Background Immunotherapy has limited efficacy in pancreatic ductal adenocarcinoma (PDAC). BXCL701, a small molecule inhibitor of DPPs 4, 8, and 9 and Fibroblast Activation Protein,¹ reverses the abilities of DPPs to block immune activation through truncation of chemokines, induction of fibrosis, and inhibition of inflammasome activation and IL-18 release. We previously demonstrated that combination therapy using BXCL701+anti-PD1 antibody significantly reduced tumor growth in a subcutaneous, syngeneic, murine mT3–2D PDAC model.² Therapy with BXCL701+anti-PD1 antibody was accompanied by significant infiltration of NK and T cells and increase in serum and intra-tumoral levels of NK attracting chemokines.² It is important to identify the critical determinants of this agent’s therapeutic benefit. We hypothesized that DPP4 inhibition contributes to the anti-tumor effects seen with BXCL701.

Methods 5x10⁴ mT3–2D (Kras^+/G12D; p53^+/-R172H; Pdx-Cre) GFP/luciferase-expressing cells were implanted orthotopically in the pancreas of C57BL/6J mice. Bioluminescence imaging confirmed established tumors and mouse cohorts were normalized by total flux (photons/second) in the designated region of interest based on pre-treatment imaging. Treatment cohorts included sitagliptin (20 μg, DPP4 inhibitor) or BXCL701 (20 μg), with both cohorts also receiving anti-PD1 antibody (200 μg). Sitagliptin or BXCL701 were administered daily via oral gavage in vehicle (PBS, 100 μL). Anti-PD1 antibody (clone RMP1–14 (BioXcell)) in vehicle was administered at a volume of 100 μL given twice weekly. Control cohort received vehicle. Blood was collected from 4–5 mice in each cohort weekly via submandibular blood draw to measure serum chemokine levels.

Results Analysis of mice at endpoint revealed no significant difference in average tumor masses between PBS and sitagliptin+anti-PD1 cohorts (0.95g vs. 0.93g, p=0.90). A significant improvement was seen with BXCL701+anti-PD1 therapy as compared to PBS (0.21g vs. 0.95g, p<0.0001) and sitagliptin+anti-PD1 therapy cohorts (0.21g vs. 0.93g, p<0.0001). Additionally, 8/9 mice treated with BXCL701+anti-PD1 showed no evidence of disease; in contrast, all sitagliptin-treated mice exhibited tumor growth.

Conclusions These findings suggest that BXCL701’s mechanisms of anti-tumor effects are not limited to DPP4 inhibition. Ongoing work will provide insight into changes in immune cell localization, chemokines and the impact of fibrosis reduction on anti-tumor effects seen with BXCL701 and the role of other targets of BXCL701, such as DPP 8 and 9, in mediating anti-tumor effects.

Acknowledgements The authors would like to thank Dr. Chunling Yi (Georgetown) for GFP/luciferase-expressing mT3–2D cells, the Animal Models Rodent Shared Resource, the Flow Cytometry and Cell Sorting Shared Resource and the Histopathology & Tissue Shared Resource at Lombardi Comprehensive Cancer Center, and the Division of Comparative Medicine at Georgetown University Medical Center for their support in this project.

References

1. Aggarwal R, Costin D, O’Neill V, et al. Abstract e17581: Phase 1b study of BXCL701, a novel small molecule inhibitor of dipeptidyl peptidases (DPP), combined with pembrolizumab (pembro), in men with metastatic castration-resistant prostate cancer (mCRPC). J Clin Oncol. 2020;38(15_suppl):e17581.

2. Fitzgerald A, Wang S, Agarwal V, et al. DPP inhibition alters the CXCR3 axis and enhances NK and CD8+ T cell infiltration to improve anti-PD1 efficacy in murine models of pancreatic ductal adenocarcinoma. J Immunother Cancer. 2021;9:e002837.

Ethics Approval This study was reviewed and approved by the Georgetown University Institutional Animal Care and Use Committee (GU IACUC), protocol number 2016–1254.