Fibroblast activation protein α-targeted CD40 agonism abrogates systemic toxicity and enables administration of high doses to induce effective anti-tumor immunity.

Purpose: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. Experimental Design: FAP-CD409s in vitro activity and FAP-specificity were validated by antigen-presenting cell (APC) activation and T cell priming assays. In addition, FAP-CD40 was tested in subcutaneous (s.c.) MC38-FAP and KPC-4662-huCEA murine tumor models. Results: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike non-targeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While non-targeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas non-targeted CD40 agonists displayed strong systemic but limited intratumoral effects. Conclusions: FAP-CD40 abrogates the systemic toxicity associated with non-targeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing anti-tumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.