Targeting natural killer cells in cancer immunotherapy

Journal name:
Nature Immunology
Year published:
Published online


Alteration in the expression of cell-surface proteins is a common consequence of malignant transformation. Natural killer (NK) cells use an array of germline-encoded activating and inhibitory receptors that scan for altered protein-expression patterns, but tumor evasion of detection by the immune system is now recognized as one of the hallmarks of cancer. NK cells display rapid and potent immunity to metastasis or hematological cancers, and major efforts are now being undertaken to fully exploit NK cell anti-tumor properties in the clinic. Diverse approaches encompass the development of large-scale NK cell–expansion protocols for adoptive transfer, the establishment of a microenvironment favorable to NK cell activity, the redirection of NK cell activity against tumor cells and the release of inhibitory signals that limit NK cell function. In this Review we detail recent advances in NK cell–based immunotherapies and discuss the advantages and limitations of these strategies.

At a glance


  1. Various approaches to therapy with the adoptive transfer of NK cells.
    Figure 1: Various approaches to therapy with the adoptive transfer of NK cells.

    NK cells can be obtained from either the patient or from a donor. (a) In autologous transfer, NK cells from the patient are activated and expanded in vitro in the presence of cytokines. Historically, IL-2 has been used for this purpose, but findings now suggest that the combination of IL-12, IL-15 and IL-18 might generate NK cells that are more functional and have memory properties. Feeder cells can be added to the culture. Irradiated human lymphoblastic K562 cells are often used as feeder cells and can be engineered to express cytokines (such as IL-15 and IL-21) and/or co-stimulatory molecules. The expanded and activated NK cells are then transferred back into the patient, who generally receives cytokine administration (IL-2, in most cases) to sustain the expansion and function of the infused NK cells. Although autologous NK cells might recognize activating signals such as stress molecules on cancer cells, their anti-tumor activity is limited by the inhibitory signal transmitted by self HLA molecules. (b) In allogeneic transfer, NK cells can be obtained from HLA-matched or haploidentical (partially matched) donors. NK cells are expanded through processes similar to those used for autologous transfer, but T cells should be removed to avoid GVHD. In this setting, the best responses are obtained when haploidentical donors do not express KIRs that recognize the patient's HLA molecules, because donor NK cells do not receive an inhibitory signal from the patient's cancer cells. (c) CARs can be engineered in autologous or allogeneic NK cells or in NK cell lines such as NK-92. CARs are designed by the fusion an antigen-binding domain (derived from a mAb scFv of known specificity) with a hinge region, a transmembrane domain and one or more stimulatory molecules. Each CAR has the CD3ζ chain (or sometimes the FcRγ chain) as its main signaling domain. Additionally, one or two co-stimulatory domain(s), usually from CD28 or CD137, can be added to the CAR construct; this leads to increased persistence and superior functionality. CARs from the first generation have no stimulatory domain, whereas CARs from the second generation and third generation have one co-stimulatory domain or two co-stimulatory domains, respectively. CAR engineering endows NK cells with antigen specificity. The binding of a CAR to the tumor antigen delivers a potent activating signal that triggers NK cell cytotoxicity, which results in elimination of the cancer cell.

  2. Targeting the tumor microenvironment to improve NK cell responses.
    Figure 2: Targeting the tumor microenvironment to improve NK cell responses.

    Various strategies can be adopted to create a microenvironment favorable to NK cells to improve the efficacy of NK cell–based therapies. Cytokines are generally administered to expand adoptively transferred NK cells, with IL-2 (1) being the most widely used in the clinic. However, IL-2 also activates Treg cells that hamper NK cell activity partly through production of the immunosuppressive cytokine TGF-β. In contrast, IL-15 (2) does not activate Treg cells and provides the additional advantage of stimulating both NK cells and cytotoxic CD8+ T cells. Other cytokines such as IL-12 (3), IL-18 (4) and IL-21 (5) can potentiate NK cell responses. Moreover, some anti-tumor agents have NK cell–modulating properties beyond their direct toxicity toward cancer cells. Genotoxic drugs (6), proteasome inhibitors (7) or immunomodulatory drug (IMiDs) (8) sensitize tumor cells to NK cell–mediated killing by altering their expression of surface molecules (downregulation of HLA molecules and upregulation of stress-induced ligands of activating receptors on NK cells). Moreover, immunomodulatory drugs (8) and the tyrosine-kinase inhibitor imatinib (9) stimulate NK cell function either directly or indirectly through the activation of other immune-cell subsets such as DCs. CD39 and CD73 are two enzymes expressed in the tumor microenvironment that contribute to production of the immunosuppressive metabolite adenosine. Blockade of these two enzymes via mAbs (10 and 11) or small-molecule inhibitors (14 and 15) restores NK cell function. Alternatively, blockade of the high-affinity adenosine receptor A2A (13) might prevent the direct suppressive effect of adenosine on NK cells. Finally, the immunosuppressive cytokine TGF-β substantially represses NK cell activity, and this pathway can be blocked by neutralizing this cytokine (12) or its receptor (16). MDSC, myeloid-derived suppressor cell.

