CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily which can activate both innate and adaptive tumor immunity, making it an attractive target for cancer immunotherapy. Systemic administration of agonistic anti-CD40 antibodies has shown signs of activity in cancer patients, but dose-limiting toxicity has prevented the exploration of the full dose range and possibly has impaired clinical efficacy. To overcome this issue, we developed a multispecific DARPin® therapeutic candidate, MP0317, intended to activate CD40 receptor locally, rather than systemically, in the tumor microenvironment through cross-linking with fibroblast activation protein (FAP) that is abundantly present in the stroma of many solid tumors, but has limited expression in healthy adult tissues.

Using primary human 1) B cells and 2) dendritic cells, MP0317 activated the CD40 pathway in vitro. Upregulation of CD80, CD86, CD69 and IL12 occurred exclusively in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking.

A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x mCD40) was generated and tested in murine cell-based in vitro assays showing comparable results as MP0317 with a strict FAP-dependent activation of CD40. The mFAP x mCD40 was also active in vivo and inhibited substantially the progression of FAP-positive tumors. Moreover, in contrast to an anti-mouse CD40 antibody (clone FGK45), the antitumor activity of mFAP x mCD40 was neither associated with elevated blood cytokine levels nor with hepatotoxicity, both of which manifest as dose limiting toxicities of some of the clinical CD40 activating antibodies. These data support a mode of action, both in vitro and in vivo, that is dependent on FAP-mediated crosslinking of CD40 receptor resulting in a tumor-localized CD40 activation without peripheral or off-tumor organ toxicity.

In conclusion, we have designed a tumor-targeted CD40 agonistic DARPin® therapeutic candidate that is able to activate the CD40 receptor locally in FAP-positive tumors producing impressive antitumor activity in the absence of systemic toxicity.

Citation Format: Nicolo Rigamonti, Sarah Jetzer, Niels Van der Velden, Omar Abdelmotaleb, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Ralph Bessey, Andrea Valeri, Heidi Poulet, Christof Zitt, Pamela A. Trail, Victor Levitsky, Niina Veitonmäki, Clara Metz. A tumor-targeted CD40 agonistic DARPin® molecule leading to antitumor activity with limited systemic toxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1073.