Abstract B170: Blockade of checkpoint programmed death 1 (PD1) delays tumor growth and induces early transferable protective immunity in a murine orthotopic pancreatic ductal adenocarcinoma model
Authors: Salazar Marcela d'Alincourt , Manuel Edwin , Tsai Weimin , D'Apuzzo Massimo , Goldstein Leanne , Blazar Bruce R. , Diamond Don J. 
Published: May 2016

Abstract
Despite the growing understanding of how PD1 signaling contributes to the immunosuppressed tumor microenvironment (TME), the mechanisms underlying tumor regression by antibody blockade remain unclear. In this study, we sought to identify the early events involved in mediating the anti-tumor effects of anti-PD1 monoclonal antibody (α-PD1 mAb) using an orthotopic mutant KrasG12D/p53R172H Pdx-Cre (KPC) pancreatic ductal adenocarcinoma (PDA) model. This model is clinically relevant because in addition to KPC cells constitutively expressing the programmed death 1 ligand 1 (PD-L1), it represents advanced tumors due to its fast growth and drug resistant phenotype.
When compared to hamster IgG isotype, single dose treatment with α-PD1 mAb (clone J43, 250µg/mouse) prolonged survival in C57BL6 mice bearing well established tumors (32 vs 76 days median survival time, respectively, p=0.0003). Mice that received α-PD1 mAb therapy presented transient moderate splenomegaly with increased spleen cellularity 72 hours following mAb administration. To determine if the transient splenomegaly represented a correlate of immune protection against tumor challenge, splenocytes from tumor bearing mice treated with IgG isotype or α-PD1 mAb for 72 hours, 7 days or 4 weeks were transferred into recipient tumor bearing mice that were pre-conditioned with cyclophosphamide (100mg/kg). Surprisingly, transferable protective immunity was only observed in the group that received splenocytes from donor mice treated with α-PD1 mAb for 72 hours (39.5 vs. 63 days median survival time for the IgG isotype vs. α-PD1 mAb treated group, p=0.042). Analysis by real time qPCR of tumor-infiltrating lymphocytes (TIL) obtained by selection of CD45+ cells using magnetic beads showed an increase in mRNA expression of IL-6 (4.6 fold), IL-8 (2.7 fold), IL-10 (6.2 fold), IFN-γ (3.8 fold) and TNF-α (4 fold) at 7 days, but not at 72 hours after PD1 blockade, suggesting that alterations in the splenic microenvironment influenced subsequent immune activation in the TME. Furthermore, individual depletion of immune cell subsets generally regarded as involved with the innate (Gr1+ cells, NK cells) or adaptive arms of the immune response (CD4 or CD8 T cells) were all capable of blocking the effect of α-PD1 mAb therapy in donor mice (p≤0.001). These results corroborate the importance of tumor recognition by the innate immune system in order to activate T cell-mediated killing, showing that stimulation of innate and/or adaptive immunity may play a direct role in controlling tumor progression.
Alternatively, gene expression analysis using the non-TIL enriched fraction (remaining CD45- cells) extracted from tumors 7 days after PD1 inhibition showed downregulation of vascular endothelial growth factor A (VEGF-A) and G1/S related cyclin mRNA, such as cyclin-A, -D2 and E, suggesting that inhibition of PD1 induced tumor cell cycle arrest occurred concomitantly with upregulation of cytokine expression. Downregulation of VEGF-A was confirmed by ELISA using whole tumor extracts (p=0.03). Even though IL-10 is an inhibitor of VEGF-A, in vitro experiments using KPC cells failed to demonstrate a direct connection. However, TME conditions are complex, and often non-reproducible in vitro. Based on our results, we challenge the notion that PD1 blockade inhibits tumor progression mainly as a result of activating canonical adaptive immune responses. In addition, induction of G1/S tumor cell cycle arrest could increase sensitivity to cytotoxic therapies, thus providing a rationale for drug combinations. Given the broad role of VEGF-A in fostering an immunosuppressive TME and as an endothelial/epithelial growth factor, further studies are underway to investigate the role of VEGF-A inhibition in α-PD1 mAb-mediated tumor regression.
Citation Format: Marcela d'Alincourt Salazar, Edwin Manuel, Weimin Tsai, Massimo D'Apuzzo, Leanne Goldstein, Bruce R. Blazar, Don J. Diamond. Blockade of checkpoint programmed death 1 (PD1) delays tumor growth and induces early transferable protective immunity in a murine orthotopic pancreatic ductal adenocarcinoma model. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B170.