  3. Therapeutic approaches that engage activating receptors on NK cells.
    Figure 3: Therapeutic approaches that engage activating receptors on NK cells.

    mAbs that target tumor-specific antigens and have been approved by the US Food and Drug Administration (FDA) (1) are widely used in the clinic. Their anti-tumor activity is attributed in part to their ability to trigger CD16 (the low-affinity receptor for IgG) on NK cells and induce ADCC. Agonistic mAbs to GITR (2), OX40 (3), CD137 (4) and CD27 (5) are currently being tested in clinical trials. These mAbs have been developed with the primary aim of stimulating T cells, but they might also positively influence NK cell functions. Enthusiasm has been growing for bispecific killer-cell engagers (BiKE) or trispecific killer-cell engagers (TriKE) that link activating receptors on NK cells to tumor antigens. Most of the bispecific engagers (5) trigger CD16, but fusion proteins that bridge NKG2D to tumor antigens (6) have also been designed. Trispecific engagers present three binding sites, and this provides the opportunity of targeting two different tumor antigens (7). The incorporation of IL-15 into a trispecific construct (8) further enhances the activation of NK cells. Alternatively, aptamers can also redirect NK cell activity toward tumor antigens (9) or can stimulate co-stimulatory molecules (10 and 11) to amplify the activation of NK cells. Finally, agonists of the activating receptor CD226 (12) have not been developed yet, but several pieces of evidence indicate that they might improve the anti-tumor activity of NK cells.

  4. Checkpoint inhibitors that 'release' NK cell functions.
    Figure 4: Checkpoint inhibitors that 'release' NK cell functions.

    The balance between activating signals and inhibitory signals received by receptors on NK cells determines the activation of NK cells. Inhibitory ligands expressed on the surface of target tumor cells or on antigen-presenting cells (APC) prevent the activation of NK cells, and abolishing these inhibitory signals by the means of blocking mAbs would restore full NK cell activity. Blocking mAbs directed against CTLA-4 (1), PD-1 (2) or PD-L1 (3) constitute one of the major advances of the past decade in terms of cancer immunotherapy. Even if T cells are considered the key mediators of the impressive efficacy of these checkpoint inhibitors, blockade of the CTLA-4 or PD-1 pathway might also enhance the anti-tumor responses of NK cells. Tim-3 is another checkpoint molecule shared by T cells and NK cells, and mAbs to Tim-3 (4) are currently being tested in cancer patients. In addition, mAbs that specifically block NK cell inhibitory receptors for MHC molecules have entered clinical trials. By blocking KIRs (5) or NKG2A (6), these mAbs release a major brake on the activation of NK cells. Finally, signaling via CD96 and TIGIT can restrain NK cell activity, and blocking mAbs to CD96 (7) or TIGIT (8) have great anti-cancer potential.


  1. Vivier, E., Tomasello, E., Baratin, M., Walzer, T. & Ugolini, S. Functions of natural killer cells. Nat. Immunol. 9, 503510 (2008).
  2. Huntington, N.D., Vosshenrich, C.A. & Di Santo, J.P. Developmental pathways that generate natural-killer-cell diversity in mice and humans. Nat. Rev. Immunol. 7, 703714 (2007).
  3. Hayakawa, Y., Huntington, N.D., Nutt, S.L. & Smyth, M.J. Functional subsets of mouse natural killer cells. Immunol. Rev. 214, 4755 (2006).
