TEG001 Insert Integrity from Vector Producer Cells until Medicinal Product
Open accessAbstract
Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of chimeric antigen receptor (CAR) T cell therapy, little information has been made available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context, we characterize αβT cells engineered to express a defined γδT cell engineered to express a defined γδT receptor (TEG) currently used in a first-in-human clinical study (NTR6541). Utilizing targeted locus amplification in combination with next generation sequencing for the vector producer clone and TEG001 products, we report on five single-nucleotide variants and nine intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001 without having an effect on cellular proliferation during extensive in vitro culture. Based on an environmental risk assessment of the five nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation in vivo.
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An insufficient number of vector copies may result in low or absent CAR receptor expression, compromising product efficacy.
“Therapeutic Efficacy: an insufficient number of vector copies may result in low or absent CAR receptor expression, compromising product efficacy [27–30].”
From Harmonisation of quality control tests for academic production of CAR-T cells: a position paper from the WP-bioproduction of the UNITC consortium (2025) · cites this paper as [30]
The Discussion (page unavailable) suggests that higher vector copy numbers may be needed for TEG001 receptor expression because its introduced TCR competes for CD3 molecules. It explicitly contrasts TEG001 with CAR T products; it does not say that insufficient copies cause low or absent CAR expression or compromise product efficacy. (LLM, conf=0.76)
Experimental — accuracy not yet measuredAn excessive number of vector copies can lead to CAR overexpression, increasing toxicity risks.
“Safety: an excessive number of vector copies can lead to CAR overexpression, increasing toxicity risks.”
From Harmonisation of quality control tests for academic production of CAR-T cells: a position paper from the WP-bioproduction of the UNITC consortium (2025) · cites this paper as [30]
The Introduction links higher vector copy numbers to potential safety risks, and the Discussion says CAR T products have generally stayed below a five-copy threshold. The paper does not say that excess copies cause CAR overexpression or that CAR overexpression increases toxicity; its transgene-copy-number section says it is unclear whether more integration sites per cell add to genotoxicity risk. The supplied passages have section labels but no page numbers. (LLM, conf=0.84)
Experimental — accuracy not yet measuredA high vector copy number raises the risk of insertional mutagenesis, where the vector integrates into genomic regions that could activate proto-oncogenes or disable tumor suppressor genes.
“Additionally, a high copy number raises the risk of insertional mutagenesis, where the vector integrates into genomic regions that could activate proto-oncogenes or disable tumor suppressor genes [28–31].”
From Harmonisation of quality control tests for academic production of CAR-T cells: a position paper from the WP-bioproduction of the UNITC consortium (2025) · cites this paper as [30]
The Introduction (page unavailable) discusses potential risks from integration sites and higher copy numbers, and gives an example of a gene disrupted by integration. But “Transgene Copy Number in TEG001 Drug Product” (page unavailable) says it is unclear whether more integration sites per cell add to genotoxicity risk; it does not establish that high copy number raises that risk. The cited passages also do not state the proposed proto-oncogene or tumor-suppressor mechanism. (LLM, conf=0.86)
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Wang, Y., Zhang, B., Wang, Y., Tan, Y., Hu, X., Che, X., & Feng, M. (2026) · Frontiers in Immunology · doi:10.3389/fimmu.2026.1755751 · Cited by 10 · see citing works
Kierkels, G. J. J., van Diest, E., Hernández-López, P., Scheper, W., de Bruin, A. C. M., Frijlink, E., Aarts‐Riemens, T., van Dooremalen, S., Beringer, D. X., Oostvogels, R., et al. (2021) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2021.06.011 · Cited by 7 · see citing works
Self-citationAdding Help to an HLA-A*24:02 Tumor-Reactive γδTCR Increases Tumor Control
Johanna, I., Hernández-López, P., Heijhuurs, S., Scheper, W., Bongiovanni, L., de Bruin, A., Beringer, D. X., Oostvogels, R., Straetemans, T., Sebestyén, Z., et al. (2021) · Frontiers in Immunology · doi:10.3389/fimmu.2021.752699 · Cited by 2 · see citing works
Self-citationTumor-reactivity of δ2 negative γδT cells and the role of the γδT cell receptorPreprint
van Dooremalen, S., Janssen, A., Bloemenkamp, L., Yonika, A., Beringer, D. X., & Kuball, J. H. E. (2021) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2021.03.17.435293
Self-citation
- Authors
- Straetemans, T., Janssen, A., Jansen, K., Doorn, R., Aarts, T., van Muyden, A. D. D., Simonis, M., Bergboer, J., de Witte, M., Sebestyen, Z., & Kuball, J.
- Affiliations
- Utrecht University (NL)
- Cergentis (Netherlands) (NL)
- Corresponding author
- Straetemans, Trudy
- Kuball, Jurgen
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2020-02
- Volume
- 28
- Issue
- 2
- Pages
- 561-571
- ISSN
- 1525-0016 (ISSN-L)
- PMC
- PMC7001055
- OpenAlex
- W2996130480
- Cited by
- 13
- References
- 59
- Citations by year
- 2026: 1 · 2025: 1 · 2023: 1 · 2022: 1 · 2021: 6 · 2020: 3
- FWCI
- 1.51
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
- Utrecht University Repository (Utrecht University) (submittedVersion)
- Funders
- ZonMw (43400003, 917.11.337)
- KWF Kankerbestrijding (UU 2010-4669, UU 2013-6426, UU 2014-6790, UU 2015-7601, UU 2018-11393)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- vector copy number, CAR T-cell therapy, single nucleotide variants, malignant transformation risk, environmental risk assessment
- MeSH terms
- Genetic Engineering, Genetic Vectors, Humans, Mutation, Phenotype, Receptors, Antigen, T-Cell, T-Lymphocytes, Genetic Therapy, Immunotherapy, Adoptive, Mutagenesis, Insertional, Virus Integration, Transgenes, Untranslated Regions
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