Intrinsically disordered insert from SH2D2A rewires CD19 CAR signaling via Tyr290

Abstract

Chimeric antigen receptor (CAR) T cells have transformed cancer immunotherapy, yet their truncated or suboptimal intracellular signaling can limit therapeutic efficacy. To enhance proximal signaling of a CD19-targeted CAR, we systematically inserted short Lck-recruiting motifs derived from Lck-adaptor proteins into the CAR intracellular tail. Six candidate sequences from four adaptor molecules (SH2D2A, SKAP1, LAT, LIME), with a sequence from CD3{varepsilon}, known to affect CAR functionality, as a positive control, were tested for expression and functional impact. Three CAR constructs (containing SH2D2A, LAT and LIME1 sequences respectively) displayed reduced surface expression, but only SH2D2A elicited a pronounced rewiring of CAR T cell phenotype following co-culture with CD19+ tumor lines. SH2D2A CAR T cells showed increased CD27 and CD56 expression and reduced expression of effector-associated mediators including granzyme B, IL-2, TNF, and IFN{gamma}. Through systematic mutagenesis and comparative phenotyping of SH2D2A CAR variants, we identified SH2D2A tyrosine 290 (Tyr290) as the critical residue mediating both the altered signaling phenotype and the low surface expression. Additionally, mutation of Tyr254 in the LIME1 CAR restored surface expression in Jurkat T cells, indicating insert- and context-dependent effects on receptor surface expression. Collectively, these results demonstrate that short, intrinsically disordered adaptor-derived sequences -- and single tyrosine residues within them -- can profoundly reprogram CAR signaling and expression.