Engineering T cells to overcome inhibitory receptor signals that limit the efficacy of adoptive cell therapy against ovarian cancer - PROJECT SUMMARY Over 20,000 women are diagnosed with ovarian cancer in the United States annually, and over half will die within five years. Outcomes have changed little in the last 20 years, highlighting the need for more effective therapies. 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. My ongoing work targets the antigen mesothelin (Msln), which contributes to malignancy and invasive progression in ovarian cancer but has limited expression in healthy cells. I showed that T cells engineered to express a human or mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill human 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, data also revealed that the ovarian tumor microenvironment (TME) limits engineered T cell persistence and anti-cancer efficacy. Tumor-specific T cells express inhibitory receptors upon encountering antigen, reducing antitumor cytokine production. I detected the ligands for the PD-1, Tim-3 and Lag-3 inhibitory receptors (immune checkpoints) expressed in human and ID8 ovarian tumors. Moreover, tumor-infiltrating TCRMsln T cells expressed PD-1, Tim- 3 and Lag-3, which correlated with reduced cytokine production. I hypothesized that immune checkpoint blockade could overcome inhibitory receptor ligation-driven suppression of engineered T cells. Therefore, I 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, but treatment also produced greater off-tumor toxicities. I now propose to use preclinical mouse models (Aim 1) and a novel human slice culture system (Aim 2) to interrogate the transcriptomic and functional changes that occur in engineered T cells and the ovarian TME after checkpoint blockade. I plan to use T cell engineering to knock down endogenous inhibitory receptor expression in tumor-specific T cells and determine if anti-tumor function is improved without the immune- related toxicities observed with systemic combination checkpoint blockade. Many solid tumors overexpress Msln and share the same T cell inhibitory pathways. Therefore, the findings from these new studies will likely inform the development of clinically-relevant T cell engineering strategies that are more resistant to immune suppression within the solid TME of many malignancies.
