Fig. 1. Hinge region optimization of HLA-restricted MANA-targeting CARs.

a Illustration summarizing the CAR designs employed in this study. Colors indicating hinge region variations: CD8α, blue; CD28s (short CD28), green; CD28l (long CD28), pink; mutFc (mutant Fc), orange. b T cells harboring each of the three MANA-targeting CARs, with the indicated hinge regions, were co-incubated with target cells loaded with a titration of the corresponding mutant and WT peptides. The following target cells were employed: T2 cells expressing endogenous HLA-A*02:01 for KRAS, T2A3 cells overexpressing HLA-A*03:01 for CTNNB1, and HLA-B*07:02-transfected COS-7 cells for IDH2. T-cell activation, as assessed by IFN-γ release, was measured by ELISA. Data are representative of three independent experiments. c T cells harboring IDH2^R140Q-HLA-B*07:02-targeting scFvs, grafted onto a CD8α-hinged CAR, were co-incubated with COS-7 cells transfected with HLA-B*07:02 and either full-length IDH2^R140Q or IDH2^WT. T-cell activation, as assessed by IFN-γ release, was measured by ELISA. Data are representative of three independent experiments. d 2Q1-Fab binding to IDH2^WT-HLA-B*07:02 was measured by multi-cycle kinetics using SPR with increasing concentrations (6.25, 12.5, 25, 50, 100, 200, 400, 800 nM) of purified 2Q1-Fab (N = 2). The curves were fit with a 1:1 binding model to calculate the listed K_D. e Same as (d) using IDH2^R140Q-HLA-B*07:02. Source data are provided as a Source data file.