Inducible Programming of CAR T Cell Intrinsic Properties for Cancer Immunotherapy

Chimeric Antigen Receptor (CAR) T cell therapy is clinically approved advanced cancer immunotherapy approach with genetically engineered autologous (patient’s own) T cells. Although CAR T cell immunotherapy is a paradigm-shifting approach to treat cancer, the therapy is not always successful as evident from non-responding or relapsed disease. CAR T cell therapy is limited in solid tumors and in hematologic malignancies that induce dysfunctional T cell phenotypes and differentiation states. Additionally, dysfunctional T cells prevent successful manufacturing of CAR T cells in a substantial number of patients seeking CD19 CAR therapy. Genetic integration of relevant accessory molecules into CAR T cells is a promising approach to improve T cell functions and therapy outcomes. However, there remain both knowledge and translational gaps in (i) understanding which accessory molecules will provide optimal therapeutic efficacy when overexpressed in CAR T cells and (ii) how to genetically integrate them into CAR T cells in a clinically feasible manner. My proposal aims to address these issues to improve CAR T cells targeting CD19+ hematologic malignancies by (i) integrating the key transcription factor (TFs) that we identified through our preliminary studies as important candidates to improve T cell intrinsic properties and (ii) genetically integrate them into CAR T cells in an inducible manner (iTF-CAR T cells). In Objective 1, we will utilize a novel genetic platform called Uni-Vect that I developed at the University of Pennsylvania (UPenn). Uni-Vect will enable transient expression of TFs in CAR T cells, while we will also test the constitutive expression of the same TFs (cTF-CAR T cells). We will create CD19 targeting CAR T cells upgraded with ectopic expression of two key transcription factors and their variants to generate CAR T cell products with fitness profiles associated with a capacity to expand, persist and mediate cancer regression after infusion. i/cTF-CAR T cell approach will be further optimized for clinical translation with the use of a single step CRISPR/Cas9-mediated targeted gene integration approach that will simultaneously improve the safety and manufacturing of i/cTF-CAR T cells. In Objective 2 we will develop and validate comprehensive in vitro systems to evaluate and compare functional and phenotypic properties of i/cTF-CAR T cells. Utilizing advanced immunological methods including deep profiling of i/cTF-CAR T cells with RNA sequencing we will examine mechanisms leading to augmented fitness. Finally, we will investigate whether i/cTF-CAR T cells can improve activity against primary chronic lymphocytic leukemia (CLL) cells from patients treated with CART19 at UPenn and whether they can improve “expansion failure” T cells. Objective 3 is designed to test i/cTF-CAR T cell products in both xenograft and syngeneic preclinical mouse models. We will determine the impact of ectopic expression of variants of TFs on CAR T cell expansion, persistence, and anti-tumor activity in vivo. Then we will perform a comprehensive analysis of CAR T cells isolated from mice and examine CAR T cell phenotype, gene expression profiles and function to gain a better understanding of which key factors contribute to improved intrinsic properties. This will inform further developments of i/cTF-CAR T cell approaches. Together, the project aims at developing i/cTF-CAR T cells genetically equipped for improved expansion, persistence, and anti-tumor activity, and improving clinical CAR T cell manufacturing. Finally, we will investigate mechanisms by which TFs may improve CAR T cells and with this new understanding of CAR T cell biology, we will establish a foundation for the development of more effective cellular immunotherapies.