Preparation and characterization of a recombinant humanized single- chain Fv antibody/human interleukin-2 fusion protein directed against the HER-2/neu (c-erbB2) proto-oncogene product, p185

High dose recombinant human interleukin 2 (rhIL-2) therapy has been used in the treatment of established tumors in both animal models and patients with advanced melanoma or renal carcinoma. However, because high dose rhIL-2 therapy causes severe systemic toxicity in normal tissues, its clinical use has been limited. Therefore, targeting interleukin-2 (IL-2) to the tumor site should improve its anti-tumor-immune response and decrease its systemic toxicity. In this study, we describe the preparation and characterization of a recombinant humanized single-chain Fv (sFv) antibody/IL-2 fusion protein. This recombinant fusion protein consists of humanized variable heavy (V(H)) and light (V(L)) domains of monoclonal antibody (mAb) 520C9 directed against the human HER-2/neu(c-erbB2) proto-oncogene product p185 and human IL-2. The fusion protein was stably expressed in baby hamster kidney cells and shown to retain the immunostimulatory activities of IL-2 as measured by IL-2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activity, this fusion protein also possesses binding specificity against the HER-2/neu (c-erbB2) proto-oncogene product, p185, as determined by enzyme linked immunosorbent assay (ELISA) using SKOV 3ip1 cells. Taken together, these findings suggest that this recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means of targeting therapeutic doses of IL- 2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.