CoChat
Sign Up
Cited by 6Virus-based gene therapy researchCAR-T cell therapy researchRNA Interference and Gene Delivery

Optimizing lentiviral vector formulation conditions for efficient ex vivo transduction of primary human T cells in chimeric antigen receptor T-cell manufacturing

Open access
Luostarinen, Annu, Kailaanmäki, Anssi, Turkki, Vesa, Köylijärvi, Marjut, Käyhty, Piia, Leinonen, Hanna, Albers-Skirdenko, Vita, Lipponen, Eevi +4 more·2024·Cytotherapy
DOI 10.1016/j.jcyt.2024.04.002
Open this paper in CoChat — free accountOpen PDF

Abstract

Background aims Chimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing. Methods To identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVO TM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 hours post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized. Results Post-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVO TM 15, and 10% trehalose-20 mM MgCl 2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 hours at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl 2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality. Conclusion A buffer consisting of 10% trehalose-20 mM MgCl 2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 hours post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1016/j.jcyt.2024.04.002

Cites

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • 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 [29]

    The Introduction (page unavailable) states that suboptimal multiplicity of infection leads to low CAR expression and impaired anticancer function, supporting the claimed concern about efficacy. It does not establish that a low vector copy number specifically causes this outcome, or explicitly describe absent CAR expression. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • A 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 [29]

    The Introduction (page not provided) calls increased vector copy number in the host-cell genome a potential risk factor for insertional mutagenesis. It does not describe integration near proto-oncogenes or tumor-suppressor genes, so the specific mechanism in the claim is not supported by this paper. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • An 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 [29]

    The Introduction (page not provided) says excessive virus may increase vector copy number, a potential risk factor for insertional mutagenesis. It separately says high cell-surface receptor density may increase tonic signaling and accelerate exhaustion, but does not establish that excessive vector copies cause CAR overexpression or that this is the route to increased toxicity risk. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

No references could be resolved yet.