  4. Martinet, L. et al. DNAM-1 expression marks an alternative program of NK cell maturation. Cell Rep. 11, 8597 (2015).
  5. Sojka, D.K., Tian, Z. & Yokoyama, W.M. Tissue-resident natural killer cells and their potential diversity. Semin. Immunol. 26, 127131 (2014).
  6. Cortez, V.S., Fuchs, A., Cella, M., Gilfillan, S. & Colonna, M. Cutting edge: Salivary gland NK cells develop independently of Nfil3 in steady-state. J. Immunol. 192, 44874491 (2014).
  7. Marquardt, N. et al. Cutting edge: identification and characterization of human intrahepatic CD49a+ NK cells. J. Immunol. 194, 24672471 (2015).
  8. Spits, H. et al. Innate lymphoid cells--a proposal for uniform nomenclature. Nat. Rev. Immunol. 13, 145149 (2013).
  9. Raulet, D.H. & Vance, R.E. Self-tolerance of natural killer cells. Nat. Rev. Immunol. 6, 520531 (2006).
  10. Romee, R., Leong, J.W. & Fehniger, T.A. Utilizing cytokines to function-enable human NK cells for the immunotherapy of cancer. Scientifica (Cairo) 2014, 205796 (2014).
  11. Guillerey, C. et al. Toll-like receptor 3 regulates NK cell responses to cytokines and controls experimental metastasis. OncoImmunology 4, e1027468 (2015).
  12. Voskoboinik, I., Smyth, M.J. & Trapani, J.A. Perforin-mediated target-cell death and immune homeostasis. Nat. Rev. Immunol. 6, 940952 (2006).
  13. Sungur, C.M. & Murphy, W.J. Positive and negative regulation by NK cells in cancer. Crit. Rev. Oncog. 19, 5766 (2014).
  14. Iannello, A., Thompson, T.W., Ardolino, M., Lowe, S.W. & Raulet, D.H. p53-dependent chemokine production by senescent tumor cells supports NKG2D-dependent tumor elimination by natural killer cells. J. Exp. Med. 210, 20572069 (2013).
  15. Guillerey, C. & Smyth, M.J. NK cells and cancer immunoediting. Curr. Top. Microbiol. Immunol. 395, 115145 (2016).
  16. Vitale, M., Cantoni, C., Pietra, G., Mingari, M.C. & Moretta, L. Effect of tumor cells and tumor microenvironment on NK-cell function. Eur. J. Immunol. 44, 15821592 (2014).
  17. Krasnova, Y., Putz, E.M., Smyth, M.J. & Souza-Fonseca-Guimaraes, F. Bench to bedside: NK cells and control of metastasis. Clin. Immunol. S1521-6616(15)30050-4 (2015).
  18. Ghiringhelli, F. et al. CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-β-dependent manner. J. Exp. Med. 202, 10751085 (2005).
  19. Smyth, M.J. et al. CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer. J. Immunol. 176, 15821587 (2006).
  20. Beavis, P.A. et al. Blockade of A2A receptors potently suppresses the metastasis of CD73+ tumors. Proc. Natl. Acad. Sci. USA 110, 1471114716 (2013).
  21. Mittal, D. et al. Antimetastatic effects of blocking PD-1 and the adenosine A2A receptor. Cancer Res. 74, 36523658 (2014).
  22. Stacey, M.A., Marsden, M., Wang, E.C., Wilkinson, G.W. & Humphreys, I.R. IL-10 restricts activation-induced death of NK cells during acute murine cytomegalovirus infection. J. Immunol. 187, 29442952 (2011).
  23. Mocellin, S. et al. IL-10 stimulatory effects on human NK cells explored by gene profile analysis. Genes Immun. 5, 621630 (2004).
  24. Viel, S. et al. TGF-β inhibits the activation and functions of NK cells by repressing the mTOR pathway. Sci. Signal. 9, ra19 (2016).
  25. Sconocchia, G. et al. Melanoma cells inhibit NK cell functions. Cancer Res. 72, 54285429 author reply 5430 (2012).
  26. Ruggeri, L. et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295, 20972100 (2002).
  27. Benson, D.M. Jr. et al. IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect. Blood 118, 63876391 (2011).
  28. Grossenbacher, S.K., Canter, R.J. & Murphy, W.J. Natural killer cell immunotherapy to target stem-like tumor cells. J. Immunother. Cancer 4, 19 (2016).
