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CAR T-cells targeting FLT3 have potent activity against FLT3ITD+AML and act synergistically with the FLT3-inhibitor crenolanib

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Abstract

FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein expressed on normal hematopoietic stem and progenitor cells (HSC) and retained on malignant blasts in acute myeloid leukemia (AML). We engineered CD8+ and CD4+ T-cells expressing a FLT3-specific chimeric antigen receptor (CAR) and demonstrate they confer potent reactivity against AML cell lines and primary AML blasts that express either wild-type FLT3 or FLT3 with internal tandem duplication (FLT3-ITD). We also show that treatment with the FLT3-inhibitor crenolanib leads to increased surface expression of FLT3 specifically on FLT3-ITD+ AML cells and consecutively, enhanced recognition by FLT3-CAR T-cells in vitro and in vivo. As anticipated, we found that FLT3-CAR T-cells recognize normal HSCs in vitro and in vivo, and disrupt normal hematopoiesis in colony-formation assays, suggesting that adoptive therapy with FLT3-CAR T-cells will require subsequent CAR T-cell depletion and allogeneic HSC transplantation to reconstitute the hematopoietic system. Collectively, our data establish FLT3 as a novel CAR target in AML with particular relevance in high-risk FLT3-ITD+ AML. Further, our data provide the first proof-of-concept that CAR T-cell immunotherapy and small molecule inhibition can be used synergistically, as exemplified by our data showing superior antileukemia efficacy of FLT3-CAR T-cells in combination with crenolanib.

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References

  1. 1.

    Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood. 2002;100:1532–42.

  2. 2.

    Kikushige Y, Yoshimoto G, Miyamoto T, Iino T, Mori Y, Iwasaki H, et al. Human Flt3 is expressed at the hematopoietic stem cell and the granulocyte/macrophage progenitor stages to maintain cell survival. J Immunol. 2008;180:7358–67.

  3. 3.

    Böiers C, Buza-Vidas N, Jensen CT, Pronk CJ, Kharazi S, Wittmann L, et al. Expression and role of FLT3 in regulation of the earliest stage of normal granulocyte-monocyte progenitor development. Blood. 2010;115:5061–8.

  4. 4.

    Rosnet O, Bühring H, Marchetto S, Rappold I, Lavagna C, Sainty D, et al. Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. Leukemia. 1996;10:238–48.

  5. 5.

    Karsunky H, Merad M, Cozzio A, Weissman IL, Manz MG. Flt3 ligand regulates dendritic cell development from Flt3+lymphoid and myeloid-committed progenitors to Flt3+dendritic cells in vivo. J Exp Med. 2003;198:305–13.

  6. 6.

    Park I-K, Trotta R, Yu J, Caligiuri MA. Axl/Gas6 pathway participates in human natural killer cell development by positively regulating FLT3 activation. Eur J Immunol 2013;43:2750–5.

  7. 7.

    Waskow C, Liu K, Darrasse-Jèze G, Guermonprez P, Ginhoux F, Merad M, et al. The receptor tyrosine kinase Flt3 is required for dendritic cell development in peripheral lymphoid tissues. Nat Immunol. 2008;9:676–83.

  8. 8.

    Carow CE, Levenstein M, Kaufmann SH, Chen J, Amin S, Rockwell P, et al. Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2) in human leukemias. Blood. 1996;87:1089–96.

  9. 9.

    Ozeki K, Kiyoi H, Hirose Y, Iwai M, Ninomiya M, Kodera Y, et al. Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia. Blood. 2004;103:1901–8.

  10. 10.

    Vora HH, Shukla SN, Brahambhatt BV, Mehta SH, Patel NA, Parikh SK, et al. Clinical relevance of FLT3 receptor protein expression in Indian patients with acute leukemia. Asia‐Pacific J Clin Oncol. 2010;6:306–19.

  11. 11.

    Kindler T, Lipka DB, Fischer T. FLT3 as a therapeutic target in AML: still challenging after all these years. Blood. 2010;116:5089–102.

  12. 12.

