Mechanisms Governing Efficacy Of Combination Cd40 Agonist And Anti-Pd-L1 In Pancreatic Ductal Adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy that is resistant to conventional therapies including monotherapy using PD-1 or PD-L1 inhibition. Combination agonistic anti-CD40 and PD-1/PD-L1 blockade have clinical promise in advanced cancer patients including PDA. The underlying mechanism(s) driving the therapeutic effects of this combination are ill-defined. Here, we create a syngeneic PDA animal model and utilize various genetic tools to assess how CD40 agonist, PD-L1 blockade or the combination impact tumor antigen-specific T cells using fluorescently-labeled peptide:MHC tetramers and cells in the tumor microenvironment. Molecular analyses of tumor cell escape variants is also performed. Methods: We recently developed a high-throughput orthotopic syngeneic KPC pancreatic cancer mouse model that expresses a novel model neoantigen in B6 mice described in Burrack et al., Cell Reports, 2019. We create fluorescently labeled peptide:H-2Db tetramers to track the fate of endogenous pancreatic tumor-antigen specific CD8+ T cells over time. Here, we use this model alone or mixed at a 1:1 ratio of KPC tumor cells that do not express the neoantigen to examine how agnostic anti-CD40 (a single dose, clone FGK145), anti-PDL1 (3 doses, clone 10F.932), or the combination impact tumor growth in the pancreas over time using bioluminescent imaging and high-resolution ultrasound. We use multiparameter flow cytometry to investigate how anti-CD40 +/- PD-L1 blockade impacts the phenotype, longevity and functionality of tetramer-binding T cells over time. We assess how other immune cell lineages are altered systemically and in the tumor microenvironment by quantifying myeloid subpopulations, B cells, NK cells and regulatory T cells following therapy. We use Batf3-/- mice and XCR1VenusDTR mice to assess the role of conventional type I dendritic cells (cDC1s) on therapeutic efficacy. We employ both cytokine and chemokine reporter strains to identify how anti-CD40 +/- PD-L1 blockade impacts inflammatory gene expression in immune cells enriched the tumor microenvironment. We examine the persistence and location of tetramer-binding T cells in the pancreas, lung and liver of mice following tumor eradication. Additionally, we re-derive resistant tumor cells from mice and evaluate the integrity of MHC class I antigen processing and presentation pathways. Finally, single cell sequencing is performed to assess the traits of subpopulations of tumor-antigen specific T cells that correlate with enhanced antitumor activity following therapy. Results: We show that anti-CD40 or anti-PD-L1 monotherapy have significant yet transient antitumor effects in mice with neoantigen+ PDA with distinct effects on tumor specific T cells. Objective responses occur in 100% of the monotherapy treated mice and survival is significantly prolonged. However, tumors recur in 100% of these animals. Tumor escape variants defective in MHC class I protein and Tap1 gene expression following IFN-gamma treatment ultimately emerge. In contrast, combination agonistic anti-CD40 + PD-L1 blockade synergize therapeutically resulting in cures in 60% of the animals and formation of pancreas resident memory T cells that specifically bind tetramer and express CD49a and CD103 following tumor eradication. Mechanistically, the combination selectively expands conventional type 1 dendritic cells (cDC1s) in the spleens and tumors of tumor-bearing animals. cDC1s in PDA are CD11c+MHCII+ and express CD8, CD103 and Xcr1. Using Batf3-/- mice or an Xcr1venusDTR transient cDC1 depletion model, we demonstrate a striking dependency on cDC1s for therapeutic benefit with anti-CD40 or PD-L1 blockade. Unexpectedly, we find that the expansion of cDC1s in pancreatic tumor-bearing animals is partially dependent on Xcr1 expression by DCs. Anti-CD40+PD-L1 blockade significantly expand the number of tetramer-binding T cells that express KLRG1 in PDA. The tetramer-binding T cells remain PD-1+ yet have lower expression of Lag3 and have heightened polyfunctionality as measured by cytokine production. Further studies using chemokine and cytokine reporter models, we uncover key differences in how anti-CD40 and anti-PD-L1 impact inflammatory gene expression by antigen presenting cells in PDA. Finally, we demonstrate the requirement for tumor neoantigen expression for efficacy because in mice that have tumors containing a 50:50 mixture of neoantigen+ pancreatic tumor cells with neoantigen- pancreatic tumor cells, combination anti-CD40 + PD-L1 blockade results in elimination of predominantly those tumor cells that express the neoantigen. Further single cell sequencing data on how this combination impacts tumor-antigen specific T cell subpopulations as well as epitope spreading will be discussed. Conclusions: These findings reveal for the first time to our knowledge that anti-CD40 + PD-L1 blockade synergize via the expansion of cDC1s in pancreatic tumor-bearing animals. Instead of anti-CD40 promoting priming of neoantigen-specific T cells, we find that this combination promotes the systemic expansion and intratumoral accumulation of KLRG1+ tumor-specific T cells that eradicate PDA and form pancreas resident CD49a+CD103+ memory T cells. Citation Format: Adam L. Burrack, Meagan R. Rollins, Ellen J. Spartz, Jackson F. Raynor, Iris Wang, Jason Mitchell, Tsuneyasu Kaisho, Brian Fife, Ross Kedl, Stephen Shen, Ingunn M. Stromnes. Mechanisms governing efficacy of combination CD40 agonist and anti-PD-L1 in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG12.