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

Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Melbourne, Victoria, 3084 Australia [A. M. S., D. G., M. R., K. C., E. C. N., F. E. S., J. R., A. K., V. K., R. M., F-T. L., Z. L., K. L., A. W. B.]; Ludwig Institute for Cancer Research, New York Branch, Memorial Sloan Kettering Cancer Center, New York, New York 10021 [E. S., E. C. R., K. K., G. R., E. H., L. J. O.]; Biovation Limited, Aberdeen AB2288GW (F. J. C.); and Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB2522D [W. J. H.], Scotland; and Department of Pathology, Immunology Division, Cambridge University, Cambridge, CB212B United Kingdom [K. L. A.]

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 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 µg/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 mm³) 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.

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