Fig. 3: Epitope editing allows for TCR-based receptors to be used in TCR SEEDs.

a, Diagram of a TCR and a HIT. b, Diagram of a TRAC exon-targeted HDRT encoding a HIT. c, Flow cytometry of anti-TCR (BW242) binding and HIT expression (anti-mouse F(ab′)₂) in T cells with a HIT at TRAC (n = 1 donor). d, Screening workflow for epitope mapping. e–g T cells were edited to express a library of mutated HITs then purified with BW242. BW242 binding and HIT expression were quantified with flow cytometry before (e) and after (f) purification. HIT⁺ BW242^– cells were sorted (blue box) for sequencing (n = 1 donor). g, Enrichment of mutations in HIT⁺BW242⁻ cells (blue box in f) compared to the original library (e). Dots represent the average of tested codons for an amino acid. h, Flow cytometry of BW242 binding and HIT expression in T cells edited with a HIT β112 saturation mutagenesis pool. Boxes indicate sorted populations. i, Enrichment of mutations in HIT⁺BW242⁻ cells versus HIT⁺BW242⁺ cells. Dots represents enrichment for a single codon (n = 1 donor). Lines display the average enrichment for an amino acid. j, BW242 binding for HIT⁺ T cells edited with HIT variants (n = 3 donors) compared with average enrichment scores (from i). The black line indicates the BW242 median fluorescence intensity (MFI) for TCR^– cells. The MFI s.e.m. is represented by bars (HIT mutants) or the shaded gray area (TCR^–). k, Diagram of a TRAC SEED HDRT encoding an epitope-edited HIT. l,m, T cells were edited with a HIT SEED (+M3814) and then purified with BW242 (n = 3 donors) l, Flow cytometry of BW242 binding and HIT expression m, Percentage of BW242⁺ and BW242⁻HIT⁺ cells. n, Cytotoxic activity of T cells with a nonmodified HIT (blue), epitope-edited HIT (red) or TCR knockout (gray) against Nalm6 lines (technical triplicate; data shown from one of four donors). Histograms show Nalm6 CD19 expression (flow cytometry). Unstained cells are shown in gray. Data are displayed as the mean ± s.e.m. T, target; E, effector.

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