    Hofmann M, Große-Hovest L, Nübling T, Pyż E, Bamberg M, Aulwurm S, et al. Generation, selection and preclinical characterization of an Fc-optimized FLT3 antibody for the treatment of myeloid leukemia. Leukemia. 2012;26:1228–37.

  13. 13.

    Stone JD, Aggen DH, Schietinger A, Schreiber H, Kranz DM. A sensitivity scale for targeting T cells with chimeric antigen receptors (CARs) and bispecific T-cell Engagers (BiTEs). Oncoimmunology. 2012;1:863–73.

  14. 14.

    Kuchenbauer F, Kern W, Schoch C, Kohlmann A, Hiddemann W, Haferlach T, et al. Detailed analysis of FLT3 expression levels in acute myeloid leukemia. Haematologica. 2005;90:1617–25.

  15. 15.

    Thiede C, Steudel C, Mohr B, Schaich M, Schäkel U, Platzbecker U, et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood. 2002;99:4326–35.

  16. 16.

    Fröhling S, Scholl C, Levine RL, Loriaux M, Boggon TJ, Bernard OA, et al. Identification of driver and passenger mutations of FLT3 by high-throughput DNA sequence analysis and functional assessment of candidate alleles. Cancer Cell. 2007;12:501–13.

  17. 17.

    Levis M, Murphy KM, Pham R, Kim K-T, Stine A, Li L, et al. Internal tandem duplications of the FLT3 gene are present in leukemia stem cells. Blood. 2005;106:673–80.

  18. 18.

    Brunet S, Labopin M, Esteve J, Cornelissen J, Socié G, Iori AP, et al. Impact of FLT3 internal tandem duplication on the outcome of related and unrelated hematopoietic transplantation for adult acute myeloid leukemia in first remission: a retrospective analysis. J Clin Oncol. 2012;30:735–41.

  19. 19.

    Schmid C, Labopin M, Socié G, Daguindau E, Volin L, Huynh A, et al. Outcome of patients with distinct molecular genotypes and cytogenetically normal AML after allogeneic transplantation. Blood. 2015;126:2062–9.

  20. 20.

    Alvarado Y, Kantarjian HM, Luthra R, Ravandi F, Borthakur G, Garcia‐Manero G, et al. Treatment with FLT3 inhibitor in patients with FLT3‐mutated acute myeloid leukemia is associated with development of secondary FLT3–tyrosine kinase domain mutations. Cancer. 2014;120:2142–9.

  21. 21.

    Heidel F, Solem FK, Breitenbuecher F, Lipka DB, Kasper S, Thiede M, et al. Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. Blood. 2006;107:293–300.

  22. 22.

    Knapper S, Burnett AK, Littlewood T, Kell WJ, Agrawal S, Chopra R, et al. A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as first-line treatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy. Blood. 2006;108:3262–70.

  23. 23.

    Weisberg E, Ray A, Nelson E, Adamia S, Barrett R, Sattler M, et al. Reversible resistance induced by FLT3 inhibition: a novel resistance mechanism in mutant FLT3-expressing cells. PloS One. 2011;6:e25351.

  24. 24.

    Smith CC, Lasater EA, Lin KC, Wang Q, McCreery MQ, Stewart WK, et al. Crenolanib is a selective type I pan-FLT3 inhibitor. Proc Natl Acad Sci. 2014;111:5319–24.

  25. 25.

    Zimmerman EI, Turner DC, Buaboonnam J, Hu S, Orwick S, Roberts MS, et al. Crenolanib is active against models of drug-resistant FLT3-ITD− positive acute myeloid leukemia. Blood. 2013;122:3607–15.

  26. 26.

    Heinrich MC, Griffith D, McKinley A, Patterson J, Presnell A, Ramachandran A, et al. Crenolanib inhibits the drug-resistant PDGFRA D842V mutation associated with imatinib-resistant gastrointestinal stromal tumors. Clin Cancer Res. 2012;18:4375–84.

  27. 27.

