An optimized bicistronic chimeric antigen receptor against GPC2 or CD276 overcomes heterogeneous expression in neuroblastoma
Meijie Tian, … , Mitchell Ho, Javed Khan
Meijie Tian, … , Mitchell Ho, Javed Khan
Published July 19, 2022
Citation Information: J Clin Invest. 2022;132(16):e155621. https://doi.org/10.1172/JCI155621.
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Research Article Immunology Therapeutics

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 2

Measurement of the expansion of 14 distinct CAR T cells in a competition assay using ddPCR.

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Measurement of the expansion of 14 distinct CAR T cells in a competition...
(A) Experimental schema of a competition assay using a pool of 14 CAR T cells cocultured with or without tumor cells to monitor each CAR T cell expansion ability over time. (B and C) CT3 CARs showed maximal expansion among CARs targeting GPC2 when cocultured with targets (red squares), and 3 anti-CD276 CARs (MGB7H3-LH, h8H9-HL, and h8H9-LH) showed higher expansion levels than did their no-target controls (black circles). Copy numbers of each CAR in the pool of 14 CAR T cells over time were measured by ddPCR using specific primers against the scFv region of CAR to quantify each CAR accurately (n = 3, error bars indicate the SD). Each CAR copy per 10,000 GFP was then normalized to the value on day 0 (D0). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA with Sidak’s multiple-comparison test.