The Dose-Dependent Tumor Targeting of Antibody-IFNγ Fusion Proteins Reveals an Unexpected Receptor-Trapping Mechanism In Vivo

Cytokines often display substantial toxicities at low concentrations, preventing their escalation for therapeutic treatment of cancer. Fusion proteins comprising cytokines and recombinant antibodies may improve the anticancer activity of proinflammatory cytokines. Murine IFN gamma was appended in the diabody format at the C-terminus of the F8 antibody, generating the F8-IFN gamma fusion protein. The F8 antibody is specific for the extra-domain A (EDA) of fibronectin, a tumor-associated antigen that is expressed in the vasculature and stroma of almost all tumor types. Tumor-targeting properties were measured in vivo using a radioiodinated preparation of the fusion protein. Therapy experiments were performed in three syngeneic murine models of cancer [F9 teratocarcinoma, WEHI-164 fibrosarcoma, and Lewis lung carcinoma (LLC)]. F8-IFN gamma retained the biologic activity of both the antibody and the cytokine moiety in vitro, but, unlike the parental F8 antibody, it did not preferentially localize to the tumors in vivo. However, when unlabeled F8-IFN gamma was administered before radioiodinated F8-IFN gamma, a selective accumulation at the tumor site was observed. F8-IFN gamma showed dose-dependent anticancer activity with a clear superiority over untargeted recombinant IFN gamma. The anticancer activity was potentiated by combining with F8-IL4 without additional toxicities, whereas combination of F8-IFN gamma with F8-TNF was lethal in all mice. Unlike other antibody-cytokine fusions, the use of IFN gamma as payload for anticancer therapy is associated with a receptor-trapping mechanism, which can be overcome by the administration of a sufficiently large amount of the fusion protein without any detectable toxicity at the doses used. (C) 2014 AACR.