Effect of cryopreservation on autologous chimeric antigen receptor T cell characteristics
Open accessAbstract
As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating cryopreservation steps becomes a logistical necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells [PBMNCs]) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservation-related subtle micro-cellular damage needs further study.
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Fresh CAR-T cells were shown earlier to have higher in vitro antitumor activity and lesser microcellular damage than frozen CAR-T cells.
“POC manufacturing enables the delivery of fresh CAR-T cells, which were shown earlier to have higher in vitro antitumor activity and lesser microcellular damage than the frozen,4,5”
From Point-of-care manufacturing of anti-CD19 CAR-T cells using a closed production platform: Experiences of an academic in Thailand (2024) · cites this paper as [5]
The Results (page unavailable) report more apoptosis- and cell-cycle-damage-related gene expression in cryo-thawed cells, supporting the comparison of cellular damage. They also report greater expression of pathways associated with T-cell cytolysis in fresh cells, but do not establish higher in vitro antitumor activity; pathway expression is not a direct activity measurement. (LLM, conf=0.87)
Experimental — accuracy not yet measuredRecovery and viability of PBMCs are significantly reduced after freezing and thawing compared to fresh apheresis products, but recovery of mononuclear cells after thawing is sufficient for consistent manufacturing of CAR T cells without evidence of significant differences in expansion, transduction efficiency or overall subset composition.
“For instance, Panch et al. showed recovery and viability of PBMCs was significantly reduced after freezing and thawing compared to fresh apheresis products (39). Nevertheless, the recovery of mononuclear cells after thawing was sufficient for consistent manufacturing of CAR T cells, without evidence of significant differences in expansion, transduction efficiency or overall subset composition (39)”
From Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy (2021) · cites this paper as [39]
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“POC manufacturing enables the delivery of fresh CAR-T cells, which were shown earlier to have higher in vitro antitumor activity and lesser microcellular damage than the frozen, 4,5 although whether fresh products would provide better clinical outcomes remains to be determined.”
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- Authors
- Panch, S. R., Srivastava, S. K., Elavia, N., McManus, A., Liu, S., Jin, P., Highfill, S. L., Li, X., Dagur, P., Kochenderfer, J. N., Fry, T. J., Mackall, C. L., Lee, D., Shah, N. N., & Stroncek, D. F.
- Affiliations
- National Institutes of Health (US)
- National Heart, Lung, and Blood Institute (US)
- Stanford University (US)
- University of Virginia (US)
- Corresponding author
- Panch, Sandhya R.
- ORCID
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2019-07
- Volume
- 27
- Issue
- 7
- Pages
- 1275-1285
- ISSN
- 1525-0016 (ISSN-L)
- PMC
- PMC6612799
- OpenAlex
- W2947301297
- Cited by
- 107
- References
- 42
- Citations by year
- 2026: 17 · 2025: 12 · 2024: 16 · 2023: 18 · 2022: 22 · 2021: 12 · 2020: 7 · 2019: 3
- FWCI
- 4.60
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- Center for Outcomes Research and Evaluation, Yale School of Medicine
- NIH Clinical Center
- National Heart, Lung, and Blood Institute
- Topics
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Keywords
- chimeric antigen receptor T cells, cryopreservation, autologous CAR T-cell therapy, CAR T cell manufacturing, cell viability, apoptosis, mitochondrial dysfunction, cell surface markers, gene expression signatures, CAR T cell persistence
- MeSH terms
- Receptors, Chimeric Antigen, Adolescent, Adult, Aged, Autoantigens, Cell Cycle, Cell Survival, Child, Female, Humans, Male, Middle Aged, Neoplasms, Phenotype, Prospective Studies, Retrospective Studies, T-Lymphocytes, Cryopreservation, Immunotherapy, Adoptive, CD4-CD8 Ratio, Apoptosis, Young Adult, Transcriptome
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