Abstract 3241: Single-cell RNA and TCR sequencing of tumor infiltrating lymphocytes identifies changes in pancreatic ductal adenocarcinomas following neoadjuvant treatment with combined anti-PD-1 blocking and anti-CD137 agonist therapy
Authors: Tandurella Joseph A. , Mitchell Jacob T. , Montagne Janelle M. , Christenson Eric , Zhu Qingfeng , Danilova Ludmilla V. , Favorov Alexander V. , Ho Won J. , Kagohara Luciane T. , Loth Melanie , Lim Su Jin , Zheng Rui , Gai Jessica , Mitchell Sarah , Sidiropoulos Dimitrios N. , Hou Wenpin , Xu Yao , Wang Hao , Zimmerman Jacquelyn W. , Yegnasubramanian Srinivasan , Anders Robert , Jaffee Elizabeth M. , Zheng Lei , Fertig Elana J. 
Published: April 2023

Abstract
Introduction: Pancreatic ductal adenocarcinomas (PDACs) are immunogenically “cold” tumors that lack effector T cell infiltration associated with immune checkpoint inhibitor (ICI) responsiveness. Neoadjuvant immunotherapy clinical trials facilitate understanding of the complexities of combination treatment strategies on immunosuppressive mechanisms. Our recent multi-omics analysis of a neoadjuvant trial comparing the GVAX vaccine alone or with nivolumab and supporting preclinical data uncovered crosstalk between CD8+CD137+ T cells and neutrophils as an axis for therapeutic intervention. These studies have formed the foundation for the addition of the CD137 agonist, urelumab, to our combination regimen to improve T cell activation in PDACs.
Methods: Patients were enrolled on NCT02451982 and initiated treatment 2 weeks prior to surgical resection. We compared specimens from 3 treatment arms for downstream analysis: Arm A: an allogeneic whole cell vaccine, GVAX (n=7); Arm B: GVAX and nivolumab (n=4); Arm C: GVAX, nivolumab, and urelumab (n=4). Samples underwent dissociation and Percoll separation to enrich for immune cells, followed by single-cell RNA- and TCR-seq using 10X Genomics. Computational analysis was performed for cell type classification with Seurat, differential expression with DESeq2 of pseudobulk data, cell-cell communication with Domino, and TCR-seq analysis with scRepertoire.
Results: Single-cell RNA-seq analysis identified changes in immune cell proportions and immune cell states across treatment arms. Alignment of scTCR-seq to RNA data showed greater CD8+ T cell expansion following triple agent therapy. Gene set enrichment identified changes in CD8+ T cell, tumor associated macrophage (TAM), and B cell responses to cytokine signaling, locomotion, amino acid metabolism, and cell adhesion pathways in Arm C. Domino analysis demonstrated increases in the naïve T cell signal CCR7 in Arm A and integrin ligand signal ICAM1 in Arm C between the CD8+ T cells and other cell types present in PDACs. CCR7 expression was significantly downregulated in CD8+ T cells in Arm C whereas CCL5, a T cell agonist cytokine, and LCP2, a TCR signaling activator and T cell receptor which is also required for integrin signaling, were significantly upregulated in CD8+ T cells in Arm C. The increased integrin ligand signaling via ICAM1 and LCP2 in Arm C is also associated with changes in CD8+ T cell motility observed from the differential expression analysis.
Conclusions: Analysis of combination GVAX, PD-1 inhibition and CD137 agonist therapy demonstrated changes in multiple immune cell proportions and their functional pathways. These data provide new evidence that PDACs can become T cell rich and respond to combination immunotherapies.
Citation Format: Joseph A. Tandurella, Jacob T. Mitchell, Janelle M. Montagne, Eric Christenson, Qingfeng Zhu, Ludmilla V. Danilova, Alexander V. Favorov, Won J. Ho, Luciane T. Kagohara, Melanie Loth, Su Jin Lim, Rui Zheng, Jessica Gai, Sarah Mitchell, Dimitrios N. Sidiropoulos, Wenpin Hou, Yao Xu, Hao Wang, Jacquelyn W. Zimmerman, Srinivasan Yegnasubramanian, Robert Anders, Elizabeth M. Jaffee, Lei Zheng, Elana J. Fertig. Single-cell RNA and TCR sequencing of tumor infiltrating lymphocytes identifies changes in pancreatic ductal adenocarcinomas following neoadjuvant treatment with combined anti-PD-1 blocking and anti-CD137 agonist therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3241.