Co-expression of immune checkpoint receptors (ICRs) PD-1, TIM-3, and LAG-3 characterizes chronically activated and exhausted tumor-infiltrating T cells (TILs), suggesting their targeting may have applicability for the treatment of multiple cancer types. We previously reported improved tumor control in various syngeneic and humanized mouse models when treated with a combination of TSR-042 (αPD-1), TSR-022 (αTIM-3), and TSR-033 (αLAG-3) as compared to single or double combinations. Here, we are characterizing TILs from ovarian cancer tissues and their functional response to triple combination treatment. Immune profiling using flow cytometry confirmed expression of all three ICRs on TILs isolated from primary resections of ovarian cancer. Ex vivo re-stimulation of immune infiltrates with S. aureus enterotoxin B in presence of ICR-targeting antibodies led to increased secretion of IFN-γ and IL-2 when treated with TSR-042. Notably, triple combination of TSR-042, TSR-022, and TSR-033 further amplified cytokine release, indicating more effective TIL reinvigoration. To further understand the differential effects of triple combination treatment over PD-1 blockade, we analyzed αCD3/αCD28-stimulated and antibody-treated ovarian cancer TILs on a single cell level using a microfluidic IsoCode chip technology that allows for parallel detection and quantification of 32 secreted proteins from live single cells. Combining the amount of each protein secreted by polyfunctional T cells (co-secreting two or more proteins per cell) with the frequency of such cells results in a measurement of polyfunctional strength (PSI), a unique IsoCode-enabled metric that has been associated with improved response to ICR inhibition. TSR-042 increased the PSI of CD4+ and CD8+ TILs 1.4 and 1.5-fold over isotype control treatment. Importantly, triple combination treatment was able to significantly increase the PSI of both subsets by 2.9 and 3.7-fold, respectively (p < 0.001). For CD8+ TILs, this increase was mainly driven by an increase in the frequency of polyfunctional subsets, while for CD4+ TILs, the absolute amounts of secreted cytokines had a larger impact. Interestingly, both classical effector cytokines like Granzyme B and IFN-γ as well as other secreted factors like chemoattractant factors, implicated in the recruitment of multiple immune cell subsets to tumor tissue, contributed to T cell polyfunctionality. Taken together, triple ICR blockade targeting PD-1, TIM-3, and LAG-3 reinvigorated ovarian cancer TILs more effectively than PD-1 inhibition alone. This was mediated by increasing T cell polyfunctionality, which has been associated with improved anti-tumor activity and response to ICR inhibition. This data further supports the concept of triple combination checkpoint blockade as a treatment option for ovarian cancer.

Citation Format: Johanna K. Kaufmann, Brianna Flynn, Kevin Morse, Maria C. Speranza, Jing Zhou, Sridhar Ramaswamy, Sean Mackay, Kevin G. Coleman. Triple checkpoint blockade targeting PD-1, TIM-3, and LAG-3 reinvigorates ovarian cancer-infiltrating T cells by increasing T cell polyfunctionality and effector function [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 3242.