Immunotherapy

Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia

Abstract

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective “off-the-shelf” immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

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References

  1. 1.

    Locatelli F, Pende D, Falco M, Della Chiesa M, Moretta A, Moretta L. NK cells mediate a crucial graft-versus-leukemia effect in haploidentical-HSCT to cure high-risk acute leukemia. Trends Immunol. 2018;39:577–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Suen WC, Lee WY, Leung KT, Pan XH, Li G. Natural killer cell-based cancer immunotherapy: a review on 10 years completed clinical trials. Cancer Investig. 2018;36:431–57.

    Article  CAS  Google Scholar 

  3. 3.

    Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, et al. Innate or adaptive immunity? The example of natural killer cells. Science. 2011;331:44–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Chiossone L, Dumas PY, Vienne M, Vivier E. Natural killer cells and other innate lymphoid cells in cancer. Nat Rev Immunol. 2018;18:671–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Chen X, Han J, Chu J, Zhang L, Zhang J, Chen C, et al. A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases. Oncotarget. 2016;7:27764–77.

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Han J, Chu J, Keung Chan W, Zhang J, Wang Y, Cohen JB, et al. CAR-engineered NK cells targeting wild-type EGFR and EGFRvIII enhance killing of glioblastoma and patient-derived glioblastoma stem cells. Sci Rep. 2015;5:11483.

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Zhang C, Burger MC, Jennewein L, Genssler S, Schonfeld K, Zeiner P, et al. ErbB2/HER2-specific NK cells for targeted therapy of glioblastoma. J Natl Cancer Inst. 2016;108:1–12.

  8. 8.

    Burga RA, Nguyen T, Zulovich J, Madonna S, Ylisastigui L, Fernandes R, et al. Improving efficacy of cancer immunotherapy by genetic modification of natural killer cells. Cytotherapy. 2016;18:1410–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Carlsten M, Childs RW. Genetic manipulation of NK cells for cancer immunotherapy: techniques and clinical implications. Front Immunol. 2015;6:266.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Rezvani K, Rouce RH. The application of natural killer cell immunotherapy for the treatment of cancer. Front Immunol. 2015;6:578.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Klingemann H. Are natural killer cells superior CAR drivers? Oncoimmunology. 2014;3:e28147.

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Liu D, Tian S, Zhang K, Xiong W, Lubaki NM, Chen Z, et al. Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV. Protein Cell. 2017;8:861–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Romanski A, Uherek C, Bug G, Seifried E, Klingemann H, Wels WS, et al. CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies. J Cell Mol Med. 2016;20:1287–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Zhang C, Oberoi P, Oelsner S, Waldmann A, Lindner A, Tonn T, et al. Chimeric antigen receptor-engineered NK-92 cells: an off-the-shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective antitumor immunity. Front Immunol. 2017;8:533.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Liu E, Tong Y, Dotti G, Shaim H, Savoldo B, Mukherjee M, et al. Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity. Leukemia. 2018;32:520–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Shah N, Martin-Antonio B, Yang H, Ku S, Lee DA, Cooper LJ, et al. Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity. PLoS ONE. 2013;8:e76781.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Moretta A, Bottino C, Vitale M, Pende D, Biassoni R, Mingari MC, et al. Receptors for HLA class-I molecules in human natural killer cells. Annu Rev Immunol. 1996;14:619–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Sivori S, Vitale M, Sanseverino L, Parolini S, Barbaresi M, Bottino C, et al. Inhibitory CD94 molecules identified by the Z199 monoclonal antibody recognize different HLA-class I molecules. Transpl Proc. 1996;28:3199–203.

    CAS  Google Scholar 

  19. 19.

