The potent antitumor activity of certain
cytokines is often achieved at the expense of unacceptable toxicity. One avenue to improve the therapeutic index of
cytokines in
cancer therapy consists of fusing them to
monoclonal antibodies capable of a selective localization at the
tumor site. We have constructed fusion
proteins of
interleukin-12 (IL-12) and
tumor necrosis factor (
TNF-alpha) with L19, an
antibody fragment specific to the extradomain B of
fibronectin which has been shown to target
tumors in animal models and in patients with
cancer. These fusions display a potent antitumor activity in several immunocompetent murine models of
cancer but do not lead to complete remissions of established aggressive
tumors. In this article, we have evaluated the
tumor-targeting properties and the anticancer activities of combinations of the two antibody-
cytokine fusion
proteins, as well as of a triple fusion
protein between
IL-12, L19, and
TNF-alpha. Although all fusion
proteins were active in vitro, the triple fusion
protein failed to localize to
tumors in vivo and to show significant
therapeutic effects. By contrast, the combination of IL-12-L19 and L19-TNF-alpha displayed potent synergistic anticancer activity and led to the eradication of F9
teratocarcinomas grafted in immunocompetent mice. When cured mice were rechallenged with
tumor cells, a delayed onset of
tumor growth was observed, indicating the induction of a partial antitumor vaccination effect. Potent anticancer effects were achieved at doses of IL-12-L19 and L19-TNF-alpha (2 micro g + 2 micro g/mouse), which were at least 5-fold lower than the maximal-tolerated dose. The combined administration of the two fusion
proteins showed only a modest increase in toxicity, compared with treatments performed with the individual fusion
proteins. These results show that the targeted delivery of
cytokines to the
tumor environment strongly potentiates their antitumor activity and that the combination treatment with IL-12-L19 and L19-TNF-alpha appears to be synergistic in vivo.