BIOLOGICAL-ACTIVITY AND INVIVO CLEARANCE OF ANTITUMOR ANTIBODY CYTOKINE FUSION PROTEINS/

Several human cytokines including IL-2, GM-CSF, and tumor necrosis fac tors alpha and beta were engineered as fusion proteins to the carboxyl terminus of a chimeric anti-ganglioside antibody, ch14.18, and expres sed in transfected hybridoma cells. All of the fusion proteins were ex pressed at high levels and were easily purified by affinity or ion-exc hange chromatography from culture supernatants. The effect of fusion o n antigen binding activity was tested and found to vary with the parti cular cytokine. No significant decreases in antigen binding were obser ved, and fusion of IL-2 had the greatest positive effect in a direct a ntigen binding assay. All fusion proteins maintained normal levels of biological activity except for GM-CSF, which was approximately 20 % ac tive, compared to recombinant GM-CSF produced in bacteria. The clearan ce of the fusion proteins was examined in normal Balb/c mice after int raperitoneal injection or in athymic (nu/nu) mice after intravenous in jection and was generally quite rapid, relative to ch14.18. This was m ainly due to a very rapid initial clearance rate (alpha phase) since t he half-lives of the beta phase of the fusion proteins (about 30 h) we re comparable to that of the free antibody (about 58 h). These results demonstrate that biologically active antibody/cytokine fusion protein s can be constructed by genetic engineering. Their relatively rapid cl earance may require constant infusion rather than bolus injection in o rder to achieve clinical efficacy.