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Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells
Non-viral platforms can be applied rapidly and cost-effectively for chimeric antigen receptor (CAR)-T cell manufacturing. In the present paper, we describe in detail a clinically relevant manufacturing process for NKG2D CAR-T cells through electroporation of CAR-encoding piggyBac transposon plasmids...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society of Gene & Cell Therapy
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005737/ https://www.ncbi.nlm.nih.gov/pubmed/33816644 http://dx.doi.org/10.1016/j.omtm.2021.02.023 |
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author | Tay, Johan C.K. Wang, Junjian Du, Zhicheng Ng, Yu Yang Li, Zhendong Ren, Yuefang Zhang, Chang Zhu, Jianqing Xu, Xue Hu Wang, Shu |
author_facet | Tay, Johan C.K. Wang, Junjian Du, Zhicheng Ng, Yu Yang Li, Zhendong Ren, Yuefang Zhang, Chang Zhu, Jianqing Xu, Xue Hu Wang, Shu |
author_sort | Tay, Johan C.K. |
collection | PubMed |
description | Non-viral platforms can be applied rapidly and cost-effectively for chimeric antigen receptor (CAR)-T cell manufacturing. In the present paper, we describe in detail a clinically relevant manufacturing process for NKG2D CAR-T cells through electroporation of CAR-encoding piggyBac transposon plasmids and in vitro expansion with K562 artificial antigen-presenting cells. With an optimized protocol, we generated the final cell therapy products with 89.2% ± 10.2% NKG2D CAR-positive cells and achieved the corresponding antigen-dependent expansion between 50,000 and 60,000 folds within 4 weeks. To facilitate repeated CAR-T cell infusions, we evaluated the practicality of cryopreservation followed by post-thaw expansion and an extended manufacturing process for up to 9 rounds of weekly K562 cell stimulation. We found that neither compromised the in vitro anti-tumor activity of NKG2D CAR-T cells. Interestingly, the expression of T cell exhaustion markers TIGIT, TIM3, and LAG3 was reduced with extended manufacturing. To enhance the safety profile of the NKG2D CAR-T cells, we incorporated a full-length CD20 transgene in tandem with the CAR construct and demonstrated that autologous NK cells could mediate efficient antibody-dependent cell-mediated cytotoxicity to remove these CAR-T cells. Collectively, our study illustrates a protocol that generates large numbers of efficacious NKG2D CAR-T cells suitable for multiple rounds of infusions. |
format | Online Article Text |
id | pubmed-8005737 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society of Gene & Cell Therapy |
record_format | MEDLINE/PubMed |
spelling | pubmed-80057372021-04-01 Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells Tay, Johan C.K. Wang, Junjian Du, Zhicheng Ng, Yu Yang Li, Zhendong Ren, Yuefang Zhang, Chang Zhu, Jianqing Xu, Xue Hu Wang, Shu Mol Ther Methods Clin Dev Original Article Non-viral platforms can be applied rapidly and cost-effectively for chimeric antigen receptor (CAR)-T cell manufacturing. In the present paper, we describe in detail a clinically relevant manufacturing process for NKG2D CAR-T cells through electroporation of CAR-encoding piggyBac transposon plasmids and in vitro expansion with K562 artificial antigen-presenting cells. With an optimized protocol, we generated the final cell therapy products with 89.2% ± 10.2% NKG2D CAR-positive cells and achieved the corresponding antigen-dependent expansion between 50,000 and 60,000 folds within 4 weeks. To facilitate repeated CAR-T cell infusions, we evaluated the practicality of cryopreservation followed by post-thaw expansion and an extended manufacturing process for up to 9 rounds of weekly K562 cell stimulation. We found that neither compromised the in vitro anti-tumor activity of NKG2D CAR-T cells. Interestingly, the expression of T cell exhaustion markers TIGIT, TIM3, and LAG3 was reduced with extended manufacturing. To enhance the safety profile of the NKG2D CAR-T cells, we incorporated a full-length CD20 transgene in tandem with the CAR construct and demonstrated that autologous NK cells could mediate efficient antibody-dependent cell-mediated cytotoxicity to remove these CAR-T cells. Collectively, our study illustrates a protocol that generates large numbers of efficacious NKG2D CAR-T cells suitable for multiple rounds of infusions. American Society of Gene & Cell Therapy 2021-03-03 /pmc/articles/PMC8005737/ /pubmed/33816644 http://dx.doi.org/10.1016/j.omtm.2021.02.023 Text en © 2021 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Original Article Tay, Johan C.K. Wang, Junjian Du, Zhicheng Ng, Yu Yang Li, Zhendong Ren, Yuefang Zhang, Chang Zhu, Jianqing Xu, Xue Hu Wang, Shu Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells |
title | Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells |
title_full | Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells |
title_fullStr | Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells |
title_full_unstemmed | Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells |
title_short | Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells |
title_sort | manufacturing nkg2d car-t cells with piggybac transposon vectors and k562 artificial antigen-presenting cells |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005737/ https://www.ncbi.nlm.nih.gov/pubmed/33816644 http://dx.doi.org/10.1016/j.omtm.2021.02.023 |
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