Extended Data Fig. 9: Endogenous TCR replacement strategy and functional characterization.

a–d, Schematic description of HDR template for endogenous TCR replacement by in-frame integration of a new TCR-β chain and a new variable region of a TCR-α chain at the TCR-α locus, and subsequent transcription and translation of the new TCR. e, HDR template for endogenous TCR replacement at the TCR-β locus. f, Multiplexed integration of a new TCR-α at the TCR-α locus and a new TCR-β at the TCR-β locus. See Supplementary Note 4 for detailed description of TCR replacement strategy. g, TCR mispair analysis after retroviral delivery or non-viral TCR replacement of an NY-ESO-1-specific TCR in gated CD4⁺ or CD8⁺ T cells. With viral introduction of the new TCR, an infected cell will potentially express at least four different TCRs (new TCR-α plus new TCR-β; new TCR-α plus endogenous TCR-β; endogenous TCR-α and new TCR-β; endogenous TCR-α plus endogenous TCR-β). Staining for the specific beta chain in the new introduced TCR (VB13.1) along with MHC-peptide multimer (NY-ESO) can provide a rough estimate of TCR mispairing by distinguishing between cells that predominantly expressed the introduced TCR (VB13.1⁺ NY-ESO⁺; new TCR-α and new TCR-β) versus those that expressed predominantly one of the potential mispaired TCRs (VB13.1⁺ NY-ESO⁻; endogenous TCR-α and new TCR-β). h, i, TCR replacement by targeting an entire new TCR into TRAC (a–d, also possible with a multiplexed knockout of TCRB), an entire new TCR into TRBC1/2 (f), or multiplexed replacement with a new TCR-α into TRAC and a new TCR-β into TRBC1/2. j, Functional cytokine production was observed selectively after antigen exposure in gated CD4⁺ T cells, similarly to gated CD8⁺ T cells (Fig. 4c). k, Non-viral TCR replacement was consistently observed at four days after electroporation in both gated CD8⁺ and CD4⁺ T cells across a cohort of six healthy blood donors. l, In a second cohort of six additional healthy blood donors, 100 million T cells from each donor were electroporated with the NY-ESO-1 TCR replacement HDR template and on-target gRNA/Cas9 (Fig. 4f). The percentage of CD4⁺ and CD8⁺ T cells that were NY-ESO-1 TCR⁺ was consistent over 10 days of expansion after electroporation. m, Over 10 days of expansion after non-viral genome targeting, CD8⁺ T cells showed a slight proliferative advantage over CD4⁺ T cells. n, The indicated melanoma cell lines were co-incubated with the indicated sorted T cell populations at a ratio of 1:5 T cells to cancer cells. At 72 h after co-incubation, the percentage cancer cell confluency was recorded with by automated microscopy (in which nuclear RFP marks the cancer cells). T cells expressing the NY-ESO-1 antigen-specific TCR, either by retroviral transduction (black) or by non-viral knock-in endogenous TCR replacement (red) both showed robust target cell killing only in the target cancer cell lines expressing both NY-ESO-1 and the HLA-A*0201 class I MHC allele. o, To ensure that target cell killing by non-viral TCR replacement T cells (red) was not due to either the gRNA or the HDR template used for TCR replacement alone, a matrix of on/off target gRNAs and on/off target HDR templates was assayed for target cell killing of the NY-ESO-1⁺ HLA-A*0201⁺ A375 cancer cell line (off-target gRNA and HDRT were specific for RAB11A–GFP fusion protein knock-in). Only cells with both the on-target gRNA as well as the on-target HDR template demonstrated target cell killing. p, Sorted NY-ESO-1⁺ TCR⁺ cells from a bulk T cell edited population (on-target gRNA, on-target HDR template) showed a strong dose–response effect for target cancer cell killing. Within 48 h, T cell to cancer cell ratios of 2:1 and greater showed almost complete killing of the target cancer cells. By 144 h, T cell to cancer cell ratios of less than 1:16 showed evidence of robust target cell killing. q, Target cell killing by non-viral TCR replacement T cells was due specifically to the NY-ESO-1-recognizing TCR⁺ cell population observed by flow cytometry after non-viral TCR replacement (Fig. 4b). Starting with the bulk edited T cell population (all of which had been electroporated with the on-target gRNA and HDR template), we separately sorted three populations of cells: the NY-ESO-1⁺ TCR⁺ cells (non-virally replaced TCR) (red), the NY-ESO-1⁻ TCR⁻ cells (TCR-knockout) (grey), and the NY-ESO-1⁻ TCR⁺ cells (those that probably retained their native TCR but did not have the NY-ESO-1-specific knock-in TCR) (orange). Only the sorted NY-ESO-1⁺ TCR⁺ population demonstrated target cell killing (4:1 T cell to cancer cell ratio). One representative donor from n = 2 (g, j) or n = 3 (h, i) independent healthy donors with mean and s.d. of technical triplicates (j). Mean and s.d. of n = 6 independent healthy donors (l, m) or of four technical replicates for n = 2 independent healthy donors (o–q) are shown. Mean and individual values for n = 2 independent healthy donors (n).