Chimeric antigen receptor T-cells (CAR T-cells) represent a novel and promising approach in cancer immunotherapy. According to the World Health Organization (WHO), the number of oncological patients is steadily growing in developed countries despite immense progress in oncological treatments, and the prognosis of individual patients is still relatively poor. Exceptional results have been recorded for CAR T-cell therapy in patients suffering from B-cell malignancies. This success opens up the possibility of using the same approach for other types of cancers. To date, the most common method for CAR T-cell generation is the use of viral vectors. However, dealing with virus-derived vectors brings possible obstacles in the CAR T-cell manufacturing process owing to strict regulations and high cost demands. Alternative approaches may facilitate further development and the transfer of the method to clinical practice. The most promising substitutes for virus-derived vectors are transposon-derived vectors, most commonly sleeping beauty, which offer great coding capability and a safe integration profile while maintaining a relatively low production cost. This review is aimed at summarizing the state of the art of nonviral approaches in CAR T-cell generation, with a unique perspective on the conditions in clinical applications and current Good Manufacturing Practice. If CAR T-cell therapy is to be routinely used in medical practice, the manufacturing cost and complexity need to be as low as possible, and transposon-based vectors seem to meet these criteria better than viral-based vectors.

Masaryk University Brno Faculty of Medicine Department of Histology and Embryology Kamenice 5 Brno 62500 Czech Republic

St Anne's University Hospital Brno International Clinical Research Center Pekarska 53 Brno 656 91 Czech Republic

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Gross G., Waks T., Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proceedings of the National Academy of Sciences of the United States of America. 1989;86(24):10024–10028. doi: 10.1073/pnas.86.24.10024. PubMed DOI PMC

Porter D. L., Levine B. L., Kalos M., Bagg A., June C. H. Chimeric antigen receptor–modified T cells in chronic lymphoid leukemia. The New England Journal of Medicine. 2011;365(8):725–733. doi: 10.1056/NEJMoa1103849. PubMed DOI PMC

Kalos M., Levine B. L., Porter D. L., et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Science Translational Medicine. 2011;3(95, article 95ra73) doi: 10.1126/scitranslmed.3002842. PubMed DOI PMC

Maher J., Brentjens R. J., Gunset G., Rivière I., Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ /CD28 receptor. Nature Biotechnology. 2002;20(1):70–75. doi: 10.1038/nbt0102-70. PubMed DOI

Imai C., Mihara K., Andreansky M., et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia. 2004;18(4):676–684. doi: 10.1038/sj.leu.2403302. PubMed DOI

Till B. G., Jensen M. C., Wang J., et al. CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results. Blood. 2012;119(17):3940–3950. doi: 10.1182/blood-2011-10-387969. PubMed DOI PMC

Zhong X.-S., Matsushita M., Plotkin J., Riviere I., Sadelain M. Chimeric antigen receptors combining 4-1BB and CD28 signaling domains augment PI3kinase/AKT/Bcl-XL activation and CD8+ T cell-mediated tumor eradication. Molecular Therapy. 2010;18(2):413–420. doi: 10.1038/mt.2009.210. PubMed DOI PMC

Zhao Y., Wang Q. J., Yang S., et al. A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity. The Journal of Immunology. 2009;183(9):5563–5574. doi: 10.4049/jimmunol.0900447. PubMed DOI PMC

Kerkar S. P., Muranski P., Kaiser A., et al. Tumor-specific CD8+T cells expressing interleukin-12 eradicate established cancers in lymphodepleted hosts. Cancer Research. 2010;70(17):6725–6734. doi: 10.1158/0008-5472.CAN-10-0735. PubMed DOI PMC

Chmielewski M., Hombach A. A., Abken H. Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma. Immunological Reviews. 2014;257(1):83–90. doi: 10.1111/imr.12125. PubMed DOI

Krenciute G., Prinzing B. L., Yi Z., et al. Transgenic expression of IL15 improves antiglioma activity of IL13Rα2-CAR T cells but results in antigen loss variants. Cancer Immunology Research. 2017;5(7):571–581. doi: 10.1158/2326-6066.CIR-16-0376. PubMed DOI PMC

