Fig. 1: Construction and functional validation of mono- or bispecific CAR T cells targeting BCMA or/and CD19 in vitro.

a Schematic diagram of the CD19/BCMA-targeting CAR constructs used in the current study. Bispecific BC19 or 19BC CAR by connecting an anti-BCMA and anti-CD19 scFv to a second-generation format containing 4-1BB. scFv Single-Chain Variable Fragment, TM Transmembrane Domain. b APC-labeled protein L was used to evaluate CAR expression in CD19-, BCMA-, BC19- or 19BC CAR T cells, results of 1 representative experiment are shown (n = 3). c Naïve, central memory (CM), effector memory (EM), and effector T cells were quantified in the infusion product (n = 3, biologically independent samples). d The growth curve of CAR T cells during in vitro manufacturing was drawn, results of 1 representative experiment are shown (n = 3). e CAR T cells were labeled with CTDR and then cocultured with Nalm6 (left, n = 3, biologically independent samples) or U266 (right, n = 3, biologically independent samples) cells. % CTDR- cells represent CAR T cell-mediated cytotoxicity. f ELISA was used to evaluate levels of interferon γ (IFN-γ) in the supernatants (n = 3, biologically independent samples). one-way ANOVA followed by Tukey’s multiple comparisons test was used for multiple comparisons with BC19 as a comparison group. g SKOV3 (left), SKOV3-BCMA (middle), and SKOV3-CD19 (right) cells were used as target cells to evaluate the specific cytotoxicity of CAR T cells. The E:T ratio is 3:1 (up) or 1:3 (down), results of 1 representative experiment are shown (n = 3). Data are presented as mean ± SD, a two-tailed Student t-test was performed for two group comparisons. One-way ANOVA analysis of variance was used to determine the statistically significant difference for multiple group comparisons. Statistics: one-way ANOVA followed by Tukey’s multiple comparisons tests (c, d, f). Source data are provided in the Source Data file.