Abstract
Cytokines driving the immune response are powerful tools for cancer immunotherapy, but their application is generally limited by severe systemic toxicity. Targeted approaches by means of antibody-cytokine fusion proteins might enable focus on the cytokine activity to the tumor site, thereby reducing unwanted side effects. Here, we investigated the possibility to improve the efficiency of interleukin (IL)-15 presentation in a targeted approach by the incorporation of an IL-15R$\alpha$ chain fragment, mimicking physiologic trans-presentation. Therefore, an antibody cytokine fusion protein (scFv\_RD\_IL-15) composed of an antibody moiety targeting the tumor stromal fibroblast activation protein (FAP), an extended IL-15R$\alpha$sushi domain (RD) and IL-15 was generated, exhibiting antibody-mediated specific binding and cytokine activity in soluble and targeted form. Comparative analysis with a corresponding antibody fusion protein devoid of RD (scFv\_IL-15) showed for scFv\_RD\_IL-15 in solution enhanced stimulatory activity on Mo7e (IL-15R$\beta$$\gamma$) cells and reduced proliferation response on CTLL-2 (IL-15R$\alpha$$\beta$$\gamma$) cells, while in FAP-targeted, that is, membrane-bound form, comparable proliferation of CTLL-2 (IL-15R$\alpha$$\beta$$\gamma$) cells was obtained. In addition, scFv\_RD\_IL-15 achieved in its soluble and target-bound form stronger proliferation and cytotoxicity on unstimulated and activated T cells, respectively. Furthermore, in vivo analysis in a lung metastasis tumor mouse model revealed a superior antitumor effect for scFv\_RD\_IL-15 in comparison with that obtained by an untargeted or RD missing version of IL-15 fusion protein. Thus, tumor-directed trans-presentation of IL-15 in association with RD in form of an antibody fusion protein seems to be a promising approach to further improve the antitumor effect of IL-15.
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