Good manufacturing practice-grade generation of CD19 and CD123-specific CAR-T cells using piggyBac transposon and allogeneic feeder cells in patients diagnosed with B-cell non-Hodgkin lymphoma and acute myeloid leukemia

. 2024 ; 15 () : 1415328. [epub] 20240813

Jazyk angličtina Země Švýcarsko Médium electronic-ecollection

Typ dokumentu časopisecké články

Perzistentní odkaz   https://www.medvik.cz/link/pmid39192973

BACKGROUND: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. METHODS: We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. RESULTS: Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. CONCLUSIONS: The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.

Erratum v

PubMed

Zobrazit více v PubMed

Jo T, Yoshihara S, Okuyama Y, Fujii K, Henzan T, Kahata K, et al. . Risk factors for CAR-T cell manufacturing failure among DLBCL patients: A nationwide survey in Japan. Br J Haematol. (2023) 202:256–66. doi: 10.1111/bjh.18831 PubMed DOI

Huang X, Guo H, Tammana S, Jung Y, Mellgren E, Bassi P, et al. . Gene transfer efficiency and genome-wide integration profiling of sleeping beauty, tol2, and piggyBac transposons in human primary T cells. Mol Ther. (2009) 18:1803–13. doi: 10.1038/mt.2010.141 PubMed DOI PMC

Galvan DL, Nakazawa Y, Kaja A, Kettlun C, Cooper LJN, Rooney CM, et al. . Genome-wide mapping of Piggybac transposon integrations in primary human T cells. J Immunother. (2009) 32:837–44. doi: 10.1097/CJI.0b013e3181b2914c PubMed DOI PMC

Manuri PVR, Wilson MH, Maiti SN, Mi T, Singh H, Olivares S, et al. . PiggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage Malignancies. Hum Gene Ther. (2010) 21:427–37. doi: 10.1089/hum.2009.114 PubMed DOI PMC

Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. (2021) 6:53. doi: 10.1038/s41392-021-00487-6 PubMed DOI PMC

Moretti A, Ponzo M, Nicolette CA, Tcherepanova IY, Biondi A, Magnani CF. The past, present, and future of non-viral CAR T cells. Front Immunol. (2022) 13:867013. doi: 10.3389/fimmu.2022.867013 PubMed DOI PMC

Kaštánková I, Štach M, Žižková H, Ptáčková P, Šmilauerová K, Mucha M, et al. . Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells. Mol Ther Methods Clin Dev. (2021) 23:119–27. doi: 10.1016/j.omtm.2021.08.006 PubMed DOI PMC

Štach M, Ptáčková P, Mucha M, Musil J, Klener P, Otáhal P. Inducible secretion of IL-21 augments anti-tumor activity of piggyBac-manufactured chimeric antigen receptor T cells. Cytotherapy. (2020) 22:744–54. doi: 10.1016/j.jcyt.2020.08.005 PubMed DOI

Ptáčková P, Musil J, Štach M, Lesný P, Němečková Š, Král V, et al. . A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21. Cytotherapy. (2018) 20:507–20. doi: 10.1016/j.jcyt.2017.10.001 PubMed DOI

Riberdy JM, Zhou S, Zheng F, Kim YI, Moore J, Vaidya A, et al. . The art and science of selecting a CD123-specific chimeric antigen receptor for clinical testing. Mol Ther Methods Clin Dev. (2020) 18:571–81. doi: 10.1016/j.omtm.2020.06.024 PubMed DOI PMC

Li X, Burnight ER, Cooney AL, Malani N, Brady T, Sander JD, et al. . piggyBac transposase tools for genome engineering. Proc Natl Acad Sci U S A. (2013) 110:2279–87. doi: 10.1073/pnas.1305987110 PubMed DOI PMC

Yusa K, Zhou L, Li MA, Bradley A, Craig NL. A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci U.S.A. (2011) 108:1531–6. doi: 10.1073/pnas.1008322108 PubMed DOI PMC

Beebe SJ, Fox PM, Rec LJ, Willis ELK, Schoenbach KH. Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells. FASEB Journal: Off Publ Fed Am Societies Exp Biol. (2003) 17:1–23. doi: 10.1096/fj.02-0859fje PubMed DOI

Escoffre JM, Portet T, Favard C, Teissié J, Dean DS, Rols MP. Electromediated formation of DNA complexes with cell membranes and its consequences for gene delivery. Biochim Biophys Acta Biomembr. (2011) 1808:1538–43. doi: 10.1016/j.bbamem.2010.10.009 PubMed DOI

Rosazza C, Deschout H, Buntz A, Braeckmans K, Rols MP, Zumbusch A. Endocytosis and endosomal trafficking of DNA after gene electrotransfer in vitro. Mol Ther Nucleic Acids. (2016) 5:e286. doi: 10.1038/mtna.2015.59 PubMed DOI PMC

Lambricht L, Lopes A, Kos S, Sersa G, Préat V, Vandermeulen G. Clinical potential of electroporation for gene therapy and DNA vaccine delivery. Expert Opin Drug Deliv. (2016) 13:295–310. doi: 10.1517/17425247.2016.1121990 PubMed DOI

Mao M, Wang L, Chang CC, Rothenberg KE, Huang J, Wang Y, et al. . Involvement of a rac1-dependent macropinocytosis pathway in plasmid DNA delivery by electrotransfection. Mol Ther. (2017) 25:803–15. doi: 10.1016/j.ymthe.2016.12.009 PubMed DOI PMC

Golzio M, Escoffre J-M, Portet T, Mauroy C, Teissie J, S. Dean D, et al. . Observations of the mechanisms of electromediated DNA uptake - from vesicles to tissues. Curr Gene Ther. (2010) 10:256–66. doi: 10.2174/156652310791823461 PubMed DOI

Nakamura K, Yagyu S, Hirota S, Tomida A, Kondo M, Shigeura T, et al. . Autologous antigen-presenting cells efficiently expand piggyBac transposon CAR-T cells with predominant memory phenotype. Mol Ther Methods Clin Dev. (2021) 21:315–24. doi: 10.1016/j.omtm.2021.03.011 PubMed DOI PMC

Numbenjapon T, Serrano LM, Chang WC, Forman SJ, Jensen MC, Cooper LJN. Antigen-independent and antigen-dependent methods to numerically expand CD19-specific CD8+ T cells. Exp Hematol. (2007) 35:1083–90. doi: 10.1016/j.exphem.2007.04.007 PubMed DOI

Morita D, Nishio N, Saito S, Tanaka M, Kawashima N, Okuno Y, et al. . Enhanced expression of anti-CD19 chimeric antigen receptor in piggyBac transposon-engineered T cells. Mol Ther Methods Clin Dev. (2018) 8:131–40. doi: 10.1016/j.omtm.2017.12.003 PubMed DOI PMC

Saito S, Nakazawa Y, Sueki A, Matsuda K, Tanaka M, Yanagisawa R, et al. . Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia. Cytother. (2014) 16:1257–69. doi: 10.1016/j.jcyt.2014.05.022 PubMed DOI PMC

Ramanayake S, Bilmon IAN, Bishop D, Dubosq M, Blyth E, Clancy L, et al. . Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials. J Cytother. (2015) 17:1251–67. doi: 10.1016/j.jcyt.2015.05.013 PubMed DOI

Najít záznam

Citační ukazatele

Nahrávání dat ...

Možnosti archivace

Nahrávání dat ...