Fibroblast Activation Protein α-Targeted CD40 Agonism Abrogates Systemic Toxicity and Enables Administration of High Doses to Induce Effective Antitumor Immunity

Abstract

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-CD40's 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 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 nontargeted 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 nontargeted 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 nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. Conclusions: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.

Validating Cell Surface Proteases as Drug Targets for Cancer Therapy: What Do We Know, and Where Do We Go?

“…First-in-human results confirmed tumour-specific uptake and a favourable safety profile, which supports further clinical exploration [79,80] (Table 1). A bispecific FAP-CD40 mAb (RO7300490) applies a similar principle to act as costimulatory signal for antigen-presenting cell (APC) activation, leading to enhanced T cell priming and tumour regression in mice xenograft models without clear signs of toxicity [137]. The FAP-directed CD40 agonist has progressed to clinical trial phase I as a single agent or in combination with ATZ (Table 1).…”

Cancer-Associated Fibroblasts: Implications for Cancer Therapy

“…The main goal of using this antibody is to activate T-cells in the TME [ 195 ]. Other strategies involve the conjugation of FAP with other immune modulators, such as IL15 [ 196 ], co-stimulatory ligands as B7.2 [ 197 ] and CD40 [ 198 ], or immunotoxins [ 199 , 200 ]. Moreover, FAP antibodies can be conjugated with agents that will directly induce apoptosis, such as the cytotoxic drug DM1, which have shown potent inhibitory activity in pre-clinical models [ 187 ].…”

Ten years in the making: application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins

“…Expression of the fibroblast activation protein (FAP) on tumor fibroblasts is found in the majority of solid tumors, making FAP an attractive antigen for tumor targeting. 119 , 120 Based on this rationale, FAP-targeted bispecific antibodies and fusion proteins have been created using CrossMab technology that rely on FAP binding with one moiety to induce, with their second moiety, hyper-clustering of tumor necrosis factor (TNF) receptor superfamily members 121 like DR5 for apoptosis induction, 85 4–1BB/CD137 for T cell activation, 89 or CD40 for activation of antigen-presenting cells, 94 , 122 as described below. The first of these conditional FAP-targeted TNFR agonistic antibodies entering Phase 1 clinical trials was the symmetric tetravalent C-terminally fused FAP/DR5 targeted 2 + 2 CrossMab CH 1 −CL RG7386 ( Figure 2g ).…”

“…This design was chosen to make FAP-CD40 (RG6189), a FAP-targeted CD40 agonistic bispecific antibody, with the goal of abrogating systemic toxicity and enabling administration of doses sufficiently high to result in highly tumor- and lymph node-specific activation of APCs with subsequent induction of antitumor immunity. 94 , 122 Phase 1 clinical trials have been initiated to validate this approach in the clinic (NCT04857138).…”