Acute myeloid leukemia

A trispecific killer engager molecule against CLEC12A effectively induces NK-cell mediated killing of AML cells


The low 5-year survival rate for patients with acute myeloid leukemia (AML), primarily caused due to disease relapse, emphasizes the need for better therapeutic strategies. Disease relapse is facilitated by leukemic stem cells (LSCs) that are resistant to standard chemotherapy and promote tumor growth. To target AML blasts and LSCs using natural killer (NK) cells, we have developed a trispecific killer engager (TriKETM) molecule containing a humanized anti-CD16 heavy chain camelid single-domain antibody (sdAb) that activates NK cells, an IL-15 molecule that drives NK-cell priming, expansion and survival, and a single-chain variable fragment (scFv) against human CLEC12A (CLEC12A TriKE). CLEC12A is a myeloid lineage antigen that is highly expressed by AML cells and LSCs, but not expressed by normal hematopoietic stem cells (HSCs), thus minimizing off-target toxicity. The CLEC12A TriKE induced robust NK-cell specific proliferation, enhanced NK-cell activation, and killing of both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Additionally, the CLEC12A TriKE was able to reduce tumor burden in preclinical mouse models. These findings highlight the clinical potential of the CLEC12A TriKE for the effective treatment of AML.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: The CLEC12A TriKE induces potent NK-cell specific proliferation.
Fig. 2: Functional validation of the CLEC12A TriKE.
Fig. 3: CLEC12A TriKE induces target cell killing in real-time imaging assay.
Fig. 4: The CLEC12A TriKE induces killing of primary AML blasts.
Fig. 5: The CLEC12A TriKE limits tumor growth in vivo.


  1. 1.

    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70:7–30.

    Article  Google Scholar 

  2. 2.

    Tamamyan G, Kadia T, Ravandi F, Borthakur G, Cortes J, Jabbour E, et al. Frontline treatment of acute myeloid leukemia in adults. Crit Rev Oncol Hematol. 2017;110:20–34.

    Article  Google Scholar 

  3. 3.

    Saygin C, Carraway HE. Emerging therapies for acute myeloid leukemia. J Hematol Oncol. 2017;10:93.

    Article  Google Scholar 

  4. 4.

    Lai C, Doucette K, Norsworthy K. Recent drug approvals for acute myeloid leukemia. J Hematol Oncol. 2019;12:100.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Griessinger E, Anjos-Afonso F, Pizzitola I, Rouault-Pierre K, Vargaftig J, Taussig D, et al. A niche-like culture system allowing the maintenance of primary human acute myeloid leukemia-initiating cells: a new tool to decipher their chemoresistance and self-renewal mechanisms. Stem Cells Transl Med. 2014;3:520–9.

    CAS  Article  Google Scholar 

  6. 6.

    Pollyea DA, Gutman JA, Gore L, Smith CA, Jordan CT. Targeting acute myeloid leukemia stem cells: a review and principles for the development of clinical trials. Haematologica. 2014;99:1277–84.

    CAS  Article  Google Scholar 

  7. 7.

    Shang Y, Zhou F. Current Advances in Immunotherapy for Acute Leukemia: An Overview of Antibody, Chimeric Antigen Receptor, Immune Checkpoint, and Natural Killer. Front Oncol. 2019;9:917.

  8. 8.

    Walter RB, Medeiros BC, Gardner KM, Orlowski KF, Gallegos L, Scott BL, et al. Gemtuzumab ozogamicin in combination with vorinostat and azacitidine in older patients with relapsed or refractory acute myeloid leukemia: a phase I/II study. Haematologica. 2014;99:54–9.

    CAS  Article  Google Scholar 

  9. 9.

    Stein EM, Walter RB, Erba HP, Fathi AT, Advani AS, Lancet JE, et al. A phase 1 trial of vadastuximab talirine as monotherapy in patients with CD33-positive acute myeloid leukemia. Blood. 2018;131:387–96.

