Abstract B48: CD40-agonist treatment can prime the inflammatory response of macrophages and reverse checkpoint inhibitor resistance in melanoma
Authors: Pizzurro Gabriela A , Bridges Kate , Kaech Susan , Bosenberg Marcus , Miller-Jensen Kathryn 
Published: December 2022

Abstract
Checkpoint inhibitors (CPIs) have revolutionized cancer treatment, but therapy resistance remains a significant clinical challenge. One strategy to restore anti-tumor activity is to target immunosuppressive tumor-associated macrophages (TAMs) and myeloid cells that suppress adaptive immune responses. Our previous results in the YUMMER mouse melanoma model demonstrated that agonistic CD40 antibody (CD40ag) and CSF1R blockade could overcome PD-1 resistance via functional activation of a specific CCL22+CCL5+ IL-12-secreting dendritic cell (DC) subset, but did not indicate a clear role for TAMs, when initiated at day 7 post-tumor injection (early treatment). We hypothesized that CD40ag-treatment efficacy is impacted by the stage of tumor growth, leading to differential responsive immune cell populations. To address this, we studied a different treatment scheme in which a single dose of CPIs anti-PD-1 and anti-CTLA4 induced complete tumor regression when applied early, but fail to be effective in delayed administration schemes (after day 10). Specifically, we added CD40ag to a single-dose CPI treatment initiated at different stages of tumor growth, in order to define key myeloid subpopulations and cell-cell interactions leading to successful TME reprogramming versus resistance. In immunocompetent mice, a Ly6Cint TAM subset exhibited a partial inflammatory response which appeared to be dependent on early tumor T-cell infiltration. Administration of early CD40ag treatment did not generate additional changes in inflammatory markers in the TAM population, despite the significant fraction of CD40+ TAMs. However, a delayed CD40ag treatment rapidly upregulated pro-inflammatory markers in a CD40+Ly6C+ TAM subset, with high iNOS, CCL3 and TNFa expression. This approach synergized with CPIs, inducing more than 90% overall survival, in contrast to the monotherapies, which showed initial tumor control but were ineffective on their own long-term. The efficay of a single-dose CPI+CD40ag decreased with treatment initiation delay. Furthermore, in Rag-/- mice, we did not detect the poorly-inflammatory TAM subset and early CD40ag treatment induced an immediate TAM response with multiple proinflammatory markers, but transient in the absence of T cells. By scRNA-seq, early treatment with CD40ag, and in combination with CPIs, increased expression of genes involved in T cell recruitment and activation, such as Il12b and Cxcl9/10, in responding subsets of DCs and TAMs, respectively. These findings supported the role of CD40ag and improved treatment schemes, priming the innate compartment and establishing an important link with adaptive immunity. Integrating scRNA-seq, flow cytometry and in vivo functional data, we determined changes in myeloid cells associated with successful immune responses as well as TAM subsets limiting immunotherapy. Future studies will focus on immune cell spatial distribution to build and refine relevant interaction networks within the TME to define successful, myeloid-driven therapeutic responses in melanoma.
Citation Format: Gabriela A Pizzurro, Kate Bridges, Susan Kaech, Marcus Bosenberg, Kathryn Miller-Jensen. CD40-agonist treatment can prime the inflammatory response of macrophages and reverse checkpoint inhibitor resistance in melanoma [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B48.