Epigenetic modulation of cellular aging to improve CAR-T therapy for solid tumors

Abstract CAR-T cell therapy has had incredible clinical success in the treatment of hematological malignancies. However, very limited activity against solid tumors has been achieved so far, despite targeting a variety of antigens and tumor types. Importantly, solid tumors account for more than 90% of all cancers, affecting more than 16 million Americans in 2020. The goal of Dorian Therapeutic’s SBIR proposal is to investigate the impact of reducing cellular senescence in CAR-T cells to improve solid tumor targeting. We have proven that T cells age the equivalent of 30 years during CAR-T manufacturing, which is detrimental to their ability to expand and kill cancer cells. Moreover, solid tumors are characterized by a strong immunosuppressive and pro-senescent microenvironment, strongly challenging T cells effector functions. Old T cells are particularly prone to be suppressed and die due to the hostile microenvironment. Rejuvenating T cells by reducing cellular aging at the epigenetic level is a novel strategy to improve T cell fitness and clinical outcomes. The goal of this project is to identify a shRNA-based genetic cassette targeting the epigenetic regulator USP16 to increase CAR-T cell fitness for the treatment of solid tumors. Dorian’s founders identified USP16 at Stanford University as an epigenetic regulator able to reduce senescence by enhancing stem cell self-renewal in multiple tissues. USP16 is a deubiquitinating enzyme responsible for the removal of ubiquitin moieties from histone H2AK119, increasing chromatin accessibility to pro-senescent programs. The company has developed in vitro and in vivo proof-of-concept that targeting USP16 by means of a shRNA co-expressed within the CD19.CAR construct reduces T cell aging and increases stem cell memory (Tscm) frequency upon manufacturing, without affecting proliferation. Most importantly, the engineered CAR-T cells resulted in improved tumor killing in a preclinical mouse model of leukemia. The Aims of this proposal are to (1) Create an in vitro model to study the effect of tumor microenvironment on T cells aging and functions, (2) Identify genetic cassettes targeting USP16 and increasing T cell fitness in vitro, and (3) Demonstrate that engineered CAR-T cells expressing a shRNA for USP16 have a better anti-tumor activity in a tumor model of osteosarcoma and in a 3D system of patient-derived tumor cells. At the successful completion of this SBIR project we will have identified the best genetic cassettes targeting USP16 and improving T cell fitness ready for toxicology studies (tumorigenicity and cytokine release syndrome (CRS) studies).Public Health Relevance CAR-T cell therapy achieved unprecedented clinical responses for hematological cancer, however success in solid tumors is very limited. We identified a novel target whose inhibition promotes self-renewal while blocking senescence, rejuvenating CAR-T cells used for therapy. The outcome of this project will be CAR-T cells that are lead to long-lasting clinical improvement in solid tumors.