    CAS  Article  Google Scholar 

  10. 10.

    Hills RK, Castaigne S, Appelbaum FR, Delaunay J, Petersdorf S, Othus M, et al. Addition of gemtuzumab ozogamicin to induction chemotherapy in adult patients with acute myeloid leukaemia: a meta-analysis of individual patient data from randomised controlled trials. Lancet Oncol. 2014;15:986–96.

    CAS  Article  Google Scholar 

  11. 11.

    Ricart AD. Antibody-drug conjugates of calicheamicin derivative: gemtuzumab ozogamicin and inotuzumab ozogamicin. Clin Cancer Res. 2011;17:6417–27.

    CAS  Article  Google Scholar 

  12. 12.

    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.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Lapusan S, Vidriales MB, Thomas X, de Botton S, Vekhoff A, Tang R, et al. Phase I studies of AVE9633, an anti-CD33 antibody-maytansinoid conjugate, in adult patients with relapsed/refractory acute myeloid leukemia. Investig N Drugs. 2012;30:1121–31.

    CAS  Article  Google Scholar 

  14. 14.

    van Rhenen A, Van Dongen GAMS, Rombouts EJ, Feller N, Moshaver B, Walsum MS, et al. The novel AML stem cell-associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells. Blood. 2007;110:2659–66.

  15. 15.

    Larsen HØ, Roug AS, Just T, Brown GD, Hokland P. Expression of the hMICL in acute myeloid leukemia—a highly reliable disease marker at diagnosis and during follow-up. Cytom B Clin Cytom. 2012;82B:3–8.

    CAS  Article  Google Scholar 

  16. 16.

    Morsink LM, Walter RB, Ossenkoppele GJ. Prognostic and therapeutic role of CLEC12A in acute myeloid leukemia. Blood Rev. 2019;34:26–33.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    van Loo PF, Hangalapura BN, Thordardottir S, Gibbins JD, Veninga H, Hendriks LJA, et al. MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis. Expert Opin Biol Ther. 2019;19:721–33.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Jiang YP, Liu BY, Zheng Q, Panuganti S, Chen R, Zhu J, et al. CLT030, a leukemic stem cell-targeting CLL1 antibody-drug conjugate for treatment of acute myeloid leukemia. Blood Adv. 2018;2:1738–49.

    CAS  Article  Google Scholar 

  19. 19.

    Laborda E, Mazagova M, Shao S, Wang X, Quirino H, Woods AK, et al. Development of a chimeric antigen receptor targeting c-type lectin-like molecule-1 for human acute myeloid leukemia. Int J Mol Sci. 2017;18:2259.

  20. 20.

    Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol. 2008;9:503–10.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Waldhauer I, Steinle A. NK cells and cancer immunosurveillance. Oncogene. 2008;27:5932–43.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Voskoboinik I, Smyth MJ, Trapani JA. Perforin-mediated target-cell death and immune homeostasis. Nat Rev Immunol. 2006;6:940–52.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Miller JS, Soignier Y, Panoskaltsis-mortari A, Mcnearney SA, Yun GH, Fautsch SK, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood. 2005;105:3051–8.

    CAS  Article  Google Scholar 

  24. 24.

    Romee R, Cooley S, Berrien-Elliott MM, Westervelt P, Verneris MR, Wagner JE, et al. First-in-human phase 1 clinical study of the IL-15 superagonist complex ALT-803 to treat relapse after transplantation. Blood. 2018;131:2515–27.

    CAS  Article  Google Scholar 

  25. 25.

    Björklund AT, Carlsten M, Sohlberg E, Liu LL, Clancy T, Karimi M, et al. Complete remission with reduction of high-risk clones following haploidentical NK-cell therapy against MDS and AML. Clin Cancer Res. 2018;24:1834–44.

    Article  Google Scholar 

  26. 26.

