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

Autor: Luostarinen A; Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland. Electronic address: annu.luostarinen@bloodservice.fi., Kailaanmäki A; Kuopio Center for Gene and Cell Therapy, Kuopio, Finland., Turkki V; Kuopio Center for Gene and Cell Therapy, Kuopio, Finland., Köylijärvi M; Kuopio Center for Gene and Cell Therapy, Kuopio, Finland., Käyhty P; Kuopio Center for Gene and Cell Therapy, Kuopio, Finland., Leinonen H; Kuopio Center for Gene and Cell Therapy, Kuopio, Finland., Albers-Skirdenko V; Kuopio Center for Gene and Cell Therapy, Kuopio, Finland., Lipponen E; Kuopio Center for Gene and Cell Therapy, Kuopio, Finland., Ylä-Herttuala S; Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland., Kaartinen T; Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland., Lesch HP; Kuopio Center for Gene and Cell Therapy, Kuopio, Finland., Kekarainen T; Kuopio Center for Gene and Cell Therapy, Kuopio, Finland.
Jazyk: angličtina
Zdroj: Cytotherapy [Cytotherapy] 2024 Sep; Vol. 26 (9), pp. 1084-1094. Date of Electronic Publication: 2024 Apr 07.
DOI: 10.1016/j.jcyt.2024.04.002
Abstrakt: 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 h 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 h 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 h 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.
Competing Interests: Declaration of competing interest AK, VT, MK, VA-S, PK, HL, EL, HPL, and TKe were employed by the Kuopio Center for Gene and Cell Therapy (KCT) during this project. The remaining authors have no commercial, proprietary, or financial interest in the products or companies described in this article.
(Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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