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. Author manuscript; available in PMC: 2021 Nov 5.
Published in final edited form as: Nat Cancer. 2021 Sep 23;2(9):904–918. doi: 10.1038/s43018-021-00244-2

Figure 8. Dual targeting, split signaling and one single CD3ζ endodomain prevent tumor escape due to antigen loss.

Figure 8.

(a) Flow cytometry histogram showing the expression of GD2 and B7-H3 in the NB cell line SH-SY5Y. Representative of 3 independent experiments. (b) Quantification of the GD2 density on the cell membrane of CHLA-255, LAN-1 and SH-SY5Y cells as measured by flow cytometry. The numbers within bars indicate the calculated number of GD2 molecules on the cell membrane of each cell line. Representative of 2 independent experiments. (c) Quantification of residual NB cells labelled with GFP and co-cultured with CD19.28ζ, GD2.28ζ and GD2.28ζ/B7-H3.BB CAR-T cells at the T cell to tumor cell ratio of 1 to 5. On day 5, NB cells (GFP+) and CAR-T cells (CD3+) were enumerated by flow cytometry. Data are shown as individual values and the mean + SD, n = 10 independent co-culture with CAR-T cells generated from 10 different donors; **p = 0.0011, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value. (d,e) Summary of IFN-γ (d) and IL-2 (e) released by CAR-T cells in the culture supernatant after 24 hours of co-culture with NB cells as measured by ELISA. Data are shown as individual values and the mean + SD, n = 10 independent co-culture with CAR-T cells generated from 10 different donors; ***p = 0.0001 for CD19.28ζ vs. GD2.28ζ/B7-H3.BB and ***p = 0.0002 for GD2.28ζ vs. GD2.28ζ/B7-H3.BB in (d), ****p <0.0001 in (e) by one-way ANOVA with Tukey’s multiple comparison test adjusted p value. (f) Schema of the SH-SY5Y metastatic xenograft NB model in NSG mice inoculated intravenously via tail vein with 5 x 105 of FFLuc-SH-SY5Y cells and treated 7 days later with 1 x 107 CD19.28ζ, GD2.28ζ and GD2.28ζ/B7-H3.BB CAR-T cells intravenously. (g,h) Representative tumor BLI images (g) and BLI kinetics (h) of FFLuc-SH-SY5Y tumor growth in the metastatic xenograft NB models shown in (f) (n = 4 or 5 mice/group). (i) Kaplan-Meier survival curve of mice in (g,h) (n = 4 or 5 mice/group); **p = 0.0027 by Log-rank test. (j) GD2 and B7-H3 expression levels in tumor cells collected from mice treated with CD19.28ζ or GD2.28ζ CAR-T cells were analyzed by flow cytometry at the time of the euthanasia. Representative of 3 independent experiments.