Avanzi M. P., Yeku O., Li X., et al. Engineered tumor-targeted T cells mediate enhanced anti-tumor efficacy both directly and through activation of the endogenous immune system. Cell Reports. 2018;23(7):2130–2141. doi: 10.1016/j.celrep.2018.04.051. PubMed DOI PMC

Hegde M., Mukherjee M., Grada Z., et al. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape. The Journal of Clinical Investigation. 2016;126(8):3036–3052. doi: 10.1172/JCI83416. PubMed DOI PMC

Morgan R. A., Boyerinas B. Genetic modification of T cells. Biomedicine. 2016;4(2):p. 9. PubMed PMC

Fesnak A. D., June C. H., Levine B. L. Engineered T cells: the promise and challenges of cancer immunotherapy. Nature Reviews. Cancer. 2016;16(9):566–581. doi: 10.1038/nrc.2016.97. PubMed DOI PMC

Cavazzana-Calvo M., Hacein-Bey S., de Saint Basile G., et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science. 2000;288(5466):669–672. doi: 10.1126/science.288.5466.669. PubMed DOI

Hacein-Bey-Abina S., Garrigue A., Wang G. P., et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. The Journal of Clinical Investigation. 2008;118(9):3132–3142. doi: 10.1172/JCI35700. PubMed DOI PMC

Naldini L., Blomer U., Gallay P., et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996;272(5259):263–267. doi: 10.1126/science.272.5259.263. PubMed DOI

Jarrosson-Wuilleme L., Goujon C., Bernaud J., Rigal D., Darlix J.-L., Cimarelli A. Transduction of nondividing human macrophages with gammaretrovirus-derived vectors. Journal of Virology. 2006;80(3):1152–1159. doi: 10.1128/JVI.80.3.1152-1159.2006. PubMed DOI PMC

Ciuffi A. Mechanisms lentivirus integration site selection. Current Gene Therapy. 2008;8:419–429. 2020, https://www.eurekaselect.com/68097/article. PubMed

Ramanayake S., Bilmon I., Bishop D., 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. Cytotherapy. 2015;17(9):1251–1267. doi: 10.1016/j.jcyt.2015.05.013. PubMed DOI

Bouard D., Alazard-Dany N., Cosset F.-L. Viral vectors: from virology to transgene expression. British Journal of Pharmacology. 2009;157(2):153–165. doi: 10.1038/bjp.2008.349. PubMed DOI PMC

Singh H., Moyes J. S. E., Huls M. H., Cooper L. J. N. Manufacture of T cells using the Sleeping Beauty system to enforce expression of a CD19-specific chimeric antigen receptor. Cancer Gene Therapy. 2015;22(2):95–100. doi: 10.1038/cgt.2014.69. PubMed DOI

Chicaybam L., Abdo L., Carneiro M., et al. CAR T cells generated using sleeping beauty transposon vectors and expanded with an EBV-transformed lymphoblastoid cell line display antitumor activity in vitro and in vivo. Human Gene Therapy. 2019;30(4):511–522. doi: 10.1089/hum.2018.218. PubMed DOI

Yant S. R., Meuse L., Chiu W., Ivics Z., Izsvak Z., Kay M. A. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nature Genetics. 2000;25(1):35–41. doi: 10.1038/75568. PubMed DOI

Izsvák Z., Hackett P. B., Cooper L. J. N., Ivics Z. Translating sleeping beauty transposition into cellular therapies: victories and challenges. BioEssays. 2010;32(9):756–767. doi: 10.1002/bies.201000027. PubMed DOI PMC

Geurts A. M., Yang Y., Clark K. J., et al. Gene transfer into genomes of human cells by the sleeping beauty transposon system. Molecular Therapy. 2003;8(1):108–117. doi: 10.1016/S1525-0016(03)00099-6. PubMed DOI

Jin Z., Maiti S., Huls H., et al. The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor. Gene Therapy. 2011;18(9):849–856. doi: 10.1038/gt.2011.40. PubMed DOI PMC

Yant S., Wu X., Huang Y., et al. 817. Nonrandom insertion site preferences for the SB transposon in vitro and in vivo. Molecular Therapy. 2004;9:S309–S310.