  29. Delgado, D.C. et al. Genotypes of NK cell KIR receptors, their ligands, and Fcγ receptors in the response of neuroblastoma patients to Hu14.18-IL2 immunotherapy. Cancer Res. 70, 95549561 (2010).
  30. Yang, R.K. et al. Intratumoral treatment of smaller mouse neuroblastoma tumors with a recombinant protein consisting of IL-2 linked to the hu14.18 antibody increases intratumoral CD8+ T and NK cells and improves survival. Cancer Immunol. Immunother. 62, 13031313 (2013).
  31. Han, J. et al. CAR-engineered NK cells targeting wild-type EGFR and EGFRvIII enhance killing of glioblastoma and patient-derived glioblastoma stem cells. Sci. Rep. 5, 11483 (2015).
  32. Zhang, C. et al. ErbB2/HER2-specific NK cells for targeted therapy of glioblastoma. J. Natl. Cancer Inst. 108, djv375 (2015).
  33. Genßler, S. et al. Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival. OncoImmunology 5, e1119354 (2015).
  34. Besse, B. et al. Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC. OncoImmunology 5, e1071008 (2015).
  35. Tonn, T. et al. Treatment of patients with advanced cancer with the natural killer cell line NK-92. Cytotherapy 15, 15631570 (2013).
  36. Becker, P.S. et al. Selection and expansion of natural killer cells for NK cell-based immunotherapy. Cancer Immunol. Immunother. 65, 477484 (2016).
  37. Knorr, D.A., Bachanova, V., Verneris, M.R. & Miller, J.S. Clinical utility of natural killer cells in cancer therapy and transplantation. Semin. Immunol. 26, 161172 (2014).
  38. Rueff, J., Medinger, M., Heim, D., Passweg, J. & Stern, M. Lymphocyte subset recovery and outcome after autologous hematopoietic stem cell transplantation for plasma cell myeloma. Biol. Blood Marrow Transplant. 20, 896899 (2014).
  39. Porrata, L.F. et al. Early lymphocyte recovery predicts superior survival after autologous stem cell transplantation in non-Hodgkin lymphoma: a prospective study. Biol. Blood Marrow Transplant. 14, 807816 (2008).
  40. Sakamoto, N. et al. Phase I clinical trial of autologous NK cell therapy using novel expansion method in patients with advanced digestive cancer. J. Transl. Med. 13, 277 (2015).
    This study established a new expansion method for obtaining large numbers of functional NK cells from small quantities of blood. Expanded populations of autologous NK cells were safe to administer to patients with cancer, and this led to enhanced cytotoxic activity against NK cell targets.
  41. Parkhurst, M.R., Riley, J.P., Dudley, M.E. & Rosenberg, S.A. Adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression. Clin. Cancer Res. 17, 62876297 (2011).
  42. Curti, A. et al. Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients. Blood 118, 32733279 (2011).
  43. Miller, J.S. et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105, 30513057 (2005).
  44. Rubnitz, J.E. 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. 28, 955959 (2010).
  45. Shah, N.N. et al. Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell-depleted stem cell transplantation. Blood 125, 784792 (2015).
  46. Bishara, A. et al. The beneficial role of inhibitory KIR genes of HLA class I NK epitopes in haploidentically mismatched stem cell allografts may be masked by residual donor-alloreactive T cells causing GVHD. Tissue Antigens 63, 204211 (2004).
  47. Davies, S.M. et al. Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Killer immunoglobulin-like receptor. Blood 100, 38253827 (2002).
  48. Gill, S. & June, C.H. Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies. Immunol. Rev. 263, 6889 (2015).
  49. Klingemann, H. Are natural killer cells superior CAR drivers? OncoImmunology 3, e28147 (2014).
  50. Glienke, W. et al. Advantages and applications of CAR-expressing natural killer cells. Front. Pharmacol. 6, 21 (2015).
  51. Klingemann, H. Challenges of cancer therapy with natural killer cells. Cytotherapy 17, 245249 (2015).
  52. Romanski, A. et al. CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies. J. Cell. Mol. Med. 20, 12871294 (2016).
  53. Schönfeld, K. et al. Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor. Mol. Ther. 23, 330338 (2015).
    This study was the first to test the effect of irradiation (a necessary safety measure) on the anti-tumor activity of NK cell lines and demonstrated that irradiated NK cells expressing an anti-ErbB2 CAR protected mice against metastases.
