Context-specific GITR agonism potentiates anti-PD-L1 and CD40-based immuno- chemotherapy combination in heterogeneous pancreatic tumors

Immunotherapy has shown limited success in pancreatic adenocarcinoma (PDAC) patients. To improve clinical management of cancer, it is crucial to identify alternative immunostimulatory targets associated with mechanisms of tumor evolution to facilitate the development of novel combination immunotherapies. Here we categorized PDACs and other cancers (n>7,500) into subgroups based on immunostimulatory glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related ligand (GITRL) and receptor (GITR) expression: GITRLhigh+GITRhigh and GITRLhigh/low+GITRlow. We characterized immune evasion mechanisms using immunotherapy preclinical trials in four representative immunocompetent mouse models, finding that the GITR agonist, DTA-1 significantly improved responses in GITRLhigh(+GITRhigh) tumors (n=2). Further characterization revealed increased activation of CD8+ T-cells (but not T-regulatory; Tregs cells) and enhanced interferon-{gamma}, immunoproteosome, antigen presentation, and T-cell receptor (TCR) gene expression in DTA-1 responders. In vivo clonal tracking using DNA barcoding showed that GITR agonist therapy significantly reduced tumor burden by targeting expansion of heterogeneous PDAC clones and not clone-initiating cells (representing potential resistance). However, emerging GITRLhigh+GITRhigh epithelial-like oligoclones from the responder model escaped immune surveillance to GITR agonist treatment via increased PD-L1, offering a combined anti-PD-L1, CD40 agonist and DTA-1 immunotherapy regimens (with/without chemotherapy) that further improved responses by decreasing PD-L1+ myeloid cells. Conversely, mesenchymal-enriched GITRLlow models exhibited primary (intrinsic) resistance to GITR agonist treatment due to reduced T-cells and increased myeloid and/or PD-L1+ non-immune cells. These results provide pre-clinical context for GITR+PD-L1+CD40- based personalized immuno-chemotherapy combinations for PDAC.