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Rapid manufacturing of non-activated potent CAR T cells

Nature Biomedical Engineering volume 6, pages 118–128 (2022)Cite this article

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Abstract

Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability.

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Fig. 1: Lentiviral vectors transduce non-activated T-cell subsets with a preference for memory subsets.
Fig. 2: CAR lentivirus mediates pseudotransduction in non-activated T cells.
Fig. 3: Non-activated T cells expressing CAR19 control leukaemia in xenograft models of ALL.
Fig. 4: The transducing conditions can enhance the transduction efficiency in non-activated T cells.
Fig. 5: Non-activated CART19 cells induce potent and durable remission of ALL at low doses.
Fig. 6: Non-activated CAR T cells generated from patient samples show potent efficacy in vivo.

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Data availability

The main data supporting the findings of this study are available within the article and its Supplementary Information. All raw data generated during the study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

References

  1. Salter, A. I., Pont, M. J. & Riddell, S. R. Chimeric antigen receptor-modified T cells: CD19 and the road beyond. Blood 131, 2621–2629 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Brudno, J. N. et al. T cells genetically modified to express an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of poor-prognosis relapsed multiple myeloma. J. Clin. Oncol. 36, 2267–2280 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cohen, A. D. et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J. Clin. Investig. 129, 2210–2221 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  4. D’Angelo, S. P. et al. Antitumor activity associated with prolonged persistence of adoptively transferred NY-ESO-1 (c259)T cells in synovial sarcoma. Cancer Discov. 8, 944–957 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Raje, N. et al. Anti-BCMA CAR T-cell therapy bb2121 in relapsed or refractory multiple myeloma. N. Engl. J. Med. 380, 1726–1737 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kalos, M. et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci. Transl. Med. 3, 95ra73 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ghassemi, S. et al. Reducing ex vivo culture improves the antileukemic activity of chimeric antigen receptor (CAR) T cells. Cancer Immunol. Res. 6, 1100–1109 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Berger, C. et al. Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates. J. Clin. Investig. 118, 294–305 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Graef, P. et al. Serial transfer of single-cell-derived immunocompetence reveals stemness of CD8+ central memory T cells. Immunity 41, 116–126 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Hinrichs, C. S. et al. Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity. Proc. Natl Acad. Sci. USA 106, 17469–17474 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hinrichs, C. S. et al. Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy. Blood 117, 808–814 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Klebanoff, C. A. et al. Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J. Clin. Invest. 126, 318–334 (2016).

    Article  PubMed  Google Scholar 

  13. Crompton, J. G. et al. Akt inhibition enhances expansion of potent tumor-specific lymphocytes with memory cell characteristics. Cancer Res. 75, 296–305 (2015).

    Article  CAS  PubMed  Google Scholar 

  14. van der Waart, A. B. et al. Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy. Blood 124, 3490–3500 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Gattinoni, L. et al. Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat. Med. 15, 808–813 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gattinoni, L. et al. A human memory T cell subset with stem cell-like properties. Nat. Med. 17, 1290–1297 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Muralidharan, S. et al. Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes. J. Immunol. 187, 5221–5232 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

    Article  CAS  PubMed  Google Scholar 

  19. Plesa, G. et al. Addition of deoxynucleosides enhances human immunodeficiency virus type 1 integration and 2LTR formation in resting CD4+ T cells. J. Virol. 81, 13938–13942 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Swiggard, W. J. et al. Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli. J. Virol. 79, 14179–14188 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Amirache, F. et al. Mystery solved: VSV-G-LVs do not allow efficient gene transfer into unstimulated T cells, B cells, and HSCs because they lack the LDL receptor. Blood 123, 1422–1424 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Finkelshtein, D., Werman, A., Novick, D., Barak, S. & Rubinstein, M. LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proc. Natl Acad. Sci. USA 110, 7306–7311 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Agosto, L. M. et al. The CXCR4-tropic human immunodeficiency virus envelope promotes more-efficient gene delivery to resting CD4+ T cells than the vesicular stomatitis virus glycoprotein G envelope. J. Virol. 83, 8153–8162 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pace, M. J., Agosto, L. & O’Doherty, U. R5 HIV env and vesicular stomatitis virus G protein cooperate to mediate fusion to naive CD4+ T cells. J. Virol. 85, 644–648 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Unutmaz, D., KewalRamani, V. N., Marmon, S. & Littman, D. R. Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes. J. Exp. Med. 189, 1735–1746 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cavalieri, S. et al. Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact immune competence. Blood 102, 497–505 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Trinite, B., Chan, C. N., Lee, C. S. & Levy, D. N. HIV-1 Vpr- and reverse transcription-induced apoptosis in resting peripheral blood CD4 T cells and protection by common gamma-chain cytokines. J. Virol. 90, 904–916 (2016).

    Article  CAS  PubMed  Google Scholar 

  28. Verhoeyen, E. et al. IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes. Blood 101, 2167–2174 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Korin, Y. D. & Zack, J. A. Nonproductive human immunodeficiency virus type 1 infection in nucleoside-treated G0 lymphocytes. J. Virol. 73, 6526–6532 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Rausell, A. et al. Innate immune defects in HIV permissive cell lines. Retrovirology 13, 43 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Descours, B. et al. SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T-cells. Retrovirology 9, 87 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Pierson, T. C. et al. Molecular characterization of preintegration latency in human immunodeficiency virus type 1 infection. J. Virol. 76, 8518–8531 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Milone, M. C. et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol. Ther. 17, 1453–1464 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Burnie, J. & Guzzo, C. The incorporation of host proteins into the external HIV-1 envelope. Viruses 11, 85 (2019).

  35. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wilen, C. B., Tilton, J. C. & Doms, R. W. HIV: cell binding and entry. Cold Spring Harb. Perspect. Med. 2, a006866 (2012).

  37. Brown, M. S. & Goldstein, J. L. A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47 (1986).

    Article  CAS  PubMed  Google Scholar 

  38. Tran, R. et al. Microfluidic transduction harnesses mass transport principles to enhance gene transfer efficiency. Mol. Ther. 25, 2372–2382 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Maude, S. L. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N. Engl. J. Med. 371, 1507–1517 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Berry, C. C. et al. INSPIIRED: quantification and visualization tools for analyzing integration site distributions. Mol. Ther. Methods Clin. Dev. 4, 17–26 (2017).

    Article  CAS  PubMed  Google Scholar 

  41. Sherman, E. et al. INSPIIRED: a pipeline for quantitative analysis of sites of new DNA integration in cellular genomes. Mol. Ther. Methods Clin. Dev. 4, 39–49 (2017).

    Article  CAS  PubMed  Google Scholar 

  42. Berry, C. C. et al. Estimating abundances of retroviral insertion sites from DNA fragment length data. Bioinformatics 28, 755–762 (2012).

  43. Nobles, C. L. et al. CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration. J. Clin. Invest. 130, 673–685 (2019).

  44. Schroder, A. R. et al. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110, 521–529 (2002).

    Article  CAS  PubMed  Google Scholar 

  45. Mitchell, R. S. et al. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol. 2, E234 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Wang, G. P., Ciuffi, A., Leipzig, J., Berry, C. C. & Bushman, F. D. HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic modifications. Genome Res. 17, 1186–1194 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Brady, T. et al. HIV integration site distributions in resting and activated CD4+ T cells infected in culture. AIDS 23, 1461–1471 (2009).

    Article  PubMed  Google Scholar 

  48. Haining, W. N. et al. Antigen-specific T-cell memory is preserved in children treated for acute lymphoblastic leukemia. Blood 106, 1749–1754 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Halliwell, B. Cell culture, oxidative stress, and antioxidants: avoiding pitfalls. Biomed. J. 37, 99–105 (2014).

    PubMed  Google Scholar 

  50. Zhang, H. et al. 4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy. J. Immunol. 179, 4910–4918 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Klebanoff, C. A. et al. Determinants of successful CD8+ T-cell adoptive immunotherapy for large established tumors in mice. Clin. Cancer Res. 17, 5343–5352 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Klebanoff, C. A. et al. Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy. JCI Insight 2, e95103 (2017).

  53. O’Doherty, U., Swiggard, W. J. & Malim, M. H. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J. Virol. 74, 10074–10080 (2000).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Baldauf, H. M. et al. SAMHD1 restricts HIV-1 infection in resting CD4+ T cells. Nat. Med. 18, 1682–1687 (2012).

    Article  CAS  PubMed  Google Scholar 

  55. Mauney, C. H., Perrino, F. W. & Hollis, T. Identification of inhibitors of the dNTP triphosphohydrolase SAMHD1 using a novel and direct high-throughput assay. Biochemistry 57, 6624–6636 (2018).

    Article  CAS  PubMed  Google Scholar 

  56. Trobridge, G. D. et al. Cocal-pseudotyped lentiviral vectors resist inactivation by human serum and efficiently transduce primate hematopoietic repopulating cells. Mol. Ther. 18, 725–733 (2010).

    Article  CAS  PubMed  Google Scholar 

  57. Klebanoff, C. A. et al. Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc. Natl Acad. Sci. USA 102, 9571–9576 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Pollok, K. E., Kim, S. H. & Kwon, B. S. Regulation of 4-1BB expression by cell–cell interactions and the cytokines, interleukin-2 and interleukin-4. Eur. J. Immunol. 25, 488–494 (1995).

    Article  CAS  PubMed  Google Scholar 

  59. Lelek, M. et al. Chromatin organization at the nuclear pore favours HIV replication. Nat. Commun. 6, 6483 (2015).

    Article  CAS  PubMed  Google Scholar 

  60. Wong, R. W., Mamede, J. I. & Hope, T. J. Impact of nucleoporin-mediated chromatin localization and nuclear architecture on HIV integration site selection. J. Virol. 89, 9702–9705 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Couzin-Frankel, J. Supply of promising T cell therapy is strained. Science 356, 1112–1113 (2017).

    Article  CAS  PubMed  Google Scholar 

  62. O’Connor, R. S. et al. Substrate rigidity regulates human T cell activation and proliferation. J. Immunol. 189, 1330–1339 (2012).

    Article  PubMed  Google Scholar 

  63. De Spiegelaere, W. et al. Quantification of integrated HIV DNA by repetitive-sampling Alu-HIV PCR on the basis of poisson statistics. Clin. Chem. 60, 886–895 (2014).

    Article  PubMed  Google Scholar 

  64. Liszewski, M. K., Yu, J. J. & O’Doherty, U. Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR. Methods 47, 254–260 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Kenderian, S. S. et al. CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia. Leukemia 29, 1637–1647 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the University of Pennsylvania Cell and Vaccine Production Facility for technical support as well as the University of Pennsylvania Stem Cell and Xenograft Core and University of Pennsylvania Flow Cytometry Core and Human Immunology Core, which is supported by NIH grants nos AI045008 and CA016520, for providing de-identified human T cells. We thank J. K. Everett and A. M. Roche for their technical expertise. This work was supported in part through funding provided by Novartis Pharmaceuticals through a research alliance with the University of Pennsylvania (M.C.M.) as well as a St. Baldrick’s Foundation Scholar Award (S.G.), a National Blood Foundation Scientific Research Grant Award (S.G.), the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Cancer Research Program under award nos W81XWH-20-1-0417 (S.G.) and RO1CA226983 (R.S.O’C.).

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Authors and Affiliations

  1. Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Saba Ghassemi, Joseph S. Durgin, Selene Nunez-Cruz, Jai Patel, John Leferovich, Feng Shen, Katherine D. Cummins, Gabriela Plesa, Saar I. Gill, Roddy S. O’Connor & Michael C. Milone

  2. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Saba Ghassemi, Selene Nunez-Cruz, John Leferovich, Marilia Pinzone, Una O’Doherty, Roddy S. O’Connor & Michael C. Milone

  3. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Vito Adrian Cantu, Shantan Reddy & Frederic D. Bushman

  4. Division of Hematology–Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Saar I. Gill

Authors
  1. Saba Ghassemi
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  2. Joseph S. Durgin
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Contributions

S.G. and M.C.M. designed the study. S.G., M.C.M., J.S.D., R.S.O’C., U.O’D., F.D.B. and S.I.G. provided conceptual guidance. S.G., J.S.D, R.S.O’C, S.N.-C., J.P., K.D.C., F.S., M.P., G.P., V.A.C., S.R. and J.L. performed the experiments. S.G. and M.C.M. analysed the data. S.G. and M.C.M. wrote the manuscript. R.S.O’C and J.S.D. read and made comments on the manuscript.

Corresponding authors

Correspondence to Saba Ghassemi or Michael C. Milone.

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Competing interests

M.C.M. is an inventor on patent applications related to CAR technology (US patents 9,481,728 and 9,499,629 B2) and has received licencing royalties from the Novartis corporation. S.G. and M.C.M. are inventors on a patent application related to methods of manufacturing non-activated CAR T cells (provisional patent PCT/US2020/027734). The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Bryon Johnson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Non-activated T cells expressing a CD33-specific CAR exhibit an antileukemic effect in vivo in the Aml xenograft model.

a, Schematic of the xenograft model and CART33 cell treatment in NSG mice. b, c, Serial quantification of disease burden by bioluminescence imaging. b, Total bioluminescence flux in mice with no treatment. c, Total bioluminescence flux in mice treated with 5×106 non-activated T cells transduced as in panel (a) (d1), 5×106 CAR + T cells stimulated with anti- CD3/CD28 microbeads and expanded over 9 days (d9), and 5×106 non-transduced (NTD) control non-activated T cells (n = 10 per group). d, e, Absolute peripheral blood CD45+ T cell counts in blood collected from mice shown in panels (c) at d, 13 days (P = 0.0065), and e, 26 days (P = 0.0012) following T cell transfer measured by a TruCount assay. The mean of each group is indicated by the solid black line. Groups were compared using the two-tailed, unpaired Mann–Whitney test.

Source data

Supplementary information

Source data

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Source data for tumour growth.

Source Data Fig. 5 (download XLSX )

Source data for tumour growth.

Source Data Fig. 6 (download XLSX )

Source data for tumour growth.

Source Data Extended Data Fig. 1 (download XLSX )

Source data for tumour growth.

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Ghassemi, S., Durgin, J.S., Nunez-Cruz, S. et al. Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 6, 118–128 (2022). https://doi.org/10.1038/s41551-021-00842-6

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