Mitchell R. S., Beitzel B. F., Schroder A. R. W., et al. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biology. 2004;2(8, article E234) doi: 10.1371/journal.pbio.0020234. PubMed DOI PMC

Kebriaei P., Singh H., Huls M. H., et al. Phase I trials using sleeping beauty to generate CD19-specific CAR T cells. The Journal of Clinical Investigation. 2016;126(9):3363–3376. doi: 10.1172/JCI86721. PubMed DOI PMC

Huang X., Guo H., Tammana S., et al. Gene transfer efficiency and genome-wide integration profiling of Sleeping Beauty , Tol2 , and piggyBac transposons in human primary T cells. Molecular Therapy. 2010;18(10):1803–1813. doi: 10.1038/mt.2010.141. PubMed DOI PMC

Tsukahara T., Iwase N., Kawakami K., et al. The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies. Gene Therapy. 2015;22(2):209–215. doi: 10.1038/gt.2014.104. PubMed DOI PMC

Urasaki A., Morvan G., Kawakami K. Functional dissection of the Tol 2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics. 2006;174(2):639–649. doi: 10.1534/genetics.106.060244. PubMed DOI PMC

Cary L. C., Goebel M., Corsaro B. G., Wang H.-G., Rosen E., Fraser M. J. Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology. 1989;172(1):156–169. doi: 10.1016/0042-6822(89)90117-7. PubMed DOI

Fraser M. J., Clszczon T., Elick T., Bauser C. Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Molecular Biology. 1996;5(2):141–151. doi: 10.1111/j.1365-2583.1996.tb00048.x. PubMed DOI

Galvan D. L., Nakazawa Y., Kaja A., et al. Genome-wide mapping of PiggyBac transposon integrations in primary human T cells. Journal of Immunotherapy. 2009;32(8):837–844. doi: 10.1097/CJI.0b013e3181b2914c. PubMed DOI PMC

Gogol-Döring A., Ammar I., Gupta S., et al. Genome-wide profiling reveals remarkable parallels between insertion site selection properties of the MLV retrovirus and the piggyBac transposon in primary human CD4+ T cells. Molecular Therapy. 2016;24(3):592–606. doi: 10.1038/mt.2016.11. PubMed DOI PMC

Wu S. C.-Y., Meir Y.-J. J., Coates C. J., et al. piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(41):15008–15013. doi: 10.1073/pnas.0606979103. PubMed DOI PMC

Nakazawa Y., Huye L. E., Dotti G., et al. Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes. Journal of Immunotherapy. 2009;32(8):826–836. doi: 10.1097/CJI.0b013e3181ad762b. PubMed DOI PMC

Morgan R. A., Yang J. C., Kitano M., Dudley M. E., Laurencot C. M., Rosenberg S. A. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Molecular Therapy. 2010;18(4):843–851. doi: 10.1038/mt.2010.24. PubMed DOI PMC

Yusa K., Zhou L., Li M. A., Bradley A., Craig N. L. A hyperactive piggyBac transposase for mammalian applications. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(4):1531–1536. doi: 10.1073/pnas.1008322108. PubMed DOI PMC

Doherty J. E., Huye L. E., Yusa K., Zhou L., Craig N. L., Wilson M. H. Hyperactive piggyBac gene transfer in human cells and in vivo. Human Gene Therapy. 2012;23(3):311–320. doi: 10.1089/hum.2011.138. PubMed DOI PMC

Cadiñanos J., Bradley A. Generation of an inducible and optimized piggyBac transposon system†. Nucleic Acids Research. 2007;35(12, article e87) doi: 10.1093/nar/gkm446. PubMed DOI PMC

Saito S., Nakazawa Y., Sueki A., et al. Anti-leukemic potency of piggyBac -mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia. Cytotherapy. 2014;16(9):1257–1269. doi: 10.1016/j.jcyt.2014.05.022. PubMed DOI PMC

Wang J., Lupo K. B., Chambers A. M., Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. Journal for ImmunoTherapy of Cancer. 2018;6(1):p. 136. doi: 10.1186/s40425-018-0441-8. PubMed DOI PMC

Zhang Z., Jiang D., Yang H., et al. Modified CAR T cells targeting membrane-proximal epitope of mesothelin enhances the antitumor function against large solid tumor. Cell Death & Disease. 2019;10(7):p. 476. doi: 10.1038/s41419-019-1711-1. PubMed DOI PMC