    Barkholt L, Alici E, Conrad R, Sutlu T, Gilljam M, Stellan B, et al. Safety analysis of ex vivo-expanded NK and NK-like T cells administered to cancer patients: a phase I clinical study. Immunotherapy. 2009;1:753–64.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Olson JA, Leveson-Gower DB, Gill S, Baker J, Beilhack A, Negrin RS. NK cells mediate reduction of GVHD by inhibiting activated, alloreactive T cells while retaining GVT effects. Blood. 2010;115:4293–301.

    CAS  Article  Google Scholar 

  28. 28.

    Liu E, Marin D, Banerjee P, MacApinlac HA, Thompson P, Basar R, et al. Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. N Engl J Med. 2020;382:545–53.

    CAS  Article  Google Scholar 

  29. 29.

    Vallera DA, Felices M, McElmurry R, McCullar V, Zhou X, Schmohl JU, et al. IL15 trispecific killer engagers (TriKE) make natural killer cells specific to CD33+ targets while also inducing persistence, in vivo expansion, and enhanced function. Clin Cancer Res. 2016;22:3440–50.

    CAS  Article  Google Scholar 

  30. 30.

    Felices M, Kodal B, Hinderlie P, Kaminski MF, Cooley S, Weisdorf DJ, et al. Novel CD19-targeted TriKE restores NK cell function and proliferative capacity in CLL. Blood Adv. 2019;3:897–907.

    CAS  Article  Google Scholar 

  31. 31.

    Sarhan D, Brandt L, Felices M, Guldevall K, Lenvik T, Hinderlie P, et al. 161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS. Blood Adv. 2018;2:1459–69.

    CAS  Article  Google Scholar 

  32. 32.

    Felices M, Lenvik TR, Kodal B, Lenvik AJ, Hinderlie P, Bendzick LE, et al. Potent cytolytic activity and specific IL15 delivery in a second-generation trispecific killer engager. Cancer Immunol Res. 2020.

  33. 33.

    Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem. 2009;284:3273–84.

    CAS  Article  Google Scholar 

  34. 34.

    Behar G, Sibéril S, Groulet A, Chames P, Pugnière M, Boix C, et al. Isolation and characterization of anti-FcγRIII (CD16) llama single-domain antibodies that activate natural killer cells. Protein Eng Des Sel. 2007;21:1–10.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Haubner S, Perna F, Köhnke T, Schmidt C, Berman S, Augsberger C, et al. Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML. Leukemia. 2019;33:64–74.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Moga E, Cantó E, Vidal S, Juarez C, Sierra J, Briones J. Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression. Exp Hematol. 2011;39:1064–71.

    CAS  Article  Google Scholar 

  37. 37.

    Rosario M, Liu B, Kong L, Collins LI, Schneider SE, Chen X, et al. The IL-15-based ALT-803 complex enhances FcγRIIIa-triggered NK cell responses and in vivo clearance of B cell lymphomas. Clin Cancer Res. 2016;22:596–608.

    CAS  Article  Google Scholar 

  38. 38.

    Khaznadar Z, Henry G, Setterblad N, Agaugue S, Raffoux E, Boissel N, et al. Acute myeloid leukemia impairs natural killer cells through the formation of a deficient cytotoxic immunological synapse. Eur J Immunol. 2014;44:3068–80.

    CAS  Article  Google Scholar 

  39. 39.

    Lion E, Willemen Y, Berneman ZN, Van Tendeloo VFI, Smits ELJ. Natural killer cell immune escape in acute myeloid leukemia. Leukemia. 2012;26:2019–26.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Miller JS, Morishima C, McNeel DG, Patel MR, Kohrt HEK, Thompson JA, et al. A first-in-human phase I study of subcutaneous outpatient recombinant human IL15 (rhIL15) in adults with advanced solid tumors. Clin Cancer Res. 2018;24:1525–35.

    CAS  Article  Google Scholar 

  41. 41.