Research works printed but not linked: 49

  1. Perry C, Rayat ACME. Lentiviral vector bioprocessing. Viruses 2021;13:268. https://doi.org/10.3390/v13020268. · DOI
  2. Wang V, Gauthier M, Decot V, Reppel L, Bensoussan D. Systematic review on CAR- T cell clinical trials up to 2022: academic center input. Cancers (Basel) 2023;15:1003. https://doi.org/10.3390/cancers15041003. · DOI
  3. Moreira AS, Cavaco DG, Faria TQ, Alves PM, Carrondo MJT, Peixoto C. Advances in lentivirus purification. Biotechnol J 2021;16:2000019. https://doi.org/10.1002/ biot.202000019. · DOI
  4. Bandeira V, Peixoto C, Rodrigues AF, Cruz PE, Alves PM, Coroadinha AS, et al. Downstream processing of lentiviral vectors: releasing bottlenecks. Hum Gene Ther Methods 2012;23:255-63. https://doi.org/10.1089/hgtb.2012.059. · DOI
  5. Kumru OS, Wang Y, Gombotz CWR, Kelley-Clarke B, Cieplak W, Kim T, et al. Physi- cal characterization and stabilization of a lentiviral vector against adsorption and freeze-thaw. J Pharm Sci 2018;107:2764-74. https://doi.org/10.1016/J. XPHS.2018.07.010. · DOI
  6. Higashikawa F, Chang LJ. Kinetic analyses of stability of simple and complex retro- viral vectors. Virology 2001;280:124-31. https://doi.org/10.1006/viro.2000.0743. · DOI
  7. Coroadinha AS, Silva AC, Pires E, Coelho A, Alves PM, Carrondo MJT. Effect of osmotic pressure on the production of retroviral vectors: enhancement in vector stability. Biotechnol Bioeng 2006;94:322-9. https://doi.org/10.1002/bit.20847. · DOI
  8. Gstraunthaler G. Alternatives to the use of fetal bovine serum: serum-free cell culture. ALTEX 2003;20:275-81. https://doi.org/10.14573/altex.2003.4.257. · DOI · Paper page
  9. Leinonen HM, Lipponen EM, Valkama AJ, Hynynen H, Oruetxebarria I, Turkki V, et al. Preclinical proof-of-concept, analytical development, and commercial scale production of lentiviral vector in adherent cells. Mol Ther Methods Clin Dev 2019;15:63-71. https://doi.org/10.1016/j.omtm.2019.08.006. · DOI
  10. van der Valk J, Bieback K, Buta C, Cochrane B, Dirks WG, Fu J, Hickman JJ, Hohen- see C, Kolar R, Liebsch M, Pistollato F, Schulz M, Thieme D, Weber T, Wiest J, Win- kler S, Gstraunthaler G. Fetal bovine serum (FBS): past À present À future. ALTEX -Alternatives to Animal Experimentation 2018;35:99-118.
  11. Broussau S, Jabbour N, Lachapelle G, Durocher Y, Tom R, Transfiguracion J, et al. Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture. Molecular Therapy 2008;16:500-7. https://doi. org/10.1038/sj.mt.6300383. · DOI
  12. Rodrigues AF, Carmo M, Alves PM, Coroadinha AS. Retroviral vector production under serum deprivation: the role of lipids. Biotechnol Bioeng 2009;104:1171- 81. https://doi.org/10.1002/bit.22499. · DOI
  13. Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci 1993;90:8033-7. https://doi.org/10.1073/pnas.90.17.8033. · DOI · Paper page
  14. Ghassemi S, Nunez-Cruz S, O'Connor RS, Fraietta JA, Patel PR, Scholler J, et al. Reducing ex vivo culture improves the antileukemic activity of chimeric antigen receptor (CAR) T cells. Cancer Immunol Res 2018;6:1100-9. https://doi.org/ 10.1158/2326-6066.cir-17-0405. · DOI · Paper page
  15. Gomes-Silva D, Mukherjee M, Srinivasan M, Krenciute G, Dakhova O, Zheng Y, et al. Tonic 4-1BB costimulation in chimeric antigen receptors impedes T cell sur- vival and is vector-dependent. Cell Rep 2017;21:17-26. https://doi.org/10.1016/j. celrep.2017.09.015. · DOI
  16. Haderbache R, Warda W, Hervouet E, da Rocha MN, Trad R, Allain V, et al. Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients. J Transl Med 2021;19:265. https://doi.org/10.1186/s12967-021-02925-z. · DOI
  17. Bauler M, Roberts JK, Wu C-C, Fan B, Ferrara F, Yip BH, et al. Production of lentivi- ral vectors using suspension cells grown in serum-free media. Mol Ther Methods Clin Dev 2020;17:58-68. https://doi.org/10.1016/j.omtm.2019.11.011. · DOI
  18. Boudeffa D, Bertin B, Biek A, Mormin M, Leseigneur F, Galy A, et al. Toward a scal- able purification protocol of GaLV-TR-pseudotyped lentiviral vectors. Hum Gene Ther Methods 2019;30:153-71. https://doi.org/10.1089/hgtb.2019.076. · DOI
  19. Merten OW, Charrier S, Laroudie N, Fauchille S, Dugu e C, Jenny C, et al. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Hum Gene Ther 2011;22:343-56. https://doi.org/ 10.1089/HUM.2010.060. · DOI
  20. Glienke W, Dragon AC, Zimmermann K, Martyniszyn-Eiben A, Mertens M, Abken H, et al. GMP-compliant manufacturing of TRUCKs: CAR T cells targeting GD2 and releasing inducible IL-18. Front Immunol 2022;13:839783. https://doi.org/ 10.3389/fimmu.2022.839783. · DOI
  21. Trist an-Manzano M, Maldonado-P erez N, Justicia-Lirio P, Muñoz P, Cortijo- Guti errez M, Pavlovic K, et al. Physiological lentiviral vectors for the generation of improved CAR-T cells. Mol Ther Oncolytics 2022;25:335-49. https://doi.org/ 10.1016/j.omto.2022.05.003. · DOI
  22. Biffi A, Montini E, Lorioli L, Cesani M, Fumagalli F, Plati T, et al. Lentiviral hemato- poietic stem cell gene therapy benefits metachromatic leukodystrophy. Science (1979) 2013;341:1233158. https://doi.org/10.1126/science.1233158. · DOI · Paper page
  23. Sutlu T, Nystr€ om S, Gilljam M, Stellan B, Applequist SE, Alici E. Inhibition of intra- cellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy. Hum Gene Ther 2012;23:1090- 100. https://doi.org/10.1089/HUM.2012.080. · DOI
  24. Valkama AJ, Oruetxebarria I, Lipponen EM, Leinonen HM, K€ ayhty P, Hynynen H, et al. Development of large-scale downstream processing for lentiviral vectors. Mol Ther Methods Clin Dev 2020;17:717-30. https://doi.org/10.1016/j.omtm. 2020.03.025. · DOI
  25. Labisch JJ, Wiese GP, Barnes K, Bollmann F, Pflanz K. Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach. PLoS One 2021;16:e0254739.
  26. Ghosh R, Koley S, Gopal S, Rodrigues AL, Dordick JS, Cramer SM. Evaluation of len- tiviral vector stability and development of ion exchange purification processes. Biotechnol Prog 2022;38:e3286. https://doi.org/10.1002/btpr.3286. · DOI
