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 4

P-COCC identifies the optimal anti-GPC2 CAR CT3 with higher activation and antitumor polyfunctionality signatures.

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P-COCC identifies the optimal anti-GPC2 CAR CT3 with higher activation a...
(A) Pie charts show the proportions of 15 clusters within 8 types of anti-GPC2 CAR T cells. Only identified CAR T cells were taken into count. Clusters are distinguished by colors in the color key. (B) Volcano plot of DEGs between the CT3 CAR and 7 other anti-GPC2 CARs 24 hours after coculturing with IMR5 cells. Genes with an adjusted P value of less than 0.05 are shown in red. The top 20 genes, ranked by average log2 fold change according to a nonparametric Wilcoxon rank-sum test, are labeled on the plot. (C) Heatmap shows averaged expression of the top 20 genes in 8 different anti-GPC2 CARs within a CAR T cell pool cocultured with IMR5 cells (STIM_24h) or without targets (CTRL_24h) for 24 hours. (D) Canonical signaling pathways regulated by the DEGs identified in B. A positive or negative z score value indicates that a pathway is predicted to be increased or decreased, respectively, in stimulated CT3 CARs relative to other 7 GPC2-targeting CARs. (E) GSEA of effector memory T cell genes in a ranked fold change list of DEGs between CT3 CARs and the other 7 anti-GPC2 CAR T cells, 24 hours after stimulation with IMR5 cells. NES, normalized enrichment score.