Shubhabrata Majumdar ^1,2 Hilda Echelibe ¹ Maria Bettini ¹ Matthew L Bettini ^1*

• ¹ The University of Utah, Salt Lake City, United States
• ² Baylor College of Medicine, Houston, Texas, United States

Chimeric antigen receptor (CAR) expressing T-cells have shown great promise for the future of cancer immunotherapy with the recent clinical successes achieved in treating different hematologic cancers. Despite these early successes, several challenges remain in the field that require to be solved for the therapy to be more efficacious. One such challenge is the lack of long-term persistence of CD28 based CAR T-cells in patients. Although, CD28 based CAR T-cells elicit a robust acute anti-tumor response, they are more prone to early exhaustion, terminal differentiation and cell death due to their strong signaling patterns. Hence attenuation of signaling strength in CD28 based CARs is an accepted strategy to improve long-term CAR T-cell function and persistence in patients. Previous studies with the conventional T-cell receptor (TCR) have suggested that manipulation of CD3 immunoreceptor tyrosine-based activation motif (ITAM) sequences can alter TCR signaling strength. Based on these studies, we have designed 2 nd generation murine anti-CD19 CD28 based CARs with restricted CD3 ITAM diversity of sequences while maintaining a multiplicity of three. They are called AAA, BBB and CCC based on which CD3 ITAM they express. The goal of the study is to understand the nonredundant signaling properties of the individual CD3 ITAMs and their effect on CAR T-cell function. We hypothesize that the individual CD3ζ ITAMs will exhibit unique signaling properties in the restricted CARs which may allow for optimization of CAR signaling and improve CAR T-cell persistence and function. Testing each of these constructs, we observed that AAA CARs had stronger signaling strength compared to BBB and CCC CARs. The signaling differences were reflected in their functional activation profiles with T-cells expressing ζCCC CARs exhibiting less exhaustion and differentiation.