Fig. 5: A CD19/CD22 Bi-ChTCR confers T cell recognition of both CD19 and CD22.

a, Bispecific CD19/CD22 ChTCR, monospecific CD19 and CD22 4-1BB/CD3ζ CARs, and a bispecific CD19/CD22 CAR⁷. The CD19-specific FMC63 scFv and the CD22-specific 9A8 scFv were used for the Bi-ChTCR; FMC63 and m971 scFvs were used in all CARs. Image created using BioRender.com. b, Flow plots of transduced and TCRαβ^KO CD8⁺ T cells stained with anti-TCRαβ and rCD19 (top) or rCD22 (bottom). c,d, Geometric mean ± s.d. of rCD19-PE (c) or rCD22-APC (d) binding to T cells expressing the indicated receptors (n = 3 independent experiments). P values were calculated using a two-way ANOVA. e, Left: TIRF microscopy of CD19/CD22 Bi-ChTCR T cells interacting with a lipid bilayer functionalized with ICAM-1-AF488 (green), CD19-AF647 (blue) and CD22-AF555 (red). Scale bar, 10 μm. Right: normalized mean intensity of ICAM-1-AF488, CD19-AF647 and CD22-AF555 staining across cell radiuses in synapses. Dots represent the mean at each position, with a trend line (n = 100 cells). f, IL-2 concentration in supernatants after coculture of T cells expressing the indicated receptors with Nalm-6 WT, Nalm-6 CD22^KO, Nalm-6 CD19^KO and Nalm-6 DKO cells. Data are shown as the mean ± s.d. for T cells from three independent healthy donors. P values were calculated using a two-way ANOVA. g, Flow histograms of CTV dilution after coculture of T cells expressing the indicated receptors with Nalm-6 cells expressing CD19 and CD22 or with KO for one or both targets. h, Frequency of divided T cells (CTV^low cells) expressing the indicated receptors. Data are shown as the mean ± s.d. from three independent donors. P values were calculated using a two-way ANOVA. i, Schematic of the NSG mouse model. Mouse icon created with BioRender.com. j, Representative bioluminescence images of Nalm-6 GFP-ffLuc⁺ tumor-bearing mice treated with 2 × 10⁶ T cells expressing the indicated CARs or the Bi-ChTCR. k, Tumor burden (mean ± s.d. radiance (photons per s per cm² per sr)) in each treatment group (n = 3 mice per control group, 5 mice per CAR treatment group and 8 mice in the Bi-ChTCR treatment group). l, Kaplan–Meier survival of mice treated with CARs or Bi-ChTCRs (n = 3–8). P values were calculated using a log-rank test m,n, Flow plots (m) and pie charts (n) showing CD19 and CD22 expression on Nalm-6 GFP-ffLuc⁺ tumor cells collected from the bone marrow at killing, for each treatment group (n = 1–4 mice per group as indicated in the figure).

Source data