Extended Data Fig. 10: In vivo functionality of T cells with non-viral TCR replacement.

a, Diagram of in vivo human antigen-specific tumour xenograft model. NSG mice (8–12 weeks old) were seeded with 1 × 10⁶ A375 cells (human melanoma cell line; NY-ESO-1 antigen⁺ and HLA-A*0201⁺) subcutaneously in a shaved flank. Primary human T cells edited to express an NY-ESO-1 antigen-specific TCR were generated (either by lentiviral transduction or non-viral TCR replacement), expanded for 10 days after transduction or electroporation, and frozen. Either a bulk-edited population was used (b, c) or an NY-ESO-1 TCR⁺-sorted population was used (d–f). At 7 days after tumour seeding, T cells were thawed and adoptively transferred via retro-orbital injection. b, Two days after transfer of 5 × 10⁶ bulk non-virally targeted T cells (~10% TCR⁺ NY-ESO-1⁺ (red), ~10% TCR⁺ NY-ESO-1⁻ (orange), and ~80% TCR⁻ NY-ESO-1⁻ (green), see Fig. 4b), NY-ESO-1⁺ non-virally edited T cells preferentially accumulated in the tumour versus the spleen. n = 5 mice for each of four human T cell donors. c, Ten days after transfer of 5 × 10⁶ bulk non-virally targeted CFSE-labelled T cells, NY-ESO-1⁺ TCR⁺ cells showed greater proliferation than TCR⁻ or TCR⁺NY-ESO-1⁻ T cells, and showed greater proliferation (CFSE low) in the tumour than in the spleen. Ten days after transfer, TCR⁻ and TCR⁺ NY-ESO-1⁻ T cells were difficult to find in the tumour (Fig. 4g). d, Individual longitudinal tumour volume tracks for data summarized in Fig. 4h. Sorted NY-ESO-1 TCR⁺ T cells (3 × 10⁶) generated either by lentiviral transduction (black) or non-viral TCR replacement (red) were transferred on day 7 after tumour seeding and compared to vehicle-only injections until 24 days after tumour seeding. Note that the same data for vehicle control data are shown for each donor in comparison to lentiviral delivery (above) and non-viral TCR replacement (below). e, f, In these experiments, 17 days after T cell transfer (d), non-virally TCR-replaced cells appeared to show greater NY-ESO-1 TCR expression and lower expression of exhaustion markers. Transfer of both lentivirally transduced and non-viral TCR replaced cells showed reductions in tumour burden on day 24. In this experimental model, non-viral TCR replacement showed further reductions compared to the lentiviral transduction (Fig. 4h), potentially due to knockout of the endogenous TCR, endogenous regulation of expression of the new TCR, some difference in the cell populations amenable to non-viral versus lentiviral editing, or confounding variables in cell handling between lentiviral transduction and non-viral genome targeting. n = 4 (b), n = 2 (d–f), or n = 1 (c) independent healthy donors in 5 (b, c) or 7 (d–f) mice per donor with mean (b, e, f) and s.d. (b).

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