The specific CD37-chimeric antigen receptor (CAR)-T cell activity against CD37-positive cells in vitro. (A). The structure of CD37-CAR. The mouse ScFv (single-chain variable fragment) was used with CD8 hinge, CD28 transmembrane/costimulatory domains, and CD3 zeta activation domain. CD8 s.p-CD8 alpha signaling peptide; TM-transmembrane. (B). FACS with mouse F(ab)2 antibody (mFAB) detected CAR-positive cells. (C). Real-time cytotoxicity assay (RTCA) showed specific killing activity of CD37-CAR-T cells against CHO-CD37 cells but not CHO cells (upper panels). Lower panels: Percent cytotoxicity calculated at the end of the experiment. Significantly high cytotoxicity was observed against CHO-CD37 for CD37-CAR-T cells. p < 0.0001, One-Way ANOVA followed by Sidak multiple comparisons test. (D). Interferon-gamma (IFN-γ) secretion by CD37-CAR-T cells against CHO-CD37 cells is significantly higher than against CHO cells. asterisk *, p < 0.0001, two-way ANOVA p <0.0001, followed by Tukey’s multiple comparison test. (E). Secretion of IFN-gamma by CD37-CAR-T cells is significantly higher with Raji cells than with CD37-negative K562 cells. asterisk *, p < 0.005, mCD37 CAR-T cells with Raji cells versus same CAR-T cells with K562 cells, Student’s t-test.