Fig. 4

Construction and characterization of bi-specific scFv antibody (BsAb)- secreting CAR-T cells. (A) Schematic representation of Mock, PD-1 scFv, TREM2 scFv, and BsAb-secreting CAR constructs targeting CEA. CART-CEA was constructed as a control. (B) Efficient transduction of all constructs in mouse primary T cells as observed by fluorescence microscopy. Scale bar: 50 μm. (C, D) Flow cytometric assay analysis (C) and bar plot summary (D) indicating the transduction efficiency of all constructs in mouse primary T cells. (E) Western blot detecting the His-tag for CAR protein in mouse primary T cells. (F) Cytotoxicity of all constructed CAR-T cells against MC38 cells. All groups of CAR-T cells were co-cultured with MC38 cells at 1:1, 4:1, 8:1, and 16:1, ratios. MC38 cell lysis was detected by an LDH cytotoxicity kit. Data are expressed as means ± SD; * P < 0.05; **P < 0.01, by one-way ANOVA. CAR-T, chimeric antigen receptor-modified-T; PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; CEA, carcinoembryonic antigen; LDH, lactate dehydrogenase