CR2/3 domain derived from viral receptor enabled the surface loading of oncolytic VSVΔ51 onto CAR-T cells. (A) Illustration of modified CAR structure for increasing specific loading of OVs on the cell surface of CAR-T cells. (B) Illustration for different strategies of EGFR-CAR structure: (1) CAR-CR2, (2) CAR-CR3, (3) CR2-CAR, (4) CR3-CAR, and (5) CR2-CR3-CAR (CR2/3-CAR). (C) Flow cytometry analysis for viral loading efficiency by measuring the viral capsid protein VSV-G onto EGFR–CAR-T model (n = 3 biological replicates). (D) Viral genes VSV-G and VSV-N were measured by qPCR in EGFR–CAR-T cells (n = 3). (E and F) Flow cytometry analysis for viral loading efficiency by measuring VSV-G expression onto the B7H3–CAR-T (E) and IL13Rα2–CAR-T (F). The left panel is the representative graph of flow cytometry analysis; the right panel is the quantitative analysis of the ratio of VSV-G–positive cells in CAR-T cells (n = 3 biological replicates). (G) Immunofluorescence analysis for viral loading efficiency by measuring the VSV-G expression in B7H3–CAR-T cells. Scale bar, 5 μm. (H) Scanning electron microscopy images of B7H3–CAR-T cells and CR2/3–B7H3–CAR-T cells loaded with VSVΔ51. Scale bar, 2 μm. (I) Illustration for analyzing the rate of viral entry. After preloading VSVΔ51 at 4°C, CAR-T cells were incubated at 37°C for the indicated time from 0 to 60 min and then were washed by protease K to clear the surface virus. (J) The rate of viral entry was calculated as the ratio of intracellular VSV-G expression to the total VSV-G expression before protease K washing. The expression of viral gene VSV-G was measured by qRT-PCR (n = 3 biological replicates). (K) The mRNA level of IFNB1, ISG15, and CXCL10 by qRT-PCR measurement. CAR-T cells were preloaded with VSVΔ51 (MOI = 10) or not for 8 h (n = 3 biological replicates). (L) Flow cytometry analysis for cell exhaustion of CAR-T cells by measuring the expression of Tim-3 and PD-1 in vitro (n = 3 biological replicates). CAR-T cells were preloaded with VSVΔ51 (MOI = 10) or not, then were incubated at 37°C for 12 h. Data represent the mean ± SD from three independent experiments (C–F and J–L). Statistics by one-way ANOVA with Tukey’s post hoc test (C, D, and J–L) or unpaired two-tailed Student’s t test (E and F). *, P < 0.05; **, P < 0.01; N.S., no significance.
Figure 1.

CR2/3 domain derived from viral receptor enabled the surface loading of oncolytic VSVΔ51 onto CAR-T cells. (A) Illustration of modified CAR structure for increasing specific loading of OVs on the cell surface of CAR-T cells. (B) Illustration for different strategies of EGFR-CAR structure: (1) CAR-CR2, (2) CAR-CR3, (3) CR2-CAR, (4) CR3-CAR, and (5) CR2-CR3-CAR (CR2/3-CAR). (C) Flow cytometry analysis for viral loading efficiency by measuring the viral capsid protein VSV-G onto EGFR–CAR-T model (n = 3 biological replicates). (D) Viral genes VSV-G and VSV-N were measured by qPCR in EGFR–CAR-T cells (n = 3). (E and F) Flow cytometry analysis for viral loading efficiency by measuring VSV-G expression onto the B7H3–CAR-T (E) and IL13Rα2–CAR-T (F). The left panel is the representative graph of flow cytometry analysis; the right panel is the quantitative analysis of the ratio of VSV-G–positive cells in CAR-T cells (n = 3 biological replicates). (G) Immunofluorescence analysis for viral loading efficiency by measuring the VSV-G expression in B7H3–CAR-T cells. Scale bar, 5 μm. (H) Scanning electron microscopy images of B7H3–CAR-T cells and CR2/3–B7H3–CAR-T cells loaded with VSVΔ51. Scale bar, 2 μm. (I) Illustration for analyzing the rate of viral entry. After preloading VSVΔ51 at 4°C, CAR-T cells were incubated at 37°C for the indicated time from 0 to 60 min and then were washed by protease K to clear the surface virus. (J) The rate of viral entry was calculated as the ratio of intracellular VSV-G expression to the total VSV-G expression before protease K washing. The expression of viral gene VSV-G was measured by qRT-PCR (n = 3 biological replicates). (K) The mRNA level of IFNB1, ISG15, and CXCL10 by qRT-PCR measurement. CAR-T cells were preloaded with VSVΔ51 (MOI = 10) or not for 8 h (n = 3 biological replicates). (L) Flow cytometry analysis for cell exhaustion of CAR-T cells by measuring the expression of Tim-3 and PD-1 in vitro (n = 3 biological replicates). CAR-T cells were preloaded with VSVΔ51 (MOI = 10) or not, then were incubated at 37°C for 12 h. Data represent the mean ± SD from three independent experiments (C–F and J–L). Statistics by one-way ANOVA with Tukey’s post hoc test (C, D, and J–L) or unpaired two-tailed Student’s t test (E and F). *, P < 0.05; **, P < 0.01; N.S., no significance.

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