An optimized bicistronic chimeric antigen receptor against GPC2 or CD276 overcomes heterogeneous expression in neuroblastoma
Meijie Tian, Adam T. Cheuk, Jun S. Wei, Abdalla Abdelmaksoud, Hsien-Chao Chou, David Milewski, Michael C. Kelly, Young K. Song, Christopher M. Dower, Nan Li, Haiying Qin, Yong Yean Kim, Jerry T. Wu, Xinyu Wen, Mehdi Benzaoui, Katherine E. Masih, Xiaolin Wu, Zhongmei Zhang, Sherif Badr, Naomi Taylor, Brad St. Croix, Mitchell Ho, Javed Khan
Meijie Tian, Adam T. Cheuk, Jun S. Wei, Abdalla Abdelmaksoud, Hsien-Chao Chou, David Milewski, Michael C. Kelly, Young K. Song, Christopher M. Dower, Nan Li, Haiying Qin, Yong Yean Kim, Jerry T. Wu, Xinyu Wen, Mehdi Benzaoui, Katherine E. Masih, Xiaolin Wu, Zhongmei Zhang, Sherif Badr, Naomi Taylor, Brad St. Croix, Mitchell Ho, Javed Khan
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Research Article Immunology

An optimized bicistronic chimeric antigen receptor against GPC2 or CD276 overcomes heterogeneous expression in neuroblastoma

Abstract

Chimeric antigen receptor (CAR) T cell therapies targeting single antigens have performed poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T cell persistence, and T cell exhaustion. Here, we aimed to identify optimal CARs against glypican 2 (GPC2) or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. First, we examined CAR T cell expansion in the presence of targets by digital droplet PCR. Next, using pooled competitive optimization of CAR by cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), termed P-COCC, we simultaneously analyzed protein and transcriptome expression of CAR T cells to identify high-activity CARs. Finally, we performed cytotoxicity assays to identify the most effective CAR against each target and combined the CARs into a bicistronic “OR” CAR (BiCisCAR). BiCisCAR T cells effectively eliminated tumor cells expressing GPC2 or CD276. Furthermore, the BiCisCAR T cells demonstrated prolonged persistence and resistance to exhaustion when compared with CARs targeting a single antigen. This study illustrated that targeting multiple TAAs with BiCisCAR may overcome heterogenous expression of target antigens in solid tumors and identified a potent, clinically relevant CAR against NB. Moreover, our multimodal approach integrating competitive expansion, P-COCC, and cytotoxicity assays is an effective strategy to identify potent CARs among a pool of candidates.

Authors

Meijie Tian, Adam T. Cheuk, Jun S. Wei, Abdalla Abdelmaksoud, Hsien-Chao Chou, David Milewski, Michael C. Kelly, Young K. Song, Christopher M. Dower, Nan Li, Haiying Qin, Yong Yean Kim, Jerry T. Wu, Xinyu Wen, Mehdi Benzaoui, Katherine E. Masih, Xiaolin Wu, Zhongmei Zhang, Sherif Badr, Naomi Taylor, Brad St. Croix, Mitchell Ho, Javed Khan

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Figure 9

BiCisCARs show superior efficacy in eradicating tumor cells heterogeneously expressing GPC2 or CD276, improved T cell persistence, and reduced T cell exhaustion.

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BiCisCARs show superior efficacy in eradicating tumor cells heterogeneou...
(A) Schema of the metastatic tumor model with 1:1 mixed NALM6-GPC2 and NALM6-CD276 cells infused with 2.5 × 106 or 5 × 106 CAR T cells on day 3 after tumor inoculation. (B and C) Representative bioluminescence images (B) and bioluminescence kinetics (C) of NALM6 cell growth before (d2) and after (≥d7) infusion with 2.5 × 106 CAR T cells. ****P < 0.0001, by 2-way RM ANOVA. (D) Kaplan-Meier survival analysis of mice treated with CAR T cells (n = 5 mice/group). **P < 0.01 and ****P < 0.0001, by log-rank test. (E) Representative flow cytometric plots of CAR T cell frequencies in spleens from the mice described above, 21 days after infusion with 5 × 106 CAR T cells. (F) The percentages of CAR T cells in spleens from mice treated with 5 × 106 CAR T cells were analyzed by flow cytometry (n = 5; data indicate the mean ± SEM). *P < 0.05 and **P < 0.01, by 1-way ANOVA with Dunnett’s multiple-comparison test. (G) Total numbers of the indicated CAR T cells in whole spleens from mice treated with 5 × 106 CAR T cells. *P < 0.05 and **P < 0.01, by nonparametric Kruskal-Wallis test. (H–J) Representative flow cytometry illustrating PD-1 (H), LAG-3 (I), and Tim-3 (J) expression in CD4+ or CD8+ CAR T cells in spleens of mice from the NALM6 metastatic model, 21 days after infusion of 5 × 106 CAR T cells (n = 5, mean ± SEM). (K–M) Percentages of PD-1 (K), LAG-3 (L), and Tim-3 (M) in CD4+ or CD8+ CAR T cells in mice 21 days after infusion with 5 × 106 CAR T cells (n = 5, mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA, Dunnett’s multiple-comparison test.