    Sivori S, Vitale M, Bottino C, Marcenaro E, Sanseverino L, Parolini S, et al. CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies. Eur J Immunol. 1996;26:2487–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, et al. p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J Exp Med. 1997;186:1129–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Pende D, Parolini S, Pessino A, Sivori S, Augugliaro R, Morelli L, et al. Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J Exp Med. 1999;190:1505–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Vitale M, Bottino C, Sivori S, Sanseverino L, Castriconi R, Marcenaro E, et al. NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis. J Exp Med. 1998;187:2065–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Sivori S, Pende D, Bottino C, Marcenaro E, Pessino A, Biassoni R, et al. NKp46 is the major triggering receptor involved in the natural cytotoxicity of fresh or cultured human NK cells. Correlation between surface density of NKp46 and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells. Eur J Immunol. 1999;29:1656–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol. 2001;19:197–223.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Braud VM, Allan DS, O’Callaghan CA, Soderstrom K, D’Andrea A, Ogg GS, et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature. 1998;391:795–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Parham P. MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol. 2005;5:201–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Biassoni R, Cantoni C, Pende D, Sivori S, Parolini S, Vitale M, et al. Human natural killer cell receptors and co-receptors. Immunol Rev. 2001;181:203–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Gras Navarro A, Kmiecik J, Leiss L, Zelkowski M, Engelsen A, Bruserud O, et al. NK cells with KIR2DS2 immunogenotype have a functional activation advantage to efficiently kill glioblastoma and prolong animal survival. J Immunol. 2014;193:6192–206.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Bono M, Pende D, Bertaina A, Moretta A, Della Chiesa M, Sivori S, et al. Analysis of KIR3DP1 polymorphism provides relevant information on centromeric KIR gene content. J Immunol. 2018;201:1460–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Della Chiesa M, Falco M, Bertaina A, Muccio L, Alicata C, Frassoni F, et al. Human cytomegalovirus infection promotes rapid maturation of NK cells expressing activating killer Ig-like receptor in patients transplanted with NKG2C-/- umbilical cord blood. J Immunol. 2014;192:1471–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Quintarelli C, Orlando D, Boffa I, Guercio M, Polito VA, Petretto A, et al. Choice of costimulatory domains and of cytokines determines CAR T-cell activity in neuroblastoma. Oncoimmunology. 2018;7:e1433518.

    Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Orlando D, Miele E, De Angelis B, Guercio M, Boffa I, Sinibaldi M, et al. Adoptive immunotherapy using PRAME-specific T cells in medulloblastoma. Cancer Res. 2018;78:3337–49.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Diaconu I, Ballard B, Zhang M, Chen Y, West J, Dotti G, et al. Inducible caspase-9 selectively modulates the toxicities of CD19-specific chimeric antigen receptor-modified T cells. Mol Ther. 2017;25:580–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Della Chiesa M, Pesce S, Muccio L, Carlomagno S, Sivori S, Moretta A, et al. Features of memory-like and PD-1(+) human NK cell subsets. Front Immunol. 2016;7:351.

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Bjorkstrom NK, Riese P, Heuts F, Andersson S, Fauriat C, Ivarsson MA, et al. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood. 2010;116:3853–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Lopez-Verges S, Milush JM, Pandey S, York VA, Arakawa-Hoyt J, Pircher H, et al. CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset. Blood. 2010;116:3865–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Pinz KG, Yakaboski E, Jares A, Liu H, Firor AE, Chen KH, et al. Targeting T-cell malignancies using anti-CD4 CAR NK-92 cells. Oncotarget. 2017;8:112783–96.

    Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Godal R, Bachanova V, Gleason M, McCullar V, Yun GH, Cooley S, et al. Natural killer cell killing of acute myelogenous leukemia and acute lymphoblastic leukemia blasts by killer cell immunoglobulin-like receptor-negative natural killer cells after NKG2A and LIR-1 blockade. Biol Blood Marrow Transplant. 2010;16:612–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Curti A, Ruggeri L, D’Addio A, Bontadini A, Dan E, Motta MR, et al. Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients. Blood. 2011;118:3273–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Rubnitz JE, Inaba H, Ribeiro RC, Pounds S, Rooney B, Bell T, et al. NKAML: a pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia. J Clin Oncol. 2010;28:955–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Matosevic S. Viral and nonviral engineering of natural killer cells as emerging adoptive cancer immunotherapies. J Immunol Res. 2018;2018:4054815.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Boissel L, Betancur-Boissel M, Lu W, Krause DS, Van Etten RA, Wels WS, et al. Retargeting NK-92 cells by means of CD19- and CD20-specific chimeric antigen receptors compares favorably with antibody-dependent cellular cytotoxicity. Oncoimmunology. 2013;2:e26527.

    Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Eng J Med 2018;378:439–48.

    Article  CAS  Google Scholar 

  44. 44.

    Park JH, Riviere I, Gonen M, Wang X, Senechal B, Curran KJ, et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Eng J Med 2018;378:449–59.

    Article  CAS  Google Scholar 

  45. 45.

    Boissel L, Betancur M, Lu W, Wels WS, Marino T, Van Etten RA, et al. Comparison of mRNA and lentiviral based transfection of natural killer cells with chimeric antigen receptors recognizing lymphoid antigens. Leuk Lymphoma. 2012;53:958–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Sutlu T, Nystrom S, Gilljam M, Stellan B, Applequist SE, Alici E. Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy. Hum Gene Ther. 2012;23:1090–100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Verneris MR, Miller JS. The phenotypic and functional characteristics of umbilical cord blood and peripheral blood natural killer cells. Br J Haematol. 2009;147:185–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Luevano M, Daryouzeh M, Alnabhan R, Querol S, Khakoo S, Madrigal A, et al. The unique profile of cord blood natural killer cells balances incomplete maturation and effective killing function upon activation. Hum Immunol. 2012;73:248–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Sarvaria A, Jawdat D, Madrigal JA, Saudemont A. Umbilical cord blood natural killer cells, their characteristics, and potential clinical applications. Front Immunol. 2017;8:329.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Pesce S, Greppi M, Tabellini G, Rampinelli F, Parolini S, Olive D, et al. Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization. J Allergy Clin Immunol. 2017;139:335–46 e3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

A heartfelt thanks for the helpful suggestions to Alessandro Moretta who passed away before the final acceptance. Supported by grants awarded by Fondazione AIRC per la Ricerca sul Cancro-Special Project 5 × 1000 no. 9962 (FL and LM), AIRC IG 2018 id. 21724 (FL), AIRC IG 2017 Id. 15704 (SS), AIRC IG 2017 Id. 15283 (LM), Ricerca Finalizzata GR-2013-02359212 (CQ), Ministero dell’Università e della Ricerca (grant PRIN 2017WC8499_004 to FL and SS) Ricerca Corrente (FL, CQ, and BDA).

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LM and FL share last authorship, CQ, SS, SC, and SC share first authorship of this paper. CQ, SS, AM, FL, LM, and BDA designed experimental studies, supervised the project conduction, analyzed the data, and wrote the manuscript. S.Caru., S.Carl., MG, ZA, SDC, and MS performed the in vitro experiments. S.Caru., BDA, AC, and BC performed the in vivo experiments. AP supported the patient sample processing. DO and IB cloned the retroviral vector. FM, SS, and S.Carl. performed Kir analysis and NK phenotype analysis. MA performed HLA typing of patient sample. LM and FL provided scientific advice and expertize in the immunotherapy field. GLP provided expertize in the optimization of the process for clinical translation. SG, LV, FDB, AM, and FL provided patient’s samples, medical advice, and expertize in the field of onco-hematology. SCaru., S.Carl., BDA, CQ, and SS contributed to the study design and to the analysis of experimental data.

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Correspondence to B. De Angelis or F. Locatelli.

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Quintarelli, C., Sivori, S., Caruso, S. et al. Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia. Leukemia 34, 1102–1115 (2020). https://doi.org/10.1038/s41375-019-0613-7

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