Ma Y., Chen Y., Yan L., et al. EGFRvIII-specific CAR-T cells produced by piggyBac transposon exhibit efficient growth suppression against hepatocellular carcinoma. International Journal of Medical Sciences. 2020;17(10):1406–1414. doi: 10.7150/ijms.45603. PubMed DOI PMC

Ptáčková P., Musil J., Štach M., 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(4):507–520. doi: 10.1016/j.jcyt.2017.10.001. PubMed DOI

Tseng W.-C., Haselton F. R., Giorgio T. D. Mitosis enhances transgene expression of plasmid delivered by cationic liposomes. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1999;1445(1):53–64. doi: 10.1016/S0167-4781(99)00039-1. PubMed DOI

Karikó K., Muramatsu H., Welsh F. A., et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Molecular Therapy. 2008;16(11):1833–1840. doi: 10.1038/mt.2008.200. PubMed DOI PMC

Zhao Y., Zheng Z., Cohen C. J., et al. High - efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Molecular Therapy. 2006;13(1):151–159. doi: 10.1016/j.ymthe.2005.07.688. PubMed DOI PMC

Harrer D. C., Simon B., Fujii S., et al. RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/β T-cell receptor: a safer alternative to genetically engineered α/β T cells for the immunotherapy of melanoma. BMC Cancer. 2017;17(1):p. 551. doi: 10.1186/s12885-017-3539-3. PubMed DOI PMC

Campillo-Davo D., Fujiki F., Van den Bergh J. M. J., et al. Efficient and nongenotoxic RNA-based engineering of human T cells using tumor-specific T cell receptors with minimal TCR mispairing. Frontiers in Immunology. 2018;9 doi: 10.3389/fimmu.2018.02503. PubMed DOI PMC

Mensali N., Myhre M. R., Dillard P., et al. Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy. Cancer Immunology, Immunotherapy. 2019;68(8):1235–1243. doi: 10.1007/s00262-019-02356-2. PubMed DOI PMC

Mensali N., Myhre M. R., Dillard P., et al. Correction to: preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy. Cancer Immunology, Immunotherapy. 2020;69(1):159–161. doi: 10.1007/s00262-019-02409-6. PubMed DOI PMC

Billingsley M. M., Singh N., Ravikumar P., Zhang R., June C. H., Mitchell M. J. Ionizable lipid nanoparticle-mediated mRNA delivery for human CAR T cell engineering. Nano Letters. 2020;20(3):1578–1589. doi: 10.1021/acs.nanolett.9b04246. PubMed DOI PMC

Grabundzija I., Irgang M., Mátés L., et al. Comparative analysis of transposable element vector systems in human cells. Molecular Therapy. 2010;18(6):1200–1209. doi: 10.1038/mt.2010.47. PubMed DOI PMC

Field A.-C., Vink C., Gabriel R., et al. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer. PLoS One. 2013;8(6, article e68201) doi: 10.1371/journal.pone.0068201. PubMed DOI PMC

Beard B. C., Dickerson D., Beebe K., et al. Comparison of HIV-derived lentiviral and MLV-based gammaretroviral vector integration sites in primate repopulating cells. Molecular Therapy. 2007;15(7):1356–1365. doi: 10.1038/sj.mt.6300159. PubMed DOI

Schröder A. R. W., Shinn P., Chen H., Berry C., Ecker J. R., Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002;110(4):521–529. doi: 10.1016/S0092-8674(02)00864-4. PubMed DOI

Hartmann J., Schüßler-Lenz M., Bondanza A., Buchholz C. J. Clinical development of CAR T cells—challenges and opportunities in translating innovative treatment concepts. EMBO Molecular Medicine. 2017;9(9):1183–1197. doi: 10.15252/emmm.201607485. PubMed DOI PMC

Vormittag P., Gunn R., Ghorashian S., Veraitch F. S. A guide to manufacturing CAR T cell therapies. Current Opinion in Biotechnology. 2018;53:164–181. doi: 10.1016/j.copbio.2018.01.025. PubMed DOI

Merten O.-W., Hebben M., Bovolenta C. Production of lentiviral vectors. Molecular Therapy - Methods & Clinical Development. 2016;3:p. 16017. doi: 10.1038/mtm.2016.17. PubMed DOI PMC