  54. Leong, J.W. et al. Preactivation with IL-12, IL-15, and IL-18 induces CD25 and a functional high-affinity IL-2 receptor on human cytokine-induced memory-like natural killer cells. Biol. Blood Marrow Transplant. 20, 463473 (2014).
  55. Burns, L.J. et al. IL-2-based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial. Bone Marrow Transplant. 32, 177186 (2003).
  56. Bachanova, V. et al. Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein. Blood 123, 38553863 (2014).
    This clinical trial report demonstrated the therapeutic benefit of eliminating Treg cells with an IL-2–diphtheria fusion protein before the infusion of haploidentical NK cells in patients with AML. This study provided evidence of the negative role of Treg cells on the population expansion of NK cells after transfer and proposed that the detection of donor NK cells 7 days after infusion could serve as a surrogate marker for a clinical response.
  57. Steele, N. et al. A phase 1 trial of recombinant human IL-21 in combination with cetuximab in patients with metastatic colorectal cancer. Br. J. Cancer 106, 793798 (2012).
  58. Waldmann, T.A. Interleukin-15 in the treatment of cancer. Expert Rev. Clin. Immunol. 10, 16891701 (2014).
  59. Porrata, L.F. et al. Interleukin-15 affects patient survival through natural killer cell recovery after autologous hematopoietic stem cell transplantation for non-Hodgkin lymphomas. Clin. Dev. Immunol. 2010, 914945 (2010).
  60. Conlon, K.C. et al. Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer. J. Clin. Oncol. 33, 7482 (2015).
    This study reported the first clinical trial in which recombinant IL-15 was administered to patients with metastatic cancer, which led to the clearance of lung lesions in two patients.
  61. Pérez-Martínez, A. et al. A phase I/II trial of interleukin-15--stimulated natural killer cell infusion after haplo-identical stem cell transplantation for pediatric refractory solid tumors. Cytotherapy 17, 15941603 (2015).
  62. Kim, P.S. et al. IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity against murine breast and colon carcinomas. Oncotarget 7, 1613016145 (2016).
  63. Sahm, C., Schönfeld, K. & Wels, W.S. Expression of IL-15 in NK cells results in rapid enrichment and selective cytotoxicity of gene-modified effectors that carry a tumor-specific antigen receptor. Cancer Immunol. Immunother. 61, 14511461 (2012).
  64. Imamura, M. et al. Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15. Blood 124, 10811088 (2014).
  65. Krieg, S. & Ullrich, E. Novel immune modulators used in hematology: impact on NK cells. Front. Immunol. 3, 388 (2013).
  66. Davies, F.E. et al. Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma. Blood 98, 210216 (2001).
  67. Borg, C. et al. Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects. J. Clin. Invest. 114, 379388 (2004).
  68. Parameswaran, R. et al. Repression of GSK3 restores NK cell cytotoxicity in AML patients. Nat. Commun. 7, 11154 (2016).
    This study identified the glycogen synthetase GSK3-β as an intrinsic inhibitor of NK cell responses in patients with AML. Small-molecule inhibitors of GSK3 increased the cytotoxicity of NK cells against AML cells and proved protective in a humanized mouse model of AML.
  69. Schmiedel, B.J. et al. Azacytidine impairs NK cell reactivity while decitabine augments NK cell responsiveness toward stimulation. Int. J. Cancer 128, 29112922 (2011).
  70. Rossi, L.E. et al. Histone deacetylase inhibitors impair NK cell viability and effector functions through inhibition of activation and receptor expression. J. Leukoc. Biol. 91, 321331 (2012).
  71. Jardine, L. et al. Sensitizing primary acute lymphoblastic leukemia to natural killer cell recognition by induction of NKG2D ligands. Leuk. Lymphoma 54, 167173 (2013).
  72. Wang, X. et al. Proteasome inhibition induces apoptosis in primary human natural killer cells and suppresses NKp46-mediated cytotoxicity. Haematologica 94, 470478 (2009).
  73. Lundqvist, A., Yokoyama, H., Smith, A., Berg, M. & Childs, R. Bortezomib treatment and regulatory T-cell depletion enhance the antitumor effects of adoptively infused NK cells. Blood 113, 61206127 (2009).
