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. 2021 Mar 31;10(1):1901434.
doi: 10.1080/2162402X.2021.1901434.

Construction of PD1/CD28 chimeric-switch receptor enhances anti-tumor ability of c-Met CAR-T in gastric cancer

Cong Chen  1   2 , Yan-Mei Gu  1 , Fan Zhang  1 , Zheng-Chao Zhang  1 , Ya-Ting Zhang  1 , Yi-Di He  1 , Ling Wang  1 , Ning Zhou  1 , Fu-Tian Tang  1 , Hong-Jian Liu  2 , Yu-Min Li  1   3
Affiliations

Construction of PD1/CD28 chimeric-switch receptor enhances anti-tumor ability of c-Met CAR-T in gastric cancer

Cong Chen et al. Oncoimmunology. .

Abstract

Chimeric antigen receptor (CAR) T cell is a promising method in cancer immunotherapy but faces many challenges in solid tumors. One of the major problems was immunosuppression caused by PD-1. In our study, the expression of c-Met in GC was analyzed from TCGA datasets, GC tissues, and cell lines. The c-Met CAR was a second-generation CAR with 4-1BB, cMet-PD1/CD28 CAR was c-Met CAR adding PD1/CD28 chimeric-switch receptor (CSR). In vitro, we measured the changes of different subgroups, phenotypes and PD-1 expression in CAR-T cells. We detected the secretion levels of different cytokines and the killing ability of CAR-Ts. In vivo, we established a xenograft GC model and observed the anti-tumor effect and off-target toxicity of different CAR-Ts. We find that the expression of c-Met was increased in GC. CD3+CD8+ T cells and CD62L+CCR7+ central memory T cells (TCM) were increased in two CAR-Ts. The stimulation of target cells could promote the expression of PD-1 in c-Met CAR-T. Compared with Mock T, the secretion of cytokines as IFN-γ, TNF-α, IL-6, IL-10 secreted by two CAR-Ts was increased, and the killing ability to c-Met positive GC cells was enhanced. The PD1/CD28 CSR could further enhance the killing ability, especially the long-term anti-tumor effect of c-Met CAR-T, and reduce the release level of IL-6. CAR-Ts target c-Met had no obvious off-target toxicity to normal organs. Thus, the PD1/CD28 CSR could further enhance the anti-tumor ability of c-Met CAR-T, and provides a promising design strategy to improve the efficacy of CAR-T in GC.

Keywords: Gastric Cancer; c-Met; car-T; immunotherapy; pd1/CD28.