Dull T., Zufferey R., Kelly M., et al. A third-generation lentivirus vector with a conditional packaging system. Journal of Virology. 1998;72(11):8463–8471. doi: 10.1128/JVI.72.11.8463-8471.1998. PubMed DOI PMC

Sanber K. S., Knight S. B., Stephen S. L., et al. Construction of stable packaging cell lines for clinical lentiviral vector production. Scientific Reports. 2015;5(1) doi: 10.1038/srep09021. PubMed DOI PMC

Turtle C. J., Hanafi L.-A., Berger C., et al. CD19 CAR–T cells of defined CD4+: CD8+ composition in adult B cell ALL patients. Journal of Clinical Investigation. 2016;126(6):2123–2138. doi: 10.1172/JCI85309. PubMed DOI PMC

Kochenderfer J. N., Somerville R., Lu L., et al. Anti-CD19 CAR T cells administered after low-dose chemotherapy can induce remissions of chemotherapy-refractory diffuse large B-cell lymphoma. Blood. 2014;124(21):550–550. doi: 10.1182/blood.V124.21.550.550. DOI

Liu E., Marin D., Banerjee P., et al. Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. The New England Journal of Medicine. 2020;382(6):545–553. doi: 10.1056/NEJMoa1910607. PubMed DOI PMC

Srour S. A., Singh H., McCarty J., et al. Long-term outcomes of Sleeping Beauty-generated CD19-specific CAR T-cell therapy for relapsed-refractory B-cell lymphomas. Blood. 2020;135(11):862–865. doi: 10.1182/blood.2019002920. PubMed DOI PMC

Magnani C. F., Gaipa G., Lussana F., et al. Sleeping Beauty–engineered CAR T cells achieve antileukemic activity without severe toxicities. The Journal of Clinical Investigation. 2020;130(11):6021–6033. doi: 10.1172/JCI138473. PubMed DOI PMC

Singh H., Figliola M. J., Dawson M. J., et al. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells. PLoS One. 2013;8(5, article e64138) doi: 10.1371/journal.pone.0064138. PubMed DOI PMC

Fielding A. K., Richards S. M., Chopra R., et al. Outcome of 609 adults after relapse of acute lymphoblastic leukemia (ALL); an MRC UKALL12/ECOG 2993 study. Blood. 2007;109(3):944–950. doi: 10.1182/blood-2006-05-018192. PubMed DOI

Raja Manuri P. V., Wilson M. H., Maiti S. N., et al. piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Human Gene Therapy. 2010;21(4):427–437. doi: 10.1089/hum.2009.114. PubMed DOI PMC

Gee A. P. GMP CAR-T cell production. Best Practice & Research Clinical Haematology. 2018;31(2):126–134. doi: 10.1016/j.beha.2018.01.002. PubMed DOI

Fernández L., Fernández A., Mirones I., et al. GMP-compliant manufacturing of NKG2D CAR memory T cells using CliniMACS prodigy. Frontiers in Immunology. 2019;10 doi: 10.3389/fimmu.2019.02361. PubMed DOI PMC

Mock U., Nickolay L., Philip B., et al. Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy. Cytotherapy. 2016;18(8):1002–1011. doi: 10.1016/j.jcyt.2016.05.009. PubMed DOI

Rojewski M. T., Fekete N., Baila S., et al. GMP-compliant isolation and expansion of bone marrow-derived MSCs in the closed, automated device quantum cell expansion system. Cell Transplantation. 2013;22(11):1981–2000. doi: 10.3727/096368912X657990. PubMed DOI

Coeshott C., Vang B., Jones M., Nankervis B. Large-scale expansion and characterization of CD3+ T-cells in the quantum® cell expansion system. Journal of Translational Medicine. 2019;17(1):p. 258. doi: 10.1186/s12967-019-2001-5. PubMed DOI PMC

Walker A., Johnson R. Commercialization of cellular immunotherapies for cancer. Biochemical Society Transactions. 2016;44(2):329–332. doi: 10.1042/BST20150240. PubMed DOI

Wiesinger M., März J., Kummer M., et al. Clinical-scale production of CAR-T cells for the treatment of melanoma patients by mRNA transfection of a CSPG4-specific CAR under full GMP compliance. Cancers. 2019;11(8):p. 1198. doi: 10.3390/cancers11081198. PubMed DOI PMC