  74. Marçais, A. et al. The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells. Nat. Immunol. 15, 749757 (2014).
  75. Weiner, L.M., Surana, R. & Wang, S. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 10, 317327 (2010).
  76. Bakema, J.E. & van Egmond, M. Fc receptor-dependent mechanisms of monoclonal antibody therapy of cancer. Curr. Top. Microbiol. Immunol. 382, 373392 (2014).
  77. Weng, W.K. & Levy, R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J. Clin. Oncol. 21, 39403947 (2003).
  78. Rodríguez, J. et al. Fc gamma receptor polymorphisms as predictive markers of Cetuximab efficacy in epidermal growth factor receptor downstream-mutated metastatic colorectal cancer. Eur. J. Cancer 48, 17741780 (2012).
  79. Clynes, R.A., Towers, T.L., Presta, L.G. & Ravetch, J.V. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat. Med. 6, 443446 (2000).
  80. Gluck, W.L. et al. Phase I studies of interleukin (IL)-2 and rituximab in B-cell non-Hodgkin's lymphoma: IL-2 mediated natural killer cell expansion correlations with clinical response. Clin. Cancer Res. 10, 22532264 (2004).
  81. Romee, R. et al. NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17). Blood 121, 35993608 (2013).
  82. Zhou, Q., Gil-Krzewska, A., Peruzzi, G. & Borrego, F. Matrix metalloproteinases inhibition promotes the polyfunctionality of human natural killer cells in therapeutic antibody-based anti-tumour immunotherapy. Clin. Exp. Immunol. 173, 131139 (2013).
  83. Bowles, J.A. et al. Anti-CD20 monoclonal antibody with enhanced affinity for CD16 activates NK cells at lower concentrations and more effectively than rituximab. Blood 108, 26482654 (2006).
  84. Gerdes, C.A. et al. GA201 (RG7160): a novel, humanized, glycoengineered anti-EGFR antibody with enhanced ADCC and superior in vivo efficacy compared with cetuximab. Clin. Cancer Res. 19, 11261138 (2013).
  85. Roberti, M.P. et al. IL-2- or IL-15-activated NK cells enhance cetuximab-mediated activity against triple-negative breast cancer in xenografts and in breast cancer patients. Breast Cancer Res. Treat. 136, 659671 (2012).
  86. Moga, E. et al. NK cells stimulated with IL-15 or CpG ODN enhance rituximab-dependent cellular cytotoxicity against B-cell lymphoma. Exp. Hematol. 36, 6977 (2008).
  87. Wu, L. et al. lenalidomide enhances natural killer cell and monocyte-mediated antibody-dependent cellular cytotoxicity of rituximab-treated CD20+ tumor cells. Clin. Cancer Res. 14, 46504657 (2008).
  88. Gleason, M.K. et al. Bispecific and trispecific killer cell engagers directly activate human NK cells through CD16 signaling and induce cytotoxicity and cytokine production. Mol. Cancer Ther. 11, 26742684 (2012).
  89. Hartmann, F. et al. Treatment of refractory Hodgkin's disease with an anti-CD16/CD30 bispecific antibody. Blood 89, 20422047 (1997).
  90. Shahied, L.S. et al. Bispecific minibodies targeting HER2/neu and CD16 exhibit improved tumor lysis when placed in a divalent tumor antigen binding format. J. Biol. Chem. 279, 5390753914 (2004).
  91. Wiernik, A. et al. Targeting natural killer cells to acute myeloid leukemia in vitro with a CD16 x 33 bispecific killer cell engager and ADAM17 inhibition. Clin. Cancer Res. 19, 38443855 (2013).
  92. Schmohl, J.U., Gleason, M.K., Dougherty, P.R., Miller, J.S. & Vallera, D.A. Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD133+ colorectal cancer cells. Target. Oncol. (2015).
    This paper reported the design of a '16x133' bispecific killer-cell engager that directs NK cell cytotoxic activity against CD133-expressing cancer cells. This has important therapeutic potential given its ability to target the drug-resistant cancer stem cell population that is CD133+ in many cancers.
  93. Vallera, D.A. 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).
    This study reports the generation of IL-15 trispecific killer-cell engagers with stable integration of IL-15 into a bispecific antibody platform that recognizes CD16 on NK cells and CD33 on myeloid cancer cells. The IL-15 linker incorporated into this construct sustained the survival and proliferation NK cells and demonstrated antitumor activity in a leukemia xenograft model.