  27. Carmo M, Alves A, Rodrigues AF, Coroadinha AS, Carrondo MJ, Alves PM, Cruz PE, et al. Stabilization of gammaretroviral and lentiviral vectors: from production to gene transfer. J Gene Med 2009;11:670-8. https://doi.org/10.1002/JGM.1353. · DOI
  28. Heemskerk MHM, Hoogeboom M, de Paus RA, Kester MGD, van der Hoorn MAWG, Goulmy E, et al. Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-spe- cific T-cell receptor complexes expressing a conserved alpha joining region. Blood 2003;102:3530-40. https://doi.org/10.1182/blood-2003-05-1524. · DOI
  29. Valkama AJ, Leinonen HM, Lipponen EM, Turkki V, Malinen J, Heikura T, et al. Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor. Gene Ther 2018;25:39-46. https://doi.org/10.1038/gt.2017.91. · DOI
  30. Koponen JK, Kekarainen T, Heinonen SE, Laitinen A, Nystedt J, Laine J, et al. Umbil- ical cord bloodÀderived progenitor cells enhance muscle regeneration in mouse hindlimb ischemia model. Molecular Therapy 2007;15:2172-7. https://doi.org/ 10.1038/sj.mt.6300302. · DOI
  31. Kaartinen T, Luostarinen A, Maliniemi P, Keto J, Arvas M, Belt H, et al. Low inter- leukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion. Cytotherapy 2017;19:689-702. https://doi.org/10.1016/j.jcyt.2017.03.067. · DOI
  32. Rasouli M. Basic concepts and practical equations on osmolality: biochemical approach. Clin Biochem 2016;49:936-41. https://doi.org/10.1016/j.clinbio- chem.2016.06.001. · DOI
  33. Figliozzi RW, Chen F, Chi A, Hsia S-CV. Using the inverse Poisson distribution to calculate multiplicity of infection and viral replication by a high-throughput fluo- rescent imaging system. Virol Sin 2016;31:180-3. https://doi.org/10.1007/ s12250-015-3662-8. · DOI
  34. Piatak M, Saag MS, Yang LC, Clark SJ, Kappes JC, Luk K-C, et al. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science (1979) 1993;259:1749-54. https://doi.org/10.1126/science. 8096089. · DOI
  35. Turkki V, Alppila E, Yl€ a-Herttuala S, Lesch HP. Experimental evaluation of an interferometric light microscopy particle counter for titering and characterization of virus preparations. Viruses 2021;13:939. https://doi.org/10.3390/v13050939. · DOI
  36. Koski J, Jahan F, Luostarinen A, Schenkwein D, Yl€ a-Herttuala S, G€ o€ os H, et al. Novel modular chimeric antigen receptor spacer for T cells derived from signal regula- tory protein alpha Ig-like domains. Front Mol Med 2022;2:1049580. https://doi. org/10.3389/fmmed.2022.1049580. · DOI
  37. Ghassemi S, Durgin JS, Nunez-Cruz S, Patel J, Leferovich J, Pinzone M, et al. Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 2022;6:118- 28. https://doi.org/10.1038/s41551-021-00842-6. · DOI
  38. Chockley P, Patil SL, Gottschalk S. Transient blockade of TBK1/IKKe allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors. Cytotherapy 2021;23:787-92. https://doi.org/ 10.1016/J.JCYT.2021.04.010. · DOI
  39. Li L, Gao Y, Srivastava R, Wang W, Xiong Q, Fang Z, et al. Lentiviral delivery of combinatorial CAR/CRISPRi circuit into human primary T cells is enhanced by TBK1/IKKɛ complex inhibitor BX795. J Transl Med 2020;18:363. https://doi.org/ 10.1186/s12967-020-02526-2. · DOI
  40. Greene MR, Lockey T, Mehta PK, Kim Y-S, Eldridge PW, Gray JT, et al. Transduction of human CD34+ repopulating cells with a self-inactivating lentiviral vector for SCID-X1 produced at clinical scale by a stable cell line. Hum Gene Ther Methods 2012;23:297-308. https://doi.org/10.1089/hgtb.2012.150. · DOI
  41. Amorij JP, Meulenaar J, Hinrichs WL, Stegmann T, Huckriede A, Coenen F, Frijlink HW. Rational design of an influenza subunit vaccine powder with sugar glass technology: preventing conformational changes of haemagglutinin during freezing and freeze- drying. Vaccine 2007;25:6447-57. https://doi.org/10.1016/J.VACCINE.2007.06.054. · DOI
  42. Sharma N, Kumar MM, Mohanty KK, Khandelwal V. Optimum use of human AB serum for monocyte-differentiated macrophages (MDMs) isolated from healthy individuals. Re:GEN Open 2022;2:145-9. https://doi.org/10.1089/regen.2022.0014. · DOI
  43. Kelly A. O'Neill, "Mitigating the risks of adventitious agents in serum: Elimination or viral inactivation" in "Advancing Manufacture of Cell and Gene Therapies VI", Dolores Baksh, GE Healthcare, USA Rod Rietze, Novartis, USA Ivan Wall, Aston University, United Kingdom Eds, ECI Symposium Series, (2019). https://dc.eng- confintl.org/cell_gene_therapies_vi/42
  44. Perry C, Mujahid N, Takeuchi Y, Rayat ACME. Insights into product and process related challenges of lentiviral vector bioprocessing. Biotechnol Bioeng 2023: 1- 16. https://doi.org/10.1002/bit.28498. · DOI
  45. Pamenter G, Davies L, Knevelman C, Miskin J, Mitrophanous K, Dikicioglu D, et al. Time-dependent sorption behavior of lentiviral vectors during anion-exchange chromatography. Biotechnol Bioeng 2023;120:2269-82. https://doi.org/10.1002/ bit.28483. · DOI
  46. Sastry L, Xu Y, Cooper R, Pollok K, Cornetta K. Evaluation of plasmid DNA removal from lentiviral vectors by benzonase treatment. Hum Gene Ther 2004;15:221-6. https://doi.org/10.1089/104303404772680029. · DOI
  47. Amaral AI, Coroadinha AS, Merten O-W, Alves PM. Improving retroviral vectors production: role of carbon sources in lipid biosynthesis. J Biotechnol 2008;138:57-66. https://doi.org/10.1016/j.jbiotec.2008.08.005. · DOI
  48. Blache U, Popp G, D€ unkel A, Koehl U, Fricke S. Potential solutions for manufacture of CAR T cells in cancer immunotherapy. Nat Commun 2022;13:5225. https://doi. org/10.1038/s41467-022-32866-0. · DOI
  49. M€ uller S, Bexte T, Gebel V, Kalensee F, Stolzenberg E, Hartmann J, et al. High cytotoxic efficiency of lentivirally and alpharetrovirally engineered CD19- specific chimeric antigen receptor natural killer cells against acute lympho- blastic leukemia. Front Immunol 2020;10:1-16. https://doi.org/10.3389/fim- mu.20219.03123. · DOI