    Wetmore C, Broniscer A, Turner D, Wright KD, Pai-Panandiker A, Kun LE. et al. First-in-pediatrics phase I study of crenolanib besylate (CP-868,596-26) administered during and after radiation therapy (RT) in newly diagnosed diffuse intrinsic pontine glioma (DIPG) and recurrent high-grade glioma (HGG). J Clin Oncol. 2014;32:10064

  28. 28.

    Randhawa JK, Kantarjian HM, Borthakur G, Thompson PA, Konopleva M, Daver N. et al. Results of a phase II study of crenolanib in relapsed/refractory acute myeloid leukemia patients (Pts) with activating FLT3 mutations. Blood. 2014;124:389

  29. 29.

    Cortes J. Results from a phase II study of crenolanib in patients with FLT3-positive acute myeloidleukemia. ASCO Annual Meeting. 2016.

  30. 30.

    Hudecek M, Sommermeyer D, Kosasih PL, Silva-Benedict A, Liu L, Rader C, et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res. 2015;3:125–35.

  31. 31.

    Hudecek M, Lupo-Stanghellini M-T, Kosasih PL, Sommermeyer D, Jensen MC, Rader C, et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin Cancer Res. 2013;19:3153–64.

  32. 32.

    Wang X, Chang W-C, Wong CW, Colcher D, Sherman M, Ostberg JR, et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood. 2011;118:1255–63.

  33. 33.

    Monjezi R, Miskey C, Gogishvili T, Schleef M, Schmeer M, Einsele H, et al. Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors. Leukemia. 2017;31:186–94.

  34. 34.

    Gill S, Tasian SK, Ruella M, Shestova O, Li Y, Porter DL, et al. Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor–modified T cells. Blood. 2014;123:2343–54.

  35. 35.

    Quentmeier H, Reinhardt J, Zaborski M, Drexler H. FLT3 mutations in acute myeloid leukemia cell lines. Leukemia. 2003;17:120.

  36. 36.

    Sanchez PV, Perry RL, Sarry JE, Perl AE, Murphy K, Swider CR, et al. A robust xenotransplantation model for acute myeloid leukemia. Leukemia. 2009;23:2109.

  37. 37.

    Galanis A, Ma H, Rajkhowa T, Ramachandran A, Small D, Cortes J, et al. Crenolanib is a potent inhibitor of FLT3 with activity against resistance-conferring point mutants. Blood. 2014;123:94–100.

  38. 38.

    Chen J, Schmitt A, Chen B, Rojewski M, Rubeler V, Fei F, et al. Nilotinib hampers the proliferation and function of CD8+T lymphocytes through inhibition of T cell receptor signalling. J Cell Mol Med. 2008;12:2107–18.

  39. 39.

    Fei F, Yu Y, Schmitt A, Rojewski MT, Chen B, Greiner J, et al. Dasatinib exerts an immunosuppressive effect on CD8+T cells specific for viral and leukemia antigens. Exp Hematol. 2008;36:1297–308.

  40. 40.

    Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, Stetler-Stevenson M, et al. 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. 2015;33:540–9.

  41. 41.

    Turtle CJ, Hanafi L-A, Berger C, Gooley TA, Cherian S, Hudecek M, et al. CD19 CAR–T cells of defined CD4+: CD8+composition in adult B cell ALL patients. J Clin Invest. 2016;126:2123–38.

  42. 42.

    Paszkiewicz PJ, Fräßle SP, Srivastava S, Sommermeyer D, Hudecek M, Drexler I, et al. Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia. J Clin Invest. 2016;126:4262.

  43. 43.

    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.

  44. 44.

    Tasian SK, Kenderian SS, Shen F, Ruella M, Shestova O, Kozlowski M, et al. Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia. Blood. 2017;129:2395–407.

  45. 45.

    Chen L, Mao H, Zhang J, Chu J, Devine S, Caligiuri M et al. Targeting FLT3 by chimeric antigen receptor T cells for the treatment of acute myeloid leukemia. Leukemia 2017;3:1830–4.

  46. 46.

    Kenderian S, Ruella M, Shestova O, Klichinsky M, Aikawa V, Morrissette J, et al. CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia. Leukemia. 2015;29:1637–47.

  47. 47.

    Gardner R, Wu D, Cherian S, Fang M, Hanafi L-A, Finney O, et al. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy. Blood. 2016;127:2406–10.

  48. 48.

    Sotillo E, Barrett DM, Black KL, Bagashev A, Oldridge D, Wu G, et al. Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov. 2015;5:1282–95.

  49. 49.

    Ruella M, Barrett DM, Kenderian SS, Shestova O, Hofmann TJ, Perazzelli J, et al. Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies. J Clin Invest. 2016;126:3814–26.

  50. 50.

    Shultz LD, Goodwin N, Ishikawa F, Hosur V, Lyons BL, Greiner DL. Human cancer growth and therapy in immunodeficient mouse models. Cold Spring Harb Protoc. 2014;2014:pdb. top073585.

  51. 51.

    Pfister O, Lorenz V, Oikonomopoulos A, Xu L, Häuselmann SP, Mbah C, et al. FLT3 activation improves post-myocardial infarction remodeling involving a cytoprotective effect on cardiomyocytes. J Am Coll Cardiol. 2014;63:1011–9. 2014/03/18/

  52. 52.

    Feldman EJ, Brandwein J, Stone R, Kalaycio M, Moore J, O’connor J, et al. Phase III randomized multicenter study of a humanized anti-CD33 monoclonal antibody, lintuzumab, in combination with chemotherapy, versus chemotherapy alone in patients with refractory or first-relapsed acute myeloid leukemia. J Clin Oncol. 2005;23:4110–6.

  53. 53.

    Roberts A, He S, Ritchie D, Hertzberg M, Kerridge I, Durrant S. et al. A phase I study of anti-CD123 monoclonal antibody (mAb) CSL360 targeting leukemia stem cells (LSC) in AML. J Clin Oncol. 2010;28:e13012

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Acknowledgements

We thank Silke Frenz and Elke Spirk for their expertise in performing the mouse experiments. H.J. was supported by a grant from the German Excellence Initiative awarded to the Graduate School of Life Sciences (GSLS), University of Würzburg. I.G.G. was supported by a grant from Fundación Alfonso Martin Escudero, Spain. M.H. is a member of the Young Scholar Program (Junges Kolleg) and Extraordinary Member of the Bavarian Academy of Sciences (Bayerische Akademie der Wissenschaften).

Author Contributions

HJ designed and performed the experiments, analyzed the data and wrote the manuscript. IG-C, TN, ST and JR designed and performed the experiments, and analyzed the data. WH, JBM and JS analyzed the data. HB provided biologic material and analyzed the data. MH and HE designed experiments, analyzed the data, wrote the manuscript and supervised the project.

Author information

Affiliations

  1. Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany

    • Hardikkumar Jetani
    • , Thomas Nerreter
    • , Julian Rydzek
    • , Hermann Einsele
    •  & Michael Hudecek
  2. Hematology Department, Hospital de la Santa Creu i Sant Pau, Sant Pau and Jose Carreras Leukemia Research Institutes, Autonomous University of Barcelona, Barcelona, Spain

    • Irene Garcia-Cadenas
    • , Javier Briones Meijide
    •  & Jordi Sierra
  3. Klinik und Poliklinik für Innere Medizin III, Universitätsklinikum Regensburg, Regensburg, Germany

    • Simone Thomas
    •  & Wolfgang Herr
  4. Institut für Transfusionsmedizin und Immunhämatologie, Goethe Universität Frankfurt, Frankfurt am Main, Germany

    • Halvard Bonig
  5. Deutsches Rote Kreuz Blutspendedienst BaWüHe, Frankfurt, Germany

    • Halvard Bonig

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Conflict of interest

MH and HJ are co-inventors on a patent related to the use of FLT3-CAR T-cells to treat AML filed by the University of Würzburg, Würzburg, Germany. MH is co-inventor on patents related to CAR-technologies filed by the Fred Hutchinson Cancer Research Center, Seattle, WA and the University of Würzburg, Würzburg, Germany. The remaining authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Michael Hudecek.

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