Bouet-Toussaint F., Genetel N., Rioux-Leclercq N., et al. Interleukin-2 expanded lymphocytes from lymph node and tumor biopsies of human renal cell carcinoma, breast and ovarian cancer. European Cytokine Network. 2000;11(2):217–224. PubMed

Crompton J. G., Sukumar M., Restifo N. P. Uncoupling T-cell expansion from effector differentiation in cell-based immunotherapy. Immunological Reviews. 2014;257(1):264–276. doi: 10.1111/imr.12135. PubMed DOI PMC

Kaartinen T., Luostarinen A., Maliniemi P., et al. Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion. Cytotherapy. 2017;19(6):689–702. doi: 10.1016/j.jcyt.2017.03.067. PubMed DOI

Sabatino M., Hu J., Sommariva M., et al. Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies. Blood. 2016;128(4):519–528. doi: 10.1182/blood-2015-11-683847. PubMed DOI PMC

Hinrichs C. S., Spolski R., Paulos C. M., et al. IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy. Blood. 2008;111(11):5326–5333. doi: 10.1182/blood-2007-09-113050. PubMed DOI PMC

Cieri N., Camisa B., Cocchiarella F., et al. IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood. 2013;121(4):573–584. doi: 10.1182/blood-2012-05-431718. PubMed DOI

Zhou J., Jin L., Wang F., Zhang Y., Liu B., Zhao T. Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects. Protein & Cell. 2019;10(10):764–769. doi: 10.1007/s13238-019-0643-y. PubMed DOI PMC

Leen A. M., Sukumaran S., Watanabe N., et al. Reversal of tumor immune inhibition using a chimeric cytokine receptor. Molecular Therapy. 2014;22(6):1211–1220. doi: 10.1038/mt.2014.47. PubMed DOI PMC

Wang Y., Jiang H., Luo H., et al. An IL-4/21 inverted cytokine receptor improving CAR-T cell potency in immunosuppressive solid-tumor microenvironment. Frontiers in Immunology. 2019;10 doi: 10.3389/fimmu.2019.01691. PubMed DOI PMC

Chavez A. T., McKenna M. K., Canestrari E., et al. Expanding CAR T cells in human platelet lysate renders T cells with in vivo longevity. Journal for Immunotherapy of Cancer. 2019;7(1):p. 330. doi: 10.1186/s40425-019-0804-9. PubMed DOI PMC

Stemberger C., Dreher S., Tschulik C., et al. Novel serial positive enrichment technology enables clinical multiparameter cell sorting. PLoS One. 2012;7(4, article e35798) doi: 10.1371/journal.pone.0035798. PubMed DOI PMC

McBride J. A., Striker R. Imbalance in the game of T cells: what can the CD4/CD8 T-cell ratio tell us about HIV and health? PLoS Pathogens. 2017;13(11, article e1006624) doi: 10.1371/journal.ppat.1006624. PubMed DOI PMC

Mackall C. L., Fleisher T. A., Brown M. R., et al. Age, thymopoiesis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy. The New England Journal of Medicine. 1995;332(3):143–149. doi: 10.1056/NEJM199501193320303. PubMed DOI

Graef P., Buchholz V. R., Stemberger C., et al. Serial transfer of single-cell-derived immunocompetence reveals stemness of CD8+ central memory T cells. Immunity. 2014;41(1):116–126. doi: 10.1016/j.immuni.2014.05.018. PubMed DOI

Buchholz V. R., Flossdorf M., Hensel I., et al. Disparate individual fates compose robust CD8+ T cell immunity. Science. 2013;340(6132):630–635. doi: 10.1126/science.1235454. PubMed DOI

Gattinoni L., Lugli E., Ji Y., et al. A human memory T cell subset with stem cell-like properties. Nature Medicine. 2011;17(10):1290–1297. doi: 10.1038/nm.2446. PubMed DOI PMC

Sommermeyer D., Hudecek M., Kosasih P. L., et al. Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia. 2016;30(2):492–500. doi: 10.1038/leu.2015.247. PubMed DOI PMC

Moeller M., Kershaw M. H., Cameron R., et al. Sustained antigen-specific antitumor recall response mediated by gene-modified CD4+ T helper-1 and CD8+ T cells. Cancer Research. 2007;67(23):11428–11437. doi: 10.1158/0008-5472.CAN-07-1141. PubMed DOI