Fig. 2: ICOS/4-1BB co-stimulatory bispecific CAR T exhibit broad antigen recognition and enhanced cytotoxicity.

a Schematic illustration of the K562 cell line model expressing individual or triple combinations of CD19 (purple), CD20 (red), and CD22 (yellow) antigens. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6. b Bar chart depicting the percentage expression of each antigen in K562 cell lines, both individually and in combination. c–f Line graph of cytotoxicity assays showing antigen-specific killing of K562 target cells. All tested constructs surpassed the performance of second-generation monospecific CD19 (m19) CAR T cells (n = 3 biologically independent samples). g Heatmap showing comparison of proliferation rates for bispecific; b20/19 or b22/19, and trispecific; t20/19/22 CAR T cells, represented as fold expansion up to Day 17 with respect to the baseline at the time of cell seeding. h Schematic of the Raji WT cell line platform expressing CD19 (purple), CD20 (red), and CD22 (yellow) antigens, edited using CRISPR-Cas9 to generate knockout variants. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6. i, j Line graph of cytotoxicity assays demonstrating the superior efficacy of b20/19 CAR T cells in eliminating antigen-negative Raji variants, compared to m19 CARs (n = 5 biologically independent samples). k Schematic representation of the tumor rechallenge (TR) model using the Raji WT cell line (Raji^WT). Gray circles represent initial engraftment and monitoring phases, pink circle shows the first incubation with Raji^WT, while purple circles indicate the timing of the RajiCD19^−/− rechallenge. l Heatmap representation of TR model showing IFN-γ secretion (pg/mL), percentage of tumor lysis (1:10; T: E), and the number of CAR T cells detected on days 7, 9, 11, 15, and 17 post-rechallenge (n = 5 biologically independent samples). Data represent mean ± SEM. Source data are provided as a Source Data file.