Abstract B59: IL-6 regulates CTLA4 expression on CD4+ T-cells and dual antibody blockade of IL-6 and CTLA4 leads to tumor regression in an orthotopic murine model of pancreatic ductal adenocarcinoma
Authors: Ware Michael B. , McQuinn Christopher , Mace Thomas A. , Bowers Jacob , Shakya Reena , Farris Brad , Young Gregory , Carson William E. , Paulos Chrystal M. , El-Rayes Bassel , Lesinski Gregory B. 
Published: March 2020

Abstract
Immune checkpoint blockade therapy has shown promise in many cancers. However, pancreatic ductal adenocarcinoma (PDAC) has proven refractory to most targeted and immune-based therapies. Previous research by our group and others demonstrated immune suppression in PDAC driven by activated fibroblasts, known as pancreatic stellate cells (PSCs). Specifically, our lab has demonstrated expansion of myeloid-derived suppressor cells (MDSCs) by PSCs in an interleukin-6 (IL-6) and STAT3 dependent manner (Mace T, Cancer Res 2013), while IL-6 blockade could enhance the in vivo efficacy of anti-PD-L1 antibody in multiple murine PDAC models (Mace T, Gut 2016). In addition, analysis of blood from n=73 treatment-naïve patients with metastatic PDAC revealed higher plasma IL-6, and circulating CD8+CTLA4+ T cells were associated with worse overall survival (Farren M, Clin Cancer Res 2015). Given these data, we hypothesized that blocking IL-6 in pancreatic cancer could shift the balance of effector and regulatory immune responses and enhance the efficacy of anti-CTLA4 based therapies. Human PBMCs were isolated from healthy donor blood and RossetteSep T-cell enrichment cocktail was used to enrich for CD3+ T cells. Enriched populations or bulk PBMCs were stimulated with IL-6. Cells were analyzed by flow cytometry for CTLA4 expression. In vivo studies were performed by orthotopically injecting KPC-luciferase cells into the pancreas of C57BL/6 mice. Tumor growth was monitored by bioluminescent imaging (BLI), and mice were treated with antibodies against IL-6 and CTLA4 or isotype controls. At the conclusion of in vivo studies, pancreas, liver, and spleen were harvested and analyzed using a combination of immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry. Stimulation of human PBMCs or enriched T cells with IL-6 resulted in upregulation of CTLA4 on CD4+CTLA4+ T cells and increased the percentage of CD4 cells positive for CTLA4. We observed no change associated with CD8+ T cells. Dual antibody blockade of IL-6 and CTLA4 in an orthotopic murine model of PDAC led to significant tumor regression as determined by BLI in comparison to isotype treatment, with significantly lower end study tumor weights. Addition of CXCR3 blocking antibodies to mice receiving the combination therapy abrogated this antitumor response. IHC analysis of tumors showed combination-treated tumors to have higher infiltration of CD8+ cells in comparison to isotype or single-agent treatment. We demonstrate, for the first time, the ability of IL-6 to regulate CTLA4 expression on CD4+ T cells, unveiling a new dynamic in PDAC-associated immune suppression. Our in vivo data demonstrate the ability of dual IL-6 and CTLA4 blockade to lead to PDAC tumor regression. These data, in combination with our previous research, strongly suggest that blocking IL-6 in the clinical setting will enhance the antitumor effect of anti-CTLA4 antibodies and other immune-based therapies in PDAC.
Citation Format: Michael B. Ware, Christopher McQuinn, Thomas A. Mace, Jacob Bowers, Reena Shakya, Brad Farris, Gregory Young, William E. Carson III, Chrystal M. Paulos, Bassel El-Rayes, Gregory B. Lesinski. IL-6 regulates CTLA4 expression on CD4+ T-cells and dual antibody blockade of IL-6 and CTLA4 leads to tumor regression in an orthotopic murine model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B59.