AbstractCertain 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 ~1500 ng/ml at early time points, indicating the absence of any in vivo trapping events, and a half-life of ~2 hours. 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.Contribution to the fieldThere is a growing interest in the antibody-based targeted delivery of pro-inflammatory cytokines for tumor therapy, which may be complementary or alternative to immune checkpoint inhibitors for immunotherapeutic applications. In this article, we have described a novel fusion protein, featuring antibody moieties specific to carbonic anhydrase IX, as well as interleukin-2 and tumor necrosis factor as pro-inflammatory cytokines, which combines the ability to recognize a tumor-associated antigen on the surface of renal cell carcinomas with the simultaneous display of two potent therapeutic payloads. The newly developed product (termed IL2-XE114-TNFmut) exhibited favorable biochemical characteristics, the ability to preferentially localize at the tumor site, a cancer growth inhibition activity and a suitable pharmacokinetic profile in Cynomolgus monkey. IL2-XE114-TNFmut may therefore represent an attractive candidate for the immunotherapy of renal cell carcinoma.
A Novel Fully-Human Potency-Matched Dual Cytokine-Antibody Fusion Protein Targets Carbonic Anhydrase IX in Renal Cell Carcinomas
