PD-L1/PD-1 blocking antibodies have demonstrated therapeutic efficacy across a range of human cancers. Extending this benefit to a greater number of patients, however, will require a better understanding of how these therapies instigate anticancer immunity. Although the PD-L1/PD-1 axis is typically associated with T cell function, we demonstrate here that dendritic cells (DCs) are an important target of PD-L1 blocking antibody. PD-L1 binds two receptors, PD-1 and B7.1 (CD80). PD-L1 is expressed much more abundantly than B7.1 on peripheral and tumor-associated DCs in patients with cancer. Blocking PD-L1 on DCs relieves B7.1 sequestration in cis by PD-L1, which allows the B7.1/CD28 interaction to enhance T cell priming. In line with this, in patients with renal cell carcinoma or non–small cell lung cancer treated with atezolizumab (PD-L1 blockade), a DC gene signature is strongly associated with improved overall survival. These data suggest that PD-L1 blockade reinvigorates DC function to generate potent anticancer T cell 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-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.

Tumor-targeted CD40 agonism represents an attractive strategy for cancer immunotherapy (CIT) as it promotes dendritic cell (DC) activation and concomitant tumor-specific T cell priming without causing systemic side effects. We developed the bispecific CD40 agonistic antibody CEA-CD40, which triggers CD40 stimulation exclusively in the presence of carcinoembryonic antigen (CEA), a glycoprotein specifically expressed on tumor cells. In this study, we demonstrate that CEA-CD40 can enable potent in vitro DC activation and consecutive T cell cross-priming in a CEA-specific manner. Furthermore, we provide evidence that CEA-CD40 increases colocalization of CEA+ tumor material and DCs. Using CEA+ tumor-derived extracellular vesicles (EVs), which are known to be an excellent tumor antigen source, we show that CEA-CD40 mediates delivery of CEA+ EVs to DCs. Importantly, our data indicates that this fosters acquisition of tumor EV major histocompatibility complex I/peptide complexes by DCs, consequently improving CD8+ T cell priming against EV-associated antigen in vitro. Thus, we provide mechanistic evidence for a dual mode of action of CEA-CD40 for CIT: we suggest that CEA-CD40 has the potential to activate DCs and in addition can promote their loading with tumor antigen derived from EVs to trigger tumor-specific T cell cross-priming.

Purpose: The incidence of human papillomavirus–associated head and neck squamous cell carcinoma (HPV+-HNSCC) is rising worldwide and although current therapeutic modalities are efficient in the majority of patients, there is a high rate of treatment failures. Thus, novel combination approaches are urgently needed to achieve better disease control in patients with HPV+-HNSCC. We investigated the safety and therapeutic efficacy of a novel fibroblast activation protein (FAP)-targeted CD40 agonist (FAP-CD40) in combination with local hypofractionated radiation in a syngeneic HPV+-HNSCC model. Experimental Design: Using an established orthotopic model, we treated tumor-bearing mice with local hypofractionated radiotherapy (2 × 6 Gy) alone or in combination with a systemic administration of the FAP-CD40 antibody. Following up the mice, we evaluated the changes in the tumor microenvironment (TME) by immunofluorescence, FACS, and NanoString RNA analysis. Results: The suboptimal radiotherapy regimen chosen failed to control tumors in the treated mice. The FAP-CD40 administered in monotherapy transiently controlled tumor growth, whereas the combined therapy induced durable complete responses in more than 80% of the tumor-bearing mice. This notable efficacy relied on the radiotherapy-induced remodeling of the TME and activation of the CD8+ T-cell-cDC1 axis and was devoid of the systemic toxicity frequently associated with CD40-targeted therapy. Moreover, the robust immunologic memory developed effectively prevented tumor relapses, a common feature in patients with HNSCC. Conclusions: Our study provides proof of concept, as well as mechanistic insights of the therapeutic efficacy of a bispecific FAP-CD40 combined with local radiotherapy in a FAP+-HNSCC model increasing overall survival and inducing long-term antitumor immunity.

CCK-octapeptide elicits both tonic and phasic activity of the pyloric sphincter. The contractile response to a dose of 0.06 mug kg(-1) h(-1) is greater than the response to 0.02 mug kg(-1) h(-1).