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Figures

Figure 1.
Figure 1.
The expression of c-Met was increased in the tissues and cells of gastric cancer. A, Differences in the expression of MET gene in gastric cancer and normal tissues, and the relationship between MET gene expression and microsatellite stability, pathological stage, Lauren classification in gastric cancer tissues. MSS: microsatellite stability, MSI: microsatellite instability. B, Immunohistochemical staining of c-Met in and adjacent tissues (a ~ c) and gastric cancer (d ~ f), and statistical analysis of AOD (g) and H-score (h), n = 50, (a ~ e) 200×, (f) 400 × . AOD: average optical density. C, Transcription levels of MET and PD-L1 genes in different gastric cancer cell lines detected by PCR, n ≥ 3. D, Expression levels of c-Met protein in different gastric cancer cell lines detected by WB, n ≥ 3. E, Expression levels of c-Met on the surface of gastric cancer cell lines detected by FCM, n ≥ 3. Column charts show mean ± SD, compared with GES-1 * p < .05, ** p < .01, *** p < .001, **** p < .0001, p values were determined by two-tailed, unpaired Student’s t test, or one-way ANOVA with Dunnett post hoc test
Figure 2.
Figure 2.
The structure and infection efficiency of CAR-Ts. A, c-Met expression in HGC27-OE cells after the infection of MET-OE lentivirus detected by (a) qRT-PCR, (b) WB, (c) FCM. B, The structures of c-Met CAR, cMet-PD1/CD28 CAR plasmids and PD1/CD28 CSR. C, The infection efficiency of c-Met CAR-T and cMet-PD1/CD28 CAR-T at MOI = 10 determined by eGFP+. D, The infection efficiency of c-Met CAR-T and cMet-PD1/CD28 CAR-T at MOI = 10 determined by Protein L+. E, Gene transcription levels of c-Met scFv and PD1/CD28 CSR after infection detected by PCR. CSR: chimeric-switch receptor, scFv: single-chain variable fragment. F, The proliferation of Mock T, c-Met CAR-T, cMet-PD1/CD28 CAR-T after lentivirus infection. Column charts represent mean ± SD, n ≥ 3, *** p < .001, **** p < .0001, p values were determined by two-tailed, unpaired Student’s t test or one-way ANOVA with Dunnett post hoc test
Figure 3.
Figure 3.
The changes of subgroups and central memory phenotype of CAR-Ts. A, Changes of CD3+CD4+ and CD3+CD8+ subgroups in two CAR-Ts. B, Changes of CD45RO+ and CD62L+CCR7+ TCM phenotype in two CAR-Ts. TCM: central memory T cell. Column charts represent mean ± SD, n ≥ 3, compared with Mock T * p < .05, ** p < .01, *** p < .001, **** p < .0001, p values were determined by one-way ANOVA with Tukey post hoc test
Figure 4.
Figure 4.
The changes of activation phenotype and PD-1 expression of CAR-Ts before or after the stimulation by target cells. A, Changes of activation phenotype of CD25, CD69, CD71, HLA-DR in c-Met CAR-T before or after MKN-45 stimulation. B, Changes of PD-1 expression in different CAR-Ts before or after MKN-45 stimulation. CAR-Ts and MKN-45 were co-cultured for 24 hours at a ratio of 2:1. The column chart shows mean ± SD, n ≥ 3, * p < .05, ** p < .01, *** p < .001. Statistical analysis was carried out by one-way ANOVA with Sidak post hoc test
Figure 5.
Figure 5.
The cytokine secretion, killing capacity, degranulation level were increased in two CAR-Ts. A, Different secretion levels of IFN-γ, TNF-α, IL-6, IL-10 in c-Met CAR-T, cMet-PD1/CD28 CAR-T after the stimulation by different target cells. The two CAR-Ts were incubated with different target cells at the ratio of 5:1 for 24 hours. B, The killing ability of c-Met CAR-T and cMet-PD1/CD28 CAR-T on different target cells under different effect:target ratios, and the effect of c-Met expression in target cells on the killing ability of CAR-Ts. CAR-Ts were co-cultured with different target cells at the ratios of 10:1, 5:1, and 1:1 for 24 hours. C, CD107a degranulation levels of c-Met CAR-T and cMet-PD1/CD28 CAR-T after stimulation by MKN-45. CAR-Ts and MKN-45 were co-cultured at the ratio of 5:1 for 12 hours. Column charts or line charts represent mean ± SD, n ≥ 3, * p < .05, *** p < .001, **** p < .0001, statistical analysis was conducted by one-way ANOVA with Sidak post hoc test
Figure 6.
Figure 6.
The two CAR-Ts have obvious anti-tumor activity in subcutaneous xenograft model of gastric cancer. A, Flow chart of in vivo experiment. B, Bioluminescence imaging of MKN-45 subcutaneous tumor model at different time points. C, Statistical analysis of total bioluminescence intensity at different time points and on d21. D, Statistical analysis of tumor volumes at different time points and on d21. E, Statistical analysis of body weight at different time points. s.c.: subcutaneously, i.t.: intratumorally, BLI: Bioluminescence Imaging. Column charts and line charts represent mean ± SD, n ≥ 3, ** p < .01, the Multiple t test or Student ’s t test was used for statistical analysis
Figure 7.
Figure 7.
HE staining of normal tissues of stomach, small intestine, heart, liver, spleen, lung and kidney in different CAR-T treatment groups
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This study was supported by National Natural Science Foundation of China (31770537, 81972523, 81870329, 81960673), Key Research and Development Project of Gansu Science and Technology Plan (18YF1WA113), Talent Innovation and Entrepreneurship Project of Lanzhou City (2020-RC-38, 2017-RC-1), China Scholarship Council (201806180088)