Ror1 artifacts12/27/2023 ![]() In vitro cytokine production and cytotoxicity experiments found that LINK CAR T cells show some degree of leakiness by responding to target cells that express only one of the two antigens. The LINK CAR design comprises an scFv (antigen A)–CD28 TM–LAT chain and a second scFv (antigen B)–CD8 TM–SLP-76 chain, which are co-transduced into T cells. 4 The authors created a split CAR design that they termed “LINK”, which encompasses two different scFvs, LAT and SLP-76, to generate AND-gated CAR T cells intended to only respond to co-expressed antigens (Fig. CRISPR-Cas9 knockout experiments showed that both LAT and SLP-76 are essential for CAR T cell effector function. These findings suggest that ZAP-70 KIDB may yield better therapeutic efficacy owing to lower tonic signaling and ensuing T cell exhaustion, which was supported by findings in a preclinical animal model.ĭuring T cell activation, ZAP-70 phosphorylates both LAT and SLP-76, which then form a scaffold for downstream signal transduction. 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 4-1BB-based second-generation CARs. 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. 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. 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. ZAP-70 is involved downstream of CD3ζ in T cell activation signaling cascade. 4 have elegantly deconstructed two complimentary signaling entities and achieved in their combination a signal strength comparable to that of a 4-1BB CAR. 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. 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. 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. 2 The AND-gate is especially attractive to achieve tumor specificity through co-recognition of two antigens, neither of which is tumor specific. 1 CAR 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. 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. Chimeric antigen receptor (CAR) T cells are powerful immune effectors that engage cell surface antigens.
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