Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis

Bispecific antibody (bsAb)-based clinical trials of cancer have been conduc ted primarily using intact murine monoclonal antibody (mAb)-derived molecul es. In some of these trials, toxicity resulting from the interactions of an tibody Fc domains with cellular Fc receptors has limited the doses of antib ody (Ab) that can be employed. Furthermore, human anti-mouse Ab responses p rohibit multiple therapy courses. These factors have decreased the efficacy of the bsAb 2B1, which targets the extracellular domains (ECD) of the HER2 /neu protooncogene product and the human Fc gamma RIII (CD16). To address t hese obstacles, we have constructed and characterized a fully human gene-fu sed bsAb from single-chain Fv (scFv) molecules specific for HER2/neu and CD 16. The human anti-CD16 scFv component, NM3E2, was: isolated from a human s cFv phage display library. As binding of NM3E2 to human neutrophil-associat ed CD16 decreased in the presence of plasma IgG, we have concluded that NM3 E2 recognizes an epitope in the vicinity of the Fe binding pocket. Furtherm ore, the NM3E2 scFv was found by surface plasmon resonance-based epitope ma pping to share an overlapping epitope with the Leu-11c mAb. The human anti- HER2/neu scFv component, C6.5, which was previously isolated from a human s cFv phage display library, was employed as fusion partner for the creation of a bispecific scFv (bs-scFv). In the presence of the C6.5 x NM3E2 bs-scFv , peripheral blood lymphocytes promoted significant lysis of human SK-OV-3 ovarian cancer cells overexpressing HER2/neu. Biodistribution studies perfo rmed in SK-OV-3 tumor-bearing scid mice revealed that 1% ID/g of I-125-labe led C6.5 x NM3E2 bs-scFv was specifically retained in tumor at 23 h followi ng injection. These results indicated that both scFv components of the bs-s cFv retained their function in the fusion protein. This bsAb should overcom e some of the problems associated with the 2B1 bsAb. C6.5 x NM3E2 bs-scFv o ffers promise as a platform for multifunctional binding proteins with poten tial clinical applications as a result of its human origin, lack of an Fe d omain, ease of production, high level of in vitro tumor cell cytotoxicity a nd highly selective tumor targeting. (C) 1999 Elsevier Science Ltd. All rig hts reserved.