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Generation of Suicide Gene-Modified Chimeric Antigen Receptor-Redirected T-Cells for Cancer Immunotherapy

  • Kentaro Minagawa
  • Mustafa Al-Obaidi
  • Antonio Di StasiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1895)

Abstract

Chimeric antigen receptor (CAR)-redirected T-cells are a powerful tool for the treatment of several type of cancers; however, they can cause several adverse effects including cytokine release syndrome, off-target effects resulting in potentially fatal organ damage or even death. Particularly, for CAR T-cells redirected toward acute myeloid leukemia (AML) antigens myelosuppression can be a challenge. The previously validated inducible Caspase9 (iC9) suicide gene system is one of the approaches to control the infused cells in vivo through its activation with a nontherapeutic chemical inducer of dimerizer (CID). We performed a preclinical validation using a model of CD33+ AML, and generated iC9 CAR T-cells co-expressing a CAR targeting the AML-associated antigen CD33 and a selectable marker (ΔCD19). ΔCD19 selected (sel.) iC9-CAR.CD33 T-cells were effective in controlling leukemia growth in vitro, and could be partially eliminated (76%) using a chemical inducer of dimerization that activates iC9. Moreover, to completely eliminate residual cells, a second targeted agent was added. Future plans with these methods are to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant. Additional strategies that we are currently validating include (1) the modulation of the suicide gene activation, using different concentrations of the inducing agent(s), to be able to eliminate CAR T-cells modified by a regulatable gene, ideally aiming at preserving a proportion of the infused cells (and their antitumor activity) for mild to moderate toxicities, or (2) the co-expression of an inhibitory CAR aiming at sparing normal cells co-expressing an antigen not shared with the tumor.

Key words

AML Chimeric antigen receptor CD33 Suicide gene Safety switch Dimerizing agent AP1903 Inducible caspase 9 

