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Automated, scaled, transposon-based production of CAR T cells

BACKGROUND: There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfe...

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Autores principales: Lock, Dominik, Monjezi, Razieh, Brandes, Caroline, Bates, Stephan, Lennartz, Simon, Teppert, Karin, Gehrke, Leon, Karasakalidou-Seidt, Rafailla, Lukic, Teodora, Schmeer, Marco, Schleef, Martin, Werchau, Niels, Eyrich, Matthias, Assenmacher, Mario, Kaiser, Andrew, Prommersberger, Sabrina, Schaser, Thomas, Hudecek, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BMJ Publishing Group 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9472140/
https://www.ncbi.nlm.nih.gov/pubmed/36096530
http://dx.doi.org/10.1136/jitc-2022-005189
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author Lock, Dominik
Monjezi, Razieh
Brandes, Caroline
Bates, Stephan
Lennartz, Simon
Teppert, Karin
Gehrke, Leon
Karasakalidou-Seidt, Rafailla
Lukic, Teodora
Schmeer, Marco
Schleef, Martin
Werchau, Niels
Eyrich, Matthias
Assenmacher, Mario
Kaiser, Andrew
Prommersberger, Sabrina
Schaser, Thomas
Hudecek, Michael
author_facet Lock, Dominik
Monjezi, Razieh
Brandes, Caroline
Bates, Stephan
Lennartz, Simon
Teppert, Karin
Gehrke, Leon
Karasakalidou-Seidt, Rafailla
Lukic, Teodora
Schmeer, Marco
Schleef, Martin
Werchau, Niels
Eyrich, Matthias
Assenmacher, Mario
Kaiser, Andrew
Prommersberger, Sabrina
Schaser, Thomas
Hudecek, Michael
author_sort Lock, Dominik
collection PubMed
description BACKGROUND: There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition. METHODS: We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR. RESULTS: We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1–3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns. CONCLUSIONS: We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.
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spelling pubmed-94721402022-09-15 Automated, scaled, transposon-based production of CAR T cells Lock, Dominik Monjezi, Razieh Brandes, Caroline Bates, Stephan Lennartz, Simon Teppert, Karin Gehrke, Leon Karasakalidou-Seidt, Rafailla Lukic, Teodora Schmeer, Marco Schleef, Martin Werchau, Niels Eyrich, Matthias Assenmacher, Mario Kaiser, Andrew Prommersberger, Sabrina Schaser, Thomas Hudecek, Michael J Immunother Cancer Immune Cell Therapies and Immune Cell Engineering BACKGROUND: There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition. METHODS: We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR. RESULTS: We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1–3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns. CONCLUSIONS: We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting. BMJ Publishing Group 2022-09-12 /pmc/articles/PMC9472140/ /pubmed/36096530 http://dx.doi.org/10.1136/jitc-2022-005189 Text en © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. https://creativecommons.org/licenses/by-nc/4.0/This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) .
spellingShingle Immune Cell Therapies and Immune Cell Engineering
Lock, Dominik
Monjezi, Razieh
Brandes, Caroline
Bates, Stephan
Lennartz, Simon
Teppert, Karin
Gehrke, Leon
Karasakalidou-Seidt, Rafailla
Lukic, Teodora
Schmeer, Marco
Schleef, Martin
Werchau, Niels
Eyrich, Matthias
Assenmacher, Mario
Kaiser, Andrew
Prommersberger, Sabrina
Schaser, Thomas
Hudecek, Michael
Automated, scaled, transposon-based production of CAR T cells
title Automated, scaled, transposon-based production of CAR T cells
title_full Automated, scaled, transposon-based production of CAR T cells
title_fullStr Automated, scaled, transposon-based production of CAR T cells
title_full_unstemmed Automated, scaled, transposon-based production of CAR T cells
title_short Automated, scaled, transposon-based production of CAR T cells
title_sort automated, scaled, transposon-based production of car t cells
topic Immune Cell Therapies and Immune Cell Engineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9472140/
https://www.ncbi.nlm.nih.gov/pubmed/36096530
http://dx.doi.org/10.1136/jitc-2022-005189
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