  94. Boltz, A. et al. Bi-specific aptamers mediating tumor cell lysis. J. Biol. Chem. 286, 2189621905 (2011).
  95. Kellner, C. et al. Fusion proteins between ligands for NKG2D and CD20-directed single-chain variable fragments sensitize lymphoma cells for natural killer cell-mediated lysis and enhance antibody-dependent cellular cytotoxicity. Leukemia 26, 830834 (2012).
  96. von Strandmann, E.P. et al. A novel bispecific protein (ULBP2-BB4) targeting the NKG2D receptor on natural killer (NK) cells and CD138 activates NK cells and has potent antitumor activity against human multiple myeloma in vitro and in vivo. Blood 107, 19551962 (2006).
  97. Groh, V., Wu, J., Yee, C. & Spies, T. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature 419, 734738 (2002).
  98. Deng, W. et al. Antitumor immunity. A shed NKG2D ligand that promotes natural killer cell activation and tumor rejection. Science 348, 136139 (2015).
    This was the first report showing a positive effect of a shed NKG2D ligand on the activation of NK cells. It demonstrated that MULT1, which binds with high affinity to NKG2D, stimulated NK cells and caused tumor rejection in mice.
  99. Lanier, L.L. NKG2D receptor and its ligands in host defense. Cancer Immunol. Res. 3, 575582 (2015).
  100. Raulet, D.H., Gasser, S., Gowen, B.G., Deng, W. & Jung, H. Regulation of ligands for the NKG2D activating receptor. Annu. Rev. Immunol. 31, 413441 (2013).
  101. Delahaye, N.F. et al. Alternatively spliced NKp30 isoforms affect the prognosis of gastrointestinal stromal tumors. Nat. Med. 17, 700707 (2011).
  102. Semeraro, M. et al. Clinical impact of the NKp30/B7-H6 axis in high-risk neuroblastoma patients. Sci. Transl. Med. 7, 283ra55 (2015).
  103. Viaud, S. et al. Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Ralpha. PLoS One 4, e4942 (2009).
  104. Guillerey, C. et al. Immunosurveillance and therapy of multiple myeloma are CD226 dependent. J. Clin. Invest. 125, 20772089 (2015).
  105. Chan, C.J. et al. DNAM-1/CD155 interactions promote cytokine and NK cell-mediated suppression of poorly immunogenic melanoma metastases. J. Immunol. 184, 902911 (2010).
  106. Makkouk, A., Chester, C. & Kohrt, H.E. Rationale for anti-CD137 cancer immunotherapy. Eur. J. Cancer 54, 112119 (2016).
  107. Baessler, T. et al. CD137 ligand mediates opposite effects in human and mouse NK cells and impairs NK-cell reactivity against human acute myeloid leukemia cells. Blood 115, 30583069 (2010).
  108. Kohrt, H.E. et al. CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies. Blood 117, 24232432 (2011).
  109. Kohrt, H.E. et al. Targeting CD137 enhances the efficacy of cetuximab. J. Clin. Invest. 124, 26682682 (2014).
    This report established the synergistic effect of cetuximab (mAb to EGFR) and stimulation of CD137 in promoting the activation of human NK cells and protecting mice against tumors in various xenograft models. This work supports the development of clinical trials combining cetuximab with agonist mAb to CD137.
  110. Sanmamed, M.F. et al. Agonists of co-stimulation in cancer immunotherapy directed against CD137, OX40, GITR, CD27, CD28, and ICOS. Semin. Oncol. 42, 640655 (2015).
  111. Romagné, F. et al. Preclinical characterization of 1-7F9, a novel human anti-KIR receptor therapeutic antibody that augments natural killer-mediated killing of tumor cells. Blood 114, 26672677 (2009).
  112. Benson, D.M. Jr. et al. A phase 1 trial of the anti-KIR antibody IPH2101 in patients with relapsed/refractory multiple myeloma. Blood 120, 43244333 (2012).
  113. Vey, N. et al. A phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission. Blood 120, 43174323 (2012).
    References 112 and 113 reported the first clinical trial of IPH2101, the antibody to human KIRs, in elderly patients with AML and patients with relapsed or refractory multiple myeloma.
  114. Korde, N. et al. A phase II trial of pan-KIR2D blockade with IPH2101 in smoldering multiple myeloma. Haematologica 99, e81e83 (2014).
