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ResearchIn-Press PreviewImmunology Open Access | 10.1172/JCI155621

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

MEIJIE TIAN,1 Adam T. Cheuk,1 Jun S. Wei,1 Abdalla Abdelmaksoud,1 Hsien-Chao Chou,1 David Milewski,1 Michael C. Kelly,2 Young K. Song,1 Christopher M. Dower,3 Nan Li,4 Haiying Qin,5 Yong Yean Kim,1 Jerry T. Wu,1 Xinyu Wen,1 Mehdi Benzaoui,5 Katherine E. Masih,1 Xiaolin Wu,6 Zhongmei Zhang,7 Sherif Badr,7 Naomi Taylor,5 Brad St. Croix,3 Mitchell Ho,4 and Javed Khan1

1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

2Single Cell Analysis Facility, Center for Cancer Research, National Institutes of Health, Bethesda, United States of America

3Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, United States of America

4Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

5Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

6Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States of America

7Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, United States of America

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Published July 19, 2022 - More info

J Clin Invest. https://doi.org/10.1172/JCI155621.
Copyright © 2022, TIAN et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Published July 19, 2022 - Version history
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Abstract

Chimeric antigen receptor (CAR) T-cell therapies targeting single antigens perform poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T-cell persistence and 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 target by digital droplet PCR. Next, using Pooled Competitive Optimization of CAR by CITE-seq (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 them into a bicistronic “OR” CAR (BiCisCAR). BiCisCAR T-cells effectively eliminated tumor cells expressing GPC2 or CD276. Furthermore, the BiCisCAR demonstrated prolonged persistence and resistance to exhaustion comparing with single antigen targeting CARs. This study illustrated that targeting multiple TAAs with BiCisCARs may overcome heterogenous expression of target antigen 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 from a pool of candidates.

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Version history
  • Version 1 (July 19, 2022): In-Press Preview
  • Version 2 (August 15, 2022): Electronic publication
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