Certain
cytokines synergize in activating anti-
cancer immunity at the site of disease and it may be desirable to generate biopharmaceutical agents, capable of simultaneous delivery of
cytokine pairs to the
tumor. In this article, we have described the cloning, expression and characterization of IL2-XE114-TNFmut, a dual-
cytokine biopharmaceutical featuring the sequential fusion of
interleukin-2 (
IL2) with the XE114 antibody in scFv format and a
tumor necrosis factor mutant (TNFmut). The fusion
protein recognized the cognate
antigen (
carbonic anhydrase IX, a marker of
hypoxia and of
renal cell carcinoma) with high affinity and specificity. IL2-XE114-TNFmut formed a stable non-covalent homotrimeric structure, displayed
cytokine activity in in vitro tests and preferentially localized to solid
tumors in vivo. The product exhibited a partial growth inhibition of murine CT26
tumors transfected for
carbonic anhydrase IX. When administered to Cynomolgus monkey as
intravenous injection, IL2-XE114-TNFmut showed the expected plasma concentration of ~1,500 ng/ml at early time points, indicating the absence of any in vivo trapping events, and a half-life of ~2 h. IL2-XE114-TNFmut may thus be considered as a promising biopharmaceutical for the treatment of metastatic
clear-cell renal cell carcinoma, since these
tumors are known to be sensitive to
IL2 and to TNF.