  115. Kohrt, H.E. et al. Anti-KIR antibody enhancement of anti-lymphoma activity of natural killer cells as monotherapy and in combination with anti-CD20 antibodies. Blood 123, 678686 (2014).
  116. Wieten, L., Mahaweni, N.M., Voorter, C.E., Bos, G.M. & Tilanus, M.G. Clinical and immunological significance of HLA-E in stem cell transplantation and cancer. Tissue Antigens 84, 523535 (2014).
  117. Nguyen, S. et al. HLA-E upregulation on IFN-γ-activated AML blasts impairs CD94/NKG2A-dependent NK cytolysis after haplo-mismatched hematopoietic SCT. Bone Marrow Transplant. 43, 693699 (2009).
  118. Martinet, L. & Smyth, M.J. Balancing natural killer cell activation through paired receptors. Nat. Rev. Immunol. 15, 243254 (2015).
  119. Chan, C.J. et al. The receptors CD96 and CD226 oppose each other in the regulation of natural killer cell functions. Nat. Immunol. 15, 431438 (2014).
  120. Blake, S.J. et al. Suppression of metastases using a new lymphocyte checkpoint target for cancer immunotherapy. Cancer Discov. 6, 446459 (2016).
    This study defined a role for the immunoglobulin family receptor CD96 in inhibiting NK cell–mediated control of tumor metastases. Pre-clinical mouse models were used to establish the anti-metastatic activity of an antibody to CD96 as a single agent or in combination with conventional checkpoint inhibitors.
  121. Lesokhin, A.M., Callahan, M.K., Postow, M.A. & Wolchok, J.D. On being less tolerant: enhanced cancer immunosurveillance enabled by targeting checkpoints and agonists of T cell activation. Sci. Transl. Med. 7, 280sr1 (2015).
  122. Stojanovic, A., Fiegler, N., Brunner-Weinzierl, M. & Cerwenka, A. CTLA-4 is expressed by activated mouse NK cells and inhibits NK Cell IFN-γ production in response to mature dendritic cells. J. Immunol. 192, 41844191 (2014).
  123. Huang, B.Y. et al. The PD-1/B7-H1 pathway modulates the natural killer cells versus mouse glioma stem cells. PLoS One 10, e0134715 (2015).
  124. Westin, J.R. et al. Safety and activity of PD1 blockade by pidilizumab in combination with rituximab in patients with relapsed follicular lymphoma: a single group, open-label, phase 2 trial. Lancet Oncol. 15, 6977 (2014).
  125. da Silva, I.P. et al. Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 blockade. Cancer Immunol. Res. 2, 410422 (2014).
  126. Xu, L. et al. Increased Tim-3 expression in peripheral NK cells predicts a poorer prognosis and Tim-3 blockade improves NK cell-mediated cytotoxicity in human lung adenocarcinoma. Int. Immunopharmacol. 29, 635641 (2015).
  127. Wang, Z. et al. the clinical significance of abnormal Tim-3 expression on NK cells from patients with gastric cancer. Immunol. Invest. 44, 578589 (2015).
  128. Nowak, J. et al. role of donor activating KIR-HLA ligand-mediated NK cell education status in control of malignancy in hematopoietic cell transplant recipients. Biol. Blood Marrow Transplant. 21, 829839 (2015).
  129. Tarek, N. et al. Unlicensed NK cells target neuroblastoma following anti-GD2 antibody treatment. J. Clin. Invest. 122, 32603270 (2012).
  130. Zamora, A.E., Grossenbacher, S.K., Aguilar, E.G. & Murphy, W.J. Models to study NK cell biology and possible clinical application. Curr. Protoc. Immunol. 110, 1114 (2015).
  131. Huntington, N.D., Mention, J., Vosshenrich, C.A., Satoh-Takayama, N. & Di Santo, J.P. in Natural Killer Cells–at the Forefront of Modern Immunology 39–61 (Springer, 2010).
  132. Li, Y. & Di Santo, J.P. Probing human NK cell biology using human immune system (HIS) mice. Curr. Top. Microbiol. Immunol. 395, 191208 (2016).
  133. Paolino, M. et al. The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature 507, 508512 (2014).
    This study highlighted the therapeutic potential of targeting the TAM–CBl-b inhibitory pathway in NK cells and demonstrated the anti-metastatic activity of newly developed small-molecule inhibitor of TAM kinase.
  134. Putz, E.M. et al. CDK8-mediated STAT1-S727 phosphