control IgG1 antibody. hu3S193 has promise for immunotherapy of Le y positive tumors and is currently entering Phase I clinical trials.