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 expres sed in a high proportion of epithelial cancers (including breast, colon. ov ary. and lung cancer) and is an attractive target for monoclonal antibody-d irected therapy The murine monoclonal 3S193 (IgG3) was generated in BALB/c mire by immunization with Le(y)-expressing cells of the MCF-7 breast carcin oma cell-line. The murine 3S193 showed high specificity for Le(y) in ELISA tests with synthetic Le(y) and Le(y)-containing glycoproteins and glycolipi ds and also reacted strongly in resetting assays and cytotoxic tests with L e(y)-expressing cells. We generated a humanized form of the murine 3S193 an tibody by linking cDNA sequences encoding the variable region of murine 3S9 13 with frameworks of the human KOL heavy chain and REI kappa chain. The ge nes 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 thr ough in vitro culture and under good manufacturing practice conditions usin g hollow-fiber bioreactors. The purified humanized 3S193 (hu3S193) was subs equently characterized and validated for use in preliminary immunotherapy i nvestigations. bu3S193 reacted specifically with Le(y) antigen, with simila r avidity to the murine form. hu3S193 demonstrated potent immune effector f unction, with higher antibody-dependent cell-mediated cytotoxicity than its murine counterpart and potent complement-dependent cytotoxicity (ED50, 1.0 mu g/ml). The in vivo immunotherapeutic potential of hu3S193 was assessed in a human breast xenograft model using MCF-7. Le(y)-positive cells. Si i.v . doses of up to 1 mg of hu3S193 were administered to animals bearing estab lished tumors (120-130 mm(3)) with no significant effect on tumor growth. I n contrast, in an MCF-7 xenograft preventive model, a I-mg hu3S193 dosage s chedule was able to significantly slow tumor growth compared with placebo a nd isotype-matched control IgG1 antibody. hu3SI93 has promise for immunothe rapy of Le(y)-positive tumors and is currently entering Phase I clinical tr ials.