Cited by

Sorted by citations

Authors
Luostarinen, A., Kailaanmäki, A., Turkki, V., Köylijärvi, M., Käyhty, P., Leinonen, H., Albers-Skirdenko, V., Lipponen, E., Ylä-Herttuala, S., Kaartinen, T., Lesch, H. P., & Kekarainen, T.
Affiliations
Finnish Red Cross (FI)
Corresponding author
Luostarinen, Annu
ORCID
Venue
Cytotherapy
Publisher
Elsevier BV
Published
2024-09
Volume
26
Issue
9
Pages
1084-1094
ISSN
1465-3249 (ISSN-L)
DOI
10.1016/j.jcyt.2024.04.002
OpenAlex
W4394060624
Cited by
6
References
0
Citations by year
2026: 4 · 2025: 2
FWCI
1.59
Type
journal article
Language
en
Open access
Licence
Locations
  • Cytotherapy (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Työväentutkimus Vuosikirja (submittedVersion)
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 › RNA Interference and Gene Delivery
Keywords
lentiviral vectors, cryopreservation, chimeric antigen receptor T cells, CAR T cell manufacturing, VSV-G pseudotyping, trehalose, sucrose, HEPES, vector stability, anti-CD19 CAR, primary T cells, vector copy number
MeSH terms
Receptors, Chimeric Antigen, Buffers, Cells, Cultured, Genetic Vectors, Humans, Sucrose, T-Lymphocytes, Transduction, Genetic, Trehalose, Magnesium Chloride, Cryopreservation, Lentivirus, Immunotherapy, Adoptive, Antigens, CD19, Green Fluorescent Proteins

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review