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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 5. MSLN and CSPG4 dual targeting CAR-T cells with split co-stimulation and shared CD3ζ show sustained T cell activation and proliferation in vitro and in vivo.

Figure 5.

(a) Schematic representation of retroviral vectors encoding CSPG4.BB, CSPG4.BBζ, MSLN.28ζ and MSLN.28ζ/CSPG4.BB CARs. scFv.763.74, single-chain variable fragment of the anti-CSPG4 monoclonal antibody 763.74; Amatuximab, single-chain variable fragment of the anti-MSLN monoclonal antibody amatuximab; CD8α, stalk and transmembrane region of human CD8α; CD28, intracellular domain of human CD28; 4-1BB, intracellular domain of human 4-1BB; CD3ζ, intracellular domain of human CD3ζ chain; F, Flag-tag. (b) Flow cytometry histograms showing the expression of MSLN and CSPG4 in the human mesothelioma cell line H2052. Representative of three independent experiments. (c, d) Summary of the number of residual H2052 cells (c) and T cells (d) in the multi-round co-culture experiments with H2052 tumor cells. Data are shown as individual values and the mean + SD, n = 3 independent experiments with CAR-T cells generated from 3 different donors for the CSPG4.BBζ group, n = 4 independent experiments with CAR-T cells generated from 4 different donors for the other groups; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value, the full list of p values can be found in the source data. (e) Schema of the H2052 intraperitoneal xenograft model in NSG mice. Eight to 10 week old female NSG mice were inoculated with 3ⅹ106 FFLuc-labelled H2052 cells by intraperitoneal injection, and treated 12 days later with 2 x 106 CD19.28ζ, CSPG4.BBζ, MSLN.28ζ or MSLN.28ζ/CSPG4.BB CAR-T cells by intraperitoneal injection. (f, g) Representative tumor BLI images (f) and BLI kinetics (g) of FFLuc-H2052 tumor growth in the mesothelioma xenograft model shown in (a). (h) Kaplan-Meier survival curve of mice in (f, g)), n = 6 mice for the CD19.28ζ group, n = 8 mice for the other groups; ***p = 0.0003, ****p < 0.0001 by Log-rank test. (i) Detection of circulating CAR-T cells (CD45+CD3+) in mice 19 days after CAR-T cell treatment by flow cytometry; data are shown as individual values and the mean + SD, n = 6 mice for CD19.28ζ group, n = 8 mice for the other groups, *p <0.05 (0.0103 for CSPG4.BBζ vs. Co, 0.0159 for MSLN.28ζ vs. Co), **p = 0.0084 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value.