The Role of CD3z ITAM (Immunoreceptor Tyrosine-Based Activation Motif) diversity in CAR T cell Force and Function

Chimeric Antigen Receptors (CARs) are a combination of an extracellular single-chain variable fragment region (scFV) that recognizes tumor protein antigens in an MHC independent manner and an intracellular region that transduces extracellular stimuli similar to T Cell Receptor (TCR) signaling. Optimization of CAR constructs is of

high importance as current CAR therapies have significant drawbacks including CAR T cell toxicity from severe cytokine release syndrome, neurotoxicity and lack of CAR T cell persistence in vivo and expansion in vitro. There is a new focus on tuning CAR intracellular signals via the immunoreceptor tyrosine-based activation motifs

(ITAMs) to increase effector function and persistence. Each of the 3-zeta chain ITAM sequences are unique with distinct kinetics of phosphorylation including a hierarchy of specificity for Zap-70 upon phosphorylation. Our previous work determined that ITAM diversity was necessary for T cell development and attenuating TCR

signaling. Importantly, ITAM diversity has also been shown to be necessary for TCR-driven proliferation and cytokine production. However, the role of CAR zeta chain diversity in CAR signaling, persistence and function has not been fully explored. Interactions between TCR and pMHC occur at the interface of two cells in a two-dimensional (2D) membrane

microenvironment. Initial binding events including affinity and kinetics of the TCR/pMHC interaction, and the CAR/protein epitope interaction, will dictate binding stability and downstream signaling events including phosphorylation of the ITAMs. We have found that CARs expressing specific ITAM sequences (zeta-AAA, zeta-

BBB or zeta-CCC) generate differing amounts of Force and bond lifetime after interacting with CD19. Therefore, a CAR may act as a mechanosensor similar to a TCR discriminating the quality of the interaction with antigen as cellular derived forces are applied to the bond. Mechanistically, we hypothesize that finetuning the

combination of ITAM sequences will differentially regulate bond lifetime, phosphorylation and signal transduction leading to differential immune activation depending on the ITAM motif. Understanding the biology of ITAM specificity has important implications in “tuning” the activation of a CAR T cell without changing specificity.