References

  1. 1.
    Minagawa K, Zhou X, Mineishi S, Di Stasi A (2015) Seatbelts in CAR therapy: how safe are CARs? Pharmaceuticals (Basel) 8(2):230–249CrossRefGoogle Scholar
  2. 2.
    Mardiros A, Dos Santos C, McDonald T, Brown CE, Wang X, Budde LE et al (2013) T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia. Blood 122(18):3138–3148CrossRefGoogle Scholar
  3. 3.
    Tettamanti S, Marin V, Pizzitola I, Magnani CF, Giordano Attianese GM, Cribioli E et al (2013) Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor. Br J Haematol 161(3):389–401CrossRefGoogle Scholar
  4. 4.
    Gill S, Tasian SK, Ruella M, Shestova O, Li Y, Porter DL et al (2014) Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells. Blood 123(15):2343–2354CrossRefGoogle Scholar
  5. 5.
    Pizzitola I, Anjos-Afonso F, Rouault-Pierre K, Lassailly F, Tettamanti S, Spinelli O et al (2014) Chimeric antigen receptors against CD33/CD123 antigens efficiently target primary acute myeloid leukemia cells in vivo. Leukemia 28(8):1596–1605CrossRefGoogle Scholar
  6. 6.
    Zhou L, Liu X, Wang X, Sun Z, Song XT (2016) CD123 redirected multiple virus-specific T cells for acute myeloid leukemia. Leuk Res 41:76–84CrossRefGoogle Scholar
  7. 7.
    Thokala R, Olivares S, Mi T, Maiti S, Deniger D, Huls H et al (2016) Redirecting specificity of T cells using the Sleeping Beauty system to express chimeric antigen receptors by mix-and-matching of VL and VH domains targeting CD123+ tumors. PLoS One 11(8):e0159477CrossRefGoogle Scholar
  8. 8.
    Cartellieri M, Feldmann A, Koristka S, Arndt C, Loff S, Ehninger A et al (2016) Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts. Blood Cancer J 6(8):e458CrossRefGoogle Scholar
  9. 9.
    Magnani CF, Turazzi N, Benedicenti F, Calabria A, Tenderini E, Tettamanti S et al (2016) Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform. Oncotarget 7(32):51581–51597CrossRefGoogle Scholar
  10. 10.
    Arcangeli S, Rotiroti MC, Bardelli M, Simonelli L, Magnani CF, Biondi A et al (2017) Balance of anti-CD123 chimeric antigen receptor binding affinity and density for the targeting of acute myeloid leukemia. Mol Ther 25(8):1933–1945CrossRefGoogle Scholar
  11. 11.
    Tasian SK, Kenderian SS, Shen F, Ruella M, Shestova O, Kozlowski M et al (2017) Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia. Blood 129(17):2395–2407CrossRefGoogle Scholar
  12. 12.
    Casucci M, Nicolis di Robilant B, Falcone L, Camisa B, Norelli M, Genovese P et al (2013) CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma. Blood 122(20):3461–3472CrossRefGoogle Scholar
  13. 13.
    Dutour A, Marin V, Pizzitola I, Valsesia-Wittmann S, Lee D, Yvon E et al (2012) In vitro and in vivo antitumor effect of anti-CD33 chimeric receptor-expressing EBV-CTL against CD33 acute myeloid leukemia. Adv Hematol 2012:683065CrossRefGoogle Scholar
  14. 14.
    Cartellieri M, Koristka S, Arndt C, Feldmann A, Stamova S, von Bonin M et al (2014) A novel ex vivo isolation and expansion procedure for chimeric antigen receptor engrafted human T cells. PLoS One 9(4):e93745CrossRefGoogle Scholar
  15. 15.
    Wang QS, Wang Y, Lv HY, Han QW, Fan H, Guo B et al (2015) Treatment of CD33-directed chimeric antigen receptor-modified T cells in one patient with relapsed and refractory acute myeloid leukemia. Mol Ther 23(1):184–191CrossRefGoogle Scholar
  16. 16.
    Minagawa K, Jamil MO, Al-Obaidi M, Pereboeva L, Salzman D, Erba HP et al (2016) In vitro pre-clinical validation of suicide gene modified anti-CD33 redirected chimeric antigen receptor T-cells for acute myeloid leukemia. PLos One 11(12):e0166891CrossRefGoogle Scholar
  17. 17.
    Ritchie DS, Neeson PJ, Khot A, Peinert S, Tai T, Tainton K et al (2013) Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia. Mol Ther 21(11):2122–2129CrossRefGoogle Scholar
  18. 18.
    Ma Q, Garber HR, Lu S, He H, Tallis E, Ding X et al (2016) A novel TCR-like CAR with specificity for PR1/HLA-A2 effectively targets myeloid leukemia in vitro when expressed in human adult peripheral blood and cord blood T cells. Cytotherapy 18(8):985–994CrossRefGoogle Scholar
  19. 19.
    Lynn RC, Poussin M, Kalota A, Feng Y, Low PS, Dimitrov DS et al (2015) Targeting of folate receptor beta on acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells. Blood 125(22):3466–3476CrossRefGoogle Scholar
  20. 20.
    Lynn RC, Feng Y, Schutsky K, Poussin M, Kalota A, Dimitrov DS et al (2016) High-affinity FRbeta-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity. Leukemia 30(6):1355–1364CrossRefGoogle Scholar
  21. 21.
    Kenderian SS, Ruella M, Shestova O, Klichinsky M, Aikawa V, Morrissette JJ et al (2015) CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia. Leukemia 29(8):1637–1647CrossRefGoogle Scholar
  22. 22.
    Fedorov VD, Themeli M, Sadelain M (2013) PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Science Transl Med 5(215):215ra172CrossRefGoogle Scholar
  23. 23.
    Di Stasi A, Tey SK, Dotti G, Fujita Y, Kennedy-Nasser A, Martinez C et al (2011) Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365(18):1673–1683CrossRefGoogle Scholar
  24. 24.
    Zhou X, Di Stasi A, Tey SK, Krance RA, Martinez C, Leung KS et al (2014) Long-term outcome after haploidentical stem cell transplant and infusion of T cells expressing the inducible caspase 9 safety transgene. Blood 123(25):3895–3905CrossRefGoogle Scholar
  25. 25.
    Zhou X, Dotti G, Krance RA, Martinez CA, Naik S, Kamble RT et al (2015) Inducible caspase-9 suicide gene controls adverse effects from alloreplete T cells after haploidentical stem cell transplantation. Blood 125(26):4103–4113CrossRefGoogle Scholar
  26. 26.
    Diaconu I, Ballard B, Zhang M, Chen Y, West J, Dotti G et al (2017) Inducible caspase-9 selectively modulates the toxicities of CD19-specific chimeric antigen receptor-modified T cells. Mol Ther 25(3):580–592CrossRefGoogle Scholar
  27. 27.
    Fu X, Tao L, Rivera A, Williamson S, Song XT, Ahmed N et al (2010) A simple and sensitive method for measuring tumor-specific T cell cytotoxicity. PLoS One 5(7):e11867CrossRefGoogle Scholar
  28. 28.
    Di Stasi A, De Angelis B, Rooney CM, Zhang L, Mahendravada A, Foster AE et al (2009) T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model. Blood 113(25):6392–6402CrossRefGoogle Scholar
  29. 29.
    Riviere I, Roy K (2017) Perspectives on manufacturing of high-quality cell therapies. Mol Ther 25(5):1067–1068CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kentaro Minagawa
    • 1
  • Mustafa Al-Obaidi
    • 1
  • Antonio Di Stasi
    • 1
    Email author
  1. 1.Department of Hematology/Oncology, Bone Marrow Transplantation and Cell Therapy UnitUniversity of Alabama at BirminghamBirminghamUSA

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