    Bakker ABH, van den Oudenrijn S, Bakker AQ, Feller N, van Meijer M, Bia JA, et al. C-type lectin-like molecule-1: a novel myeloid cell surface marker associated with acute myeloid leukemia. Cancer Res. 2004;64:8443–50.,

    CAS  Article  Google Scholar 

  42. 42.

    Zeijlemaker W, Grob T, Meijer R, Hanekamp D, Kelder A, Carbaat-Ham JC, et al. CD34+CD38− leukemic stem cell frequency to predict outcome in acute myeloid leukemia. Leukemia. 2019;33:1102–12.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Eckel AM, Cherian S, Miller V, Soma L. CD33 expression on natural killer cells is a potential confounder for residual disease detection in acute myeloid leukemia by flow cytometry. Cytom Part B - Clin Cytom. 2019;1–5.

  44. 44.

    Kantarjian H, Stein A, Gökbuget N, Fielding AK, Schuh AC, Ribera JM, et al. Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia. N Engl J Med. 2017;376:836–47.

    CAS  Article  Google Scholar 

  45. 45.

    Leong SR, Sukumaran S, Hristopoulos M, Totpal K, Stainton S, Lu E, et al. An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia. Blood. 2017;129:609–18.

    CAS  Article  Google Scholar 

  46. 46.

    Schuster SJ, Bishop MR, Tam CS, Waller EK, Borchmann P, McGuirk JP, et al. Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma. N Engl J Med. 2018;380:45–56.

    Article  PubMed  Google Scholar 

  47. 47.

    Xu X, Sun Q, Liang X, Chen Z, Zhang X, Zhou X, et al. Mechanisms of relapse after CD19 CAR T-cell therapy for acute lymphoblastic leukemia and its prevention and treatment strategies. Front Immunol. 2019;10:2664.

    Article  Google Scholar 

  48. 48.

    Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson CA, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377:2531–44.

    CAS  Article  Google Scholar 

  49. 49.

    Perna F, Berman SH, Soni RK, Mansilla-Soto J, Eyquem J, Hamieh M, et al. Integrating proteomics and transcriptomics for systematic combinatorial chimeric antigen receptor therapy of AML. Cancer Cell. 2017;32:506–19.e5.

    CAS  Article  Google Scholar 

  50. 50.

    Toft-Petersen M, Nederby L, Kjeldsen E, Kerndrup GB, Brown GD, Hokland P, et al. Unravelling the relevance of CLEC12A as a cancer stem cell marker in myelodysplastic syndrome. Br J Haematol. 2016;175:393–401.

    CAS  Article  Google Scholar 

Download references


We would like to acknowledge the Translational Therapy Laboratory, Flow Cytometry, and Imaging cores at the University of Minnesota for their services. This work was supported in part by NCI P01 CA111412 (JSM, BRB, DAV, MF), NCI P01 65493 (JSM, BRB), R35 CA197292 (JSM), P30 CA077598 (JSM, MF), R01 HL56067 (BRB), Minnesota Masonic Charities, and the Killebrew-Thompson Memorial Fund. We would also like to thank Xianzheng Zhou, at New York Medical College, for use of his HL-60luc cells.


This work was supported in part by DoD CA150085 (MF), NCI P01 CA111412 (MF and JSM), P01 CA65493 (MF and JSM), and R35 CA197292 (MF and JSM). The TriKE™ work was also supported in part by funding provided by GT Biopharma, Inc (MF, DAV, and JSM). The University of Minnesota has licensed the technology covered in this work to GT Biopharma. All conflicts have been declared and managed in accordance with the University’s conflict management plan.

Author information



Corresponding author

Correspondence to Jeffrey S. Miller.

Ethics declarations

Conflict of interest

MF, JSM, and DAV consult for and hold stock options in GT Biopharma, a company which may commercially benefit from the results of this research project. These interests have been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policy.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Arvindam, U.S., van Hauten, P.M.M., Schirm, D. et al. A trispecific killer engager molecule against CLEC12A effectively induces NK-cell mediated killing of AML cells. Leukemia (2020).

Download citation