Abstract B043: CD3ζ ITAM diversity determines chimeric antigen receptor signaling and function
Authors: Majumdar Shubhabrata , Echelibe Hilda , Bettini Matthew L. 
Published: October 2024

Abstract
CAR T-cells have shown great potential for the future of cancer immunotherapy after showing promising clinical results in treating several hematologic tumors. Despite these early successes, several challenges remain in the field. One such challenge is a lack of long-term persistence of CD28-based CAR T-cells in patients. Although CD28-based CAR T-cells mount a robust acute tumor killing response, they are more prone to early exhaustion, terminal differentiation and cell death due to their strong signaling patterns. Hence, attenuation of signaling strength of CD28-based CARs is considered a viable strategy for prolonging persistence. Previous work in conventional T-cells has suggested that restricting ITAM diversity to single ITAM sequences while maintaining same ITAM number can attenuate signaling strength to different extents. Based on this, we have designed ITAM diversity restricted CD28-based murine anti-CD19 CARs with the goals of optimizing CAR signaling strength and studying the individual signaling properties of the individual CD3ζ ITAM sequences. They are named as ζWT (conventional CD28-CD3ζ CAR), ζA, ζB and ζC CARs based on which individual CD3ζ ITAM they express in three copies. Our approach is slightly different from other ITAM related design manipulations where ITAM number has been reduced. We hypothesize that the individual CD3ζ ITAMs will exhibit non-redundant signaling properties in the diversity restricted CARs which will allow for optimization of CAR signaling and improve CAR T-cell persistence and function. To test this, we first interrogated the signaling differences of the individual CD3ζ ITAMs using western blots and Ca2+ signaling by flow cytometry in CAR T-cells stimulated with CD19+ beads or tumor cells. Our observations suggest ζB and ζC CARs have a lower signaling strength compared to ζWT and ζA. To understand how the signaling differences affect CAR-T function, we performed in vitro CAR-T:CD19+ tumor cell co-culture functional assays to analyze their specific cytotoxicity, degranulation and cytokine expression profiles. We observed ζB and ζC CAR-Ts have a weaker activation profile based on the CD107a degranulation assay and their cytokine expression profile. To follow this up, we performed an in vitro seven-day exhaustion assay with repeat stimulation using CD19+ beads to analyze their propensity for exhaustion. Again, ζB and ζC CAR-Ts had lower expression of exhaustion related inhibitory receptors suggesting they are less prone to exhaustion. In conclusion, we observe individual CD3ζ ITAM sequences have non-redundant signaling properties in terms of signaling strength. ζB and ζC CAR-Ts which have lower signaling strength have a weaker activation profile and are less prone to exhaustion. Based on these observations, we predict ζB and ζC will perform better long-term in vivo and provide better protection against tumor growth.
Citation Format: Shubhabrata Majumdar, Hilda Echelibe, Matthew L. Bettini. CD3ζ ITAM diversity determines chimeric antigen receptor signaling and function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B043.