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Gene Modification and Immunological Analyses for the Development of Immunotherapy Utilizing T Cells Redirected with Antigen-Specific Receptors

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2048))

Abstract

Cancer immunotherapy has been developed and established as a new treatment modality. Recently, adoptive transfer therapy using T cells redirected with antigen-specific antitumor receptors, such as T-cell receptor (TCR) and chimeric antigen receptor (CAR), has demonstrated clinical benefits even in patients with refractory malignancies. To advance this treatment modality, both generation of gene-modified T cells and evaluation of their reactivity with high quality in vitro are required. To achieve this, it is important to establish the ways (1) to generate optimal viral particle for T-cell transduction, (2) to transduce antitumor receptors into T cells and expand redirected T cells efficiently, and (3) to assess the functionality of antigen-specific gene-modified T cells precisely. Here, we summarize established protocols to generate and analyze antitumor receptor-transduced T cells. These procedures help to further assess characteristics of gene-modified T cells, resulting in promotion of translational research for cancer immunotherapy.

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References

  1. Couzin-Frankel J (2013) Breakthrough of the year 2013. Cancer immunotherapy. Science 342:1432–1433

    Article  CAS  Google Scholar 

  2. Morgan RA, Dudley ME, Wunderlich JR et al (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314:126–129

    Article  CAS  Google Scholar 

  3. Johnson LA, Morgan RA, Dudley ME et al (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 114:535–546

    Article  CAS  Google Scholar 

  4. Parkhurst MR, Yang JC, Langan RC et al (2011) T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 19:620–626

    Article  CAS  Google Scholar 

  5. Robbins PF, Morgan RA, Feldman SA et al (2011) Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 29:917–924

    Article  Google Scholar 

  6. Rapoport AP, Stadtmauer EA, Binder-Scholl GK et al (2015) NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med 21:914–921

    Article  CAS  Google Scholar 

  7. Tawara I, Kageyama S, Miyahara Y et al (2017) Safety and persistence of WT1-specific T-cell receptor gene-transduced lymphocytes in patients with AML and MDS. Blood 130:1985–1994

    Article  CAS  Google Scholar 

  8. Maude SL, Frey N, Shaw PA et al (2014) Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371:1507–1517

    Article  Google Scholar 

  9. Kochenderfer JN, Dudley ME, Kassim SH et al (2015) Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 33:540–549

    Article  CAS  Google Scholar 

  10. Schuster SJ, Svoboda J, Chong EA et al (2017) Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 377:2545–2554

    Article  CAS  Google Scholar 

  11. Park JH, Riviere I, Gonen M et al (2018) Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med 378:449–459

    Article  CAS  Google Scholar 

  12. Khalil DN, Smith EL, Brentjens RJ et al (2016) The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev Clin Oncol 13:273–290

    Article  CAS  Google Scholar 

  13. Brudno JN, Kochenderfer JN (2018) Chimeric antigen receptor T-cell therapies for lymphoma. Nat Rev Clin Oncol 15:31–46

    Article  CAS  Google Scholar 

  14. June CH, O'Connor RS, Kawalekar OU et al (2018) CAR T cell immunotherapy for human cancer. Science 359:1361–1365

    Article  CAS  Google Scholar 

  15. Guo T, Ochi T, Nakatsugawa M et al (2016) Generating de novo antigen-specific human T cell receptors by retroviral transduction of centric hemichain. J Vis Exp (116). https://doi.org/10.3791/54697

  16. Nakatsugawa M, Yamashita Y, Ochi T et al (2015) Specific roles of each TCR hemichain in generating functional chain-centric TCR. J Immunol 194:3487–3500

    Article  CAS  Google Scholar 

  17. Ochi T, Nakatsugawa M, Chamoto K et al (2015) Optimization of T-cell reactivity by exploiting TCR chain centricity for the purpose of safe and effective antitumor TCR gene therapy. Cancer Immunol Res 3:1070–1081

    Article  CAS  Google Scholar 

  18. Chamoto K, Guo T, Imataki O et al (2016) CDR3beta sequence motifs regulate autoreactivity of human invariant NKT cell receptors. J Autoimmun 68:39–51

    Article  CAS  Google Scholar 

  19. Kagoya Y, Tanaka S, Guo T et al (2018) A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects. Nat Med 24:352–359

    Article  CAS  Google Scholar 

  20. Kitamura T (1998) New experimental approaches in retrovirus-mediated expression screening. Int J Hematol 67:351–359

    Article  CAS  Google Scholar 

  21. Butler MO, Hirano N (2014) Human cell-based artificial antigen-presenting cells for cancer immunotherapy. Immunol Rev 257:191–209

    Article  CAS  Google Scholar 

  22. Heemskerk MH, Hoogeboom M, de Paus RA et al (2003) Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region. Blood 102:3530–3540

    Article  CAS  Google Scholar 

  23. Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7:1063–1066

    Article  CAS  Google Scholar 

  24. Yang S, Cohen CJ, Peng PD et al (2008) Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition. Gene Ther 15:1411–1423

    Article  CAS  Google Scholar 

  25. Kim JH, Lee SR, Li LH et al (2011) High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6:e18556

    Article  CAS  Google Scholar 

  26. Hirano N, Butler MO, Xia Z et al (2006) Engagement of CD83 ligand induces prolonged expansion of CD8+ T cells and preferential enrichment for antigen specificity. Blood 107:1528–1536

    Article  CAS  Google Scholar 

  27. Lissina A, Ladell K, Skowera A et al (2009) Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers. J Immunol Methods 340:11–24

    Article  CAS  Google Scholar 

  28. Robbins PF, Li YF, El-Gamil M et al (2008) Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. J Immunol 180:6116–6131

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by Grant-in-Aid for Research Activity Start-up of JSPS (to T.O.) and Research Scholarship of The Uehara Memorial Foundation (to T.O.). Plat-A cell line is kindly provided by Dr. Toshio Kitamura, The Institute of Medical Science, The University of Tokyo. Jurkat 76 cell line is a generous gift from Dr. Mirjam Heemskerk, Leiden University Medical Center. We thank Drs. Hiroshi Fujiwara and Masaki Yasukawa for helpful discussions.

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Correspondence to Naoto Hirano .

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Ochi, T., Maruta, M., Hirano, N. (2019). Gene Modification and Immunological Analyses for the Development of Immunotherapy Utilizing T Cells Redirected with Antigen-Specific Receptors. In: Kaneko, S. (eds) In Vitro Differentiation of T-Cells. Methods in Molecular Biology, vol 2048. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9728-2_3

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  • DOI: https://doi.org/10.1007/978-1-4939-9728-2_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9727-5

  • Online ISBN: 978-1-4939-9728-2

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