Background Pancreatic ductal adenocarcinoma (PDAC) features a fibrotic stroma that obstructs immune infiltration. We hypothesize that dysregulated CD26/DPP4 enzymatic activity in PDAC constrains the efficacy of immunotherapy. We also posit that in vivo pharmacological targeting of CD26 enzyme activity, via FDA-approved inhibitors (sitagliptin or STG) for Type 2 Diabetes, influences immune infiltration and enhances immunotherapy response in murine PDAC models.

Methods CD26 protein was evaluated in immortalized cancer associated fibroblasts (CAFs) and PDAC cells by immunoblot and flow, while soluble CD26 was measured by ELISA. In vivo studies used C57BL/6 mice orthotopically implanted with syngeneic luciferase-expressing KPC-tumor cells in the pancreas. Tumors were verified and monitored by bioluminescence imaging (BLI) and mice randomized to treatment: STG (75mg/kg in drinking water), anti-PD-L1 Ab (200ug 3x/week), or both for 9 days. Controls received vehicle and/or isotype Ab. Tumors were harvested at endpoint and tumor infiltrating lymphocytes (TIL) and checkpoint expression was evaluated. Two in vivo studies evaluated survival (treated 3 weeks) or TIL (treated 9 days) for PDAC-bearing mice treated with STG, anti-PD-L1, and anti-Lag3 (including single, double, and triple therapy combinations). A combination study evaluating survival in mice orthotopically implanted with MT5 cells is ongoing.

Results CD26 was among the most highly expressed genes in patient PDAC-derived CAFs vs. normal fibroblasts. CD26 protein was observed in human CAFs (HT137 and h-iPSC-PDAC-1) and PDAC cells (HPAC and PANC1) via immunoblot and flow, however, soluble CD26 was only observed in CAF supernatants (180 pg/mL and 587.6 pg/mL). Concurrent administration of STG and anti-PD-L1 Ab limited tumor progression and increased CD4, as well as increased CD8 (p’s<0.04). CD4+ TIL from STG/anti-PD-L1 had high Lag-3 expression (p’s<0.025). Triple therapy (STG, anti-PD-L1 Ab, and anti-Lag-3) increased overall survival (p=0.005) of mice bearing KPC-luc tumors and increased CD3 vs all groups (p’s<p=0.02). Our ongoing survival study in mice bearing MT5 tumors initially demonstrates similar benefit from treatment with STG administered in combination with immune checkpoint blockade.

Conclusions Our results are the first to demonstrate CD26 enzyme inhibition (STG), augments in vivo efficacy of anti-PD-L1 Ab and anti-Lag-3 in relevant orthotopic PDAC models. This work identifies immune populations, including T cells, that provide mechanistic insight for efficacy. We demonstrate versatility of CD26 inhibition and its capacity to modulate checkpoint molecules and efficacy in PDAC.

Acknowledgements The authors would like to thank Emory University, the Winship Cancer Institute, the Pediatrics/Winship Advanced Flow Cytometry Core, the Winship Cancer Animal Models Shared Resource, and the Winship Integrated Cellular Imaging Core for all of their help. Funding from the Morningside Foundation and the Department of Hematology and Medical Oncology at the Winship Cancer Institute of Emory University. Supported by NIH grants R01CA208253, R01CA228406, and P30CA138292. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Ethics Approval In vivo murine studies obtained ethics approval through the Institutional Animal Care and Use Committee (IACUC) at Emory University.