Abstract
The development of chimeric antigen receptor (CAR) T cells has been a revolutionary technology for the treatment of relapsed and refractory leukemias and lymphomas. The synthetic CAR molecule redirects T cell function toward tumor surface-expressed antigens through a single-chain variable fragment (scFv) fused to CD3z and intracellular costimulatory domains. Here, we describe a protocol for the generation of CAR T cells using primary mouse T cells and a gammaretroviral vector encoding a CAR transgene. This protocol outlines several transduction and expansion methods based on the use of two transduction enhancers, RetroNectin® and Vectofusin®-1, and cell culture systems such as conventional plates or G-Rex® devices.
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References
Baumann JG, Unutmaz D, Miller MD, Breun SK, Grill SM, Mirro J, Littman DR, Rein A, Kewal Ramani VN (2004) Murine T cells potently restrict human immunodeficiency virus infection. J Virol 78(22):12537–12547. https://doi.org/10.1128/JVI.78.22.12537-12547.2004
Tsurutani N, Yasuda J, Yamamoto N, Choi BI, Kadoki M, Iwakura Y (2007) Nuclear import of the preintegration complex is blocked upon infection by human immunodeficiency virus type 1 in mouse cells. J Virol 81(2):677–688. https://doi.org/10.1128/JVI.00870-06
Kerkar SP, Sanchez-Perez L, Yang S, Borman ZA, Muranski P, Ji Y, Chinnasamy D, Kaiser AD, Hinrichs CS, Klebanoff CA, Scott CD, Gattinoni L, Morgan RA, Rosenberg SA, Restifo NP (2011) Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies. J Immunother 34(4):343–352. https://doi.org/10.1097/CJI.0b013e3182187600
Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, Wunderlich J, Hawley RG, Moayeri M, Rosenberg SA, Morgan RA (2005) Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 16(4):457–472. https://doi.org/10.1089/hum.2005.16.457
Naviaux RK, Costanzi E, Haas M, Verma IM (1996) The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J Virol 70(8):5701–5705. https://doi.org/10.1128/JVI.70.8.5701-5705.1996
Evgin L, Kottke T, Tonne J, Thompson J, Huff AL, van Vloten J, Moore M, Michael J, Driscoll C, Pulido J, Swanson E, Kennedy R, Coffey M, Loghmani H, Sanchez-Perez L, Olivier G, Harrington K, Pandha H, Melcher A, Diaz RM, Vile RG (2022) Oncolytic virus-mediated expansion of dual-specific CAR T cells improves efficacy against solid tumors in mice. Sci Transl Med 14(640):eabn2231. https://doi.org/10.1126/scitranslmed.abn2231
Evgin L, Huff AL, Wongthida P, Thompson J, Kottke T, Tonne J, Schuelke M, Ayasoufi K, Driscoll CB, Shim KG, Reynolds P, Monie DD, Johnson AJ, Coffey M, Young SL, Archer G, Sampson J, Pulido J, Perez LS, Vile R (2020) Oncolytic virus-derived type I interferon restricts CAR T cell therapy. Nat Commun 11(1):3187. https://doi.org/10.1038/s41467-020-17011-z
Hanenberg H, Xiao XL, Dilloo D, Hashino K, Kato I, Williams DA (1996) Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med 2(8):876–882. https://doi.org/10.1038/nm0896-876
Majdoul S, Seye AK, Kichler A, Holic N, Galy A, Bechinger B, Fenard D (2016) Molecular determinants of Vectofusin-1 and its derivatives for the enhancement of lentivirally mediated gene transfer into hematopoietic stem/progenitor cells. J Biol Chem 291(5):2161–2169. https://doi.org/10.1074/jbc.M115.675033
Vermeer LS, Hamon L, Schirer A, Schoup M, Cosette J, Majdoul S, Pastre D, Stockholm D, Holic N, Hellwig P, Galy A, Fenard D, Bechinger B (2017) Vectofusin-1, a potent peptidic enhancer of viral gene transfer forms pH-dependent alpha-helical nanofibrils, concentrating viral particles. Acta Biomater 64:259–268. https://doi.org/10.1016/j.actbio.2017.10.009
Radek C, Bernadin O, Drechsel K, Cordes N, Pfeifer R, Strasser P, Mormin M, Gutierrez-Guerrero A, Cosset FL, Kaiser AD, Schaser T, Galy A, Verhoeyen E, Johnston ICD (2019) Vectofusin-1 improves transduction of primary human cells with diverse retroviral and lentiviral pseudotypes, enabling robust, automated closed-system manufacturing. Hum Gene Ther 30(12):1477–1493. https://doi.org/10.1089/hum.2019.157
Vera JF, Brenner LJ, Gerdemann U, Ngo MC, Sili U, Liu H, Wilson J, Dotti G, Heslop HE, Leen AM, Rooney CM (2010) Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex). J Immunother 33(3):305–315. https://doi.org/10.1097/CJI.0b013e3181c0c3cb
Jin J, Sabatino M, Somerville R, Wilson JR, Dudley ME, Stroncek DF, Rosenberg SA (2012) Simplified method of the growth of human tumor infiltrating lymphocytes in gas-permeable flasks to numbers needed for patient treatment. J Immunother 35(3):283–292. https://doi.org/10.1097/CJI.0b013e31824e801f
Bajgain P, Mucharla R, Wilson J, Welch D, Anurathapan U, Liang B, Lu X, Ripple K, Centanni JM, Hall C, Hsu D, Couture LA, Gupta S, Gee AP, Heslop HE, Leen AM, Rooney CM, Vera JF (2014) Optimizing the production of suspension cells using the G-Rex “M” series. Mol Ther Methods Clin Dev 1:14015. https://doi.org/10.1038/mtm.2014.15
Gagliardi C, Khalil M, Foster AE (2019) Streamlined production of genetically modified T cells with activation, transduction and expansion in closed-system G-Rex bioreactors. Cytotherapy 21(12):1246–1257. https://doi.org/10.1016/j.jcyt.2019.10.006
Ludwig J, Hirschel M (2086) Methods and process optimization for large-scale CAR T expansion using the G-Rex cell culture platform. Methods Mol Biol 2020:165–177. https://doi.org/10.1007/978-1-0716-0146-4_12
Gotti E, Tettamanti S, Zaninelli S, Cuofano C, Cattaneo I, Rotiroti MC, Cribioli S, Alzani R, Rambaldi A, Introna M, Golay J (2022) Optimization of therapeutic T cell expansion in G-Rex device and applicability to large-scale production for clinical use. Cytotherapy 24(3):334–343. https://doi.org/10.1016/j.jcyt.2021.11.004
Acknowledgements
The authors thank Steven A. Rosenberg, Richard A. Morgan, and Steven Feldman of the Surgery Branch at the National Cancer Institute for providing us with the MSGV1 retroviral construct into which the mouse CD19 CAR was cloned. This work was funded by BC Cancer Research, the BC Cancer Foundation, CIHR, and an MSHRBC Scholar Award (LE), as well as the Fondation Léon Fredericq (grant to PL) and a Canadian Cancer Society Research Training Award (GS).
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Loos, P., Short, L., Savage, G., Evgin, L. (2024). Expansion and Retroviral Transduction of Primary Murine T Cells for CAR T-Cell Therapy. In: Siciliano, V., Ceroni, F. (eds) Cancer Immunotherapy. Methods in Molecular Biology, vol 2748. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3593-3_4
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DOI: https://doi.org/10.1007/978-1-0716-3593-3_4
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