Fig. 1
The Sd/Gv-based CAR effectively redirected T cells to eliminate target cells. A Schematic representation of the design of modular CAR by Sd/Gv platform. Sd: SDCatcher, Gv: GVoptiTag. B and C Coomassie blue staining and western blotting analysis of Gv-VRC01 scFv and Gv-CD5-CD30 scFvs. Anti-His antibody was used to confirm the expression and purity of each protein. D The Sd-28BBZ3-P2A-tCD19 elements were transduced into HEK293T cells, and the expression of CD19 indirectly indicated the expression of Sd-28BBZ3. Meanwhile, Gv-scFvs (VRC01 scFv or CD5-CD30 scFvs) were expressed as fusion proteins with a 6-Histidine tag. The cells expressing Sd-28BBZ3-P2A-tCD19 were incubated with Gv-scFv proteins, allowing for indirect detection of Gv-scFV binding to Sd on the cell surface through the presence of His tag. Immunofluorescence staining analysis of His-tagged Gv-scFv proteins conjugating to Sd-28BBZ3-P2A-tCD19 on HEK293T cells with anti-CD19 (red) and anti-His (green) antibodies. DAPI was used to stain the nuclei (blue). Scale bars represented 10 μm. E The Sd-28BBZ3-P2A-tCD19 elements were transduced into HEK293T cells. After 12 h, the culture medium was removed and the cells were cultured with fresh medium supplemented with 100 nM Gv-scFVs (VRC01 scFv or CD5-CD30 scFvs) proteins. Following an additional 12 h, the membrane proteins were extracted and the covalent binding of Sd-Gv was detected using western blot with anti-Gv/Sd antibodies. F and G Direct killing of target cell lines was performed utilizing the Cyto-Tox nonradioactive cytotoxicity kit with Jurkatgp160 (F) or Karpas 299 (G) cells as targets. The effector cells included the following cells. Control CD8+ T: CD8+ T cells transduced with empty vector. Sd-28BBZ3 CD8+ T: CD8+ T cells transduced with Sd-28BBZ3. Sd-28BBZ3 + Gv-scFv: CD8+ T cells transduced with Sd-28BBZ3 and incubated with proteins of Gv-scFvs (VRC01 scFv or CD5-CD30 scFvs). VRC01-CAR: CD8+ T cells transduced with conventional VRC01-CAR. CD5-CD30-CAR: CD8+ T cells transduced with conventional CD5-CD30-CAR. Different ratios of effector cells and target cells were co-incubated. Data represented as mean ± SD. N = 3 independent biological replicates. Statistical analysis was performed by two-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01