Combinatorial immunotherapy with agonistic CD40 activates dendritic cells to express IL-12 and overcome PD-1 resistance II

Checkpoint inhibitors have revolutionized cancer treatment, but resistance remains a significant clinical challenge. Myeloid cells within the tumor microenvironment can modulate checkpoint resistance by either supporting or suppressing adaptive immune responses. Using an anti-PD-1 resistant mouse melanoma model, we show that targeting the myeloid compartment via CD40 activation and CSF1R blockade in combination with anti-PD-1 results in complete tumor regression in a majority of mice. This triple therapy combination is primarily CD40 agonist-driven in the first 24 hours post-therapy and shows a similar systemic cytokine profile in human patients as in mice. Functional single-cell cytokine secretion profiling of dendritic cells (DCs) using a novel microwell assay identified a CCL22+CCL5+ IL-12-secreting DC subset as important early-stage effectors of triple therapy. CD4+ and CD8+ T cells are both critical effectors of treatment, and systems analysis of single-cell RNA-sequencing data supports a role for DC-secreted IL-12 in priming T cell activation and recruitment. Finally, we show that treatment with a novel IL-12 mRNA therapeutic alone is sufficient to overcome PD-1 resistance and cause tumor regression. Overall, we conclude that combining myeloid-based innate immune activation and enhancement of adaptive immunity is a viable strategy to overcome anti-PD-1 resistance. C57BL6/J mice were injected with 0.5x10^6 YUMMER1.7 cells on day 0, and then were either left untreated or treated with the combination of anti-PD-1, CSF1R blockade, and agonistic CD40 (i.e., TTx) by i.p. injection on day 7. To assess cellular and molecular heterogeneity in the tumor in response to treatment, we performed scTCR-seq on cells isolated from untreated and TTx-treated tumors 24 hours after treatment on day 8. One tumor was used per treatment condition.