CAR T cell design: approaching the elusive AND-gate

Logic gating is a strategy for chimeric antigen receptor (CAR) T cell therapy to target tumor cells that lack tumor-specific antigens and prevent on-target/off-tumor toxicity.In a recent Nature article, Tousley et al. present an elegant CAR T cell ANDgate design to enhance CAR T cell safety without compromising efficacy.Chimeric antigen receptor (CAR) T cells are powerful immune effectors that engage cell surface antigens.The relative scarcity of broadly expressed, tumor-associated CAR targets has led many to investigate combinatorial antigen recognition patterns to address and reconcile the challenges of tumor heterogeneity and on-target/ off-tumor toxicity. 1CAR T cells may engage two antigens in different ways, resulting in T cell activation upon binding to either one of two antigens (OR-gate), to one antigen depending on the presence of the second (NOT and IF-BETTER gates), or to both antigens simultaneously (AND-gate) (Fig. 1a). 2 The AND-gate is especially attractive to achieve tumor specificity through co-recognition of two antigens, neither of which is tumor specific.This is a tall order, requiring two independent receptors for antigen which, bound in isolation, do not trigger T cell activation (thus averting undue toxicities), but together produce a signal that is sufficient to elicit an effective anti-tumor response. 2n early approach by Kloss et al. attempted to achieve this goal by designing a "weakened" CD3ζ-based CAR specific for antigen A, which was "rescued" by a chimeric costimulatory receptor (CCR) specific for antigen B. 3 In mice bearing three anatomically distant tumors expressing A only, B only or A + B, these CAR + CCR T cells preferentially eliminated the A + B tumor with a minimal impact on the other two.In a recent study, Tousley et al. 4 have elegantly deconstructed two complimentary signaling entities and achieved in their combination a signal strength comparable to that of a 4-1BB CAR.ZAP-70 is involved downstream of CD3ζ in T cell activation signaling cascade.ZAP-70 has been previously used as an intracellular CAR activation domain, but those CARs proved to be inefficient in lysing tumor cells in vitro, 5,6 possibly due to the difficulty of achieving adequate cell surface CAR expression when incorporating the whole ZAP-70 molecule 4 and to the self-inhibitory function of the SH2 domain. 7Tousley et al. successfully designed a ZAP-70-based CAR that bypasses the need for the CD3ζ module and maintains equal in vitro IL-2 production and cytotoxicity compared to a 4-1BB/CD3ζ-based second-generation CAR.This new ZAP-70 KIDB CAR design uses an intracellular structure containing a native linker, interdomain B, and kinase domains of Zap-70 with exclusion of the SH2 domain.Key T cell proximal (PLCγ1 and SLP-76), distal (AKT and ERK) and NFκB signaling activation studies show that ZAP-70 KIDB CAR T cells exhibit similar levels of phosphorylation during target cell stimulation and reduced base levels relative to CD28-and