Abstract 3208: Using T cell engineering plus triple checkpoint blockade to enhance the efficacy of adoptive immunotherapy in ovarian cancer
Authors: Anderson Kristin G. , Burnett Madison G. , Voillet Valentin , Chiu Edison Y. , Bates Breanna M. , Garcia Nicolas M. , Gottado Raphael , Greenberg Philip D. 
Published: August 2019

Abstract
Over 20,000 women are diagnosed with ovarian cancer in the United States annually, and over half will die within 5 years. Outcomes have changed little in the last 20 years, highlighting the need for therapy innovation. One promising new strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors; such T cell immunotherapies have the potential to control tumor growth without toxicity to healthy tissues. In considering candidate immunotherapy targets, we focused on mesothelin (MSLN), which contributes to invasive progression and malignancy in ovarian cancer but has limited expression in healthy cells. We showed that T cells engineered to express a human or mouse MSLN-specific high-affinity T cell receptor (TCRMSLN) can kill human patient-derived ovarian cancer cell lines or the murine ID8 cell line, respectively. In a disseminated ID8 tumor model, adoptively transferred TCRMSLN T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that the ovarian tumor microenvironment (TME) limits engineered T cell persistence and cancer cell killing.
To identify immunosuppressive features active in both the human and murine ovarian TME, we performed gene expression analyses. Deep transcriptome profiling confirmed similar gene expression signatures in human cancers and in the preclinical ID8 model. Among these, RNA sequencing detected PD-L1, Galectin-9 and Galectin-3, ligands for CD8 T cell-expressed PD-1, Tim-3 and Lag-3 ‘checkpoint’ receptors, respectively. We also measured PD-L1, Galectin-9 and Galectin-3 expression in human and mouse ovarian cancers by flow cytometry and immunohistochemistry, and multiplex immunohistochemistry of human ovarian tumors confirmed the presence of endogenous CD8 T cells expressing one, two or all three inhibitory receptors. Moreover, flow cytometry revealed that TCRMSLN-transduced T cells increase expression of the inhibitory receptors in ID8 tumors relative to cells in the spleen as early as three weeks after transfer, in association with decreased production of anti-tumor cytokines.
Based on our results, we hypothesized that we could overcome engineered T cell suppression via inhibitory receptor ligation. We treated tumor-bearing mice with TCRMSLN T cells plus anti-PD-1, anti-Tim-3 and/or anti-Lag-3 checkpoint-blocking antibodies, targeting up to three inhibitory receptors simultaneously. Triple checkpoint blockade dramatically increased anti-tumor cytokine production by intratumoral TCRMSLN T cells. As many solid tumors both overexpress MSLN as well as PD-1, Tim-3 and Lag-3, the use of multi-checkpoint blockade with engineered T cells has real potential to also enhance the efficacy of engineered adoptive T cell therapy against other malignancies.
Citation Format: Kristin G. Anderson, Madison G. Burnett, Valentin Voillet, Edison Y. Chiu, Breanna M. Bates, Nicolas M. Garcia, Raphael Gottado, Philip D. Greenberg. Using T cell engineering plus triple checkpoint blockade to enhance the efficacy of adoptive immunotherapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3208.