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Automated generation of gene-edited CAR T cells at clinical scale
Open accessAbstract
The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a TRAC locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding TRAC locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/β-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/β-free CAR19 T cells at clinical scale.
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Fully automated closed processes eliminate any handling of the product during manufacturing but are restricted to a single product in each production run.
“Fully automated closed processes eliminate any handling of the product during manufacturing but are restricted to a single product in each production run (77-79).”
From Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy (2021) · cites this paper as [79]
The Introduction and Results describe an automated, closed CAR T-cell manufacturing process. They do not say that it eliminates all product handling or that such processes are restricted to one product per production run; the Results describe product transfer and in-process sampling. (LLM, conf=0.85)
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Schwarze, L. I., Nuppenau, N., Müller, J. H., Uhde, A., Ashkar, N. A., Schröder, T., Riecken, K., & Fehse, B. (2026) · Human Gene Therapy · doi:10.1177/10430342261492461
Metadata
- Authors
- Alzubi, J., Lock, D., Rhiel, M., Schmitz, S., Wild, S., Mussolino, C., Hildenbeutel, M., Brandes, C., Rositzka, J., Lennartz, S., Haas, S. A., Chmielewski, K. O., Schaser, T., Kaiser, A., Cathomen, T., & Cornu, T. I.
- Affiliations
- University of Freiburg (DE)
- Miltenyi Biotec (Germany) (DE)
- Corresponding author
- Cathomen, Toni
- ORCID
- Venue
- Molecular Therapy - Methods & Clinical Development
- Publisher
- Elsevier BV
- Published
- 2021-03
- Volume
- 20
- Pages
- 379-388
- ISSN
- 2329-0501 (ISSN-L)
- PMC
- PMC7848723
- OpenAlex
- W3117758135
- Cited by
- 53
- References
- 35
- Citations by year
- 2026: 4 · 2025: 9 · 2024: 9 · 2023: 13 · 2022: 12 · 2021: 6
- FWCI
- 2.80
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecular Therapy — Methods & Clinical Development (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Topics
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- chimeric antigen receptor T cells, genome editing, TALEN, CD19, lentiviral transduction, electroporation, CliniMACS Prodigy, good manufacturing practices, adoptive immunotherapy, B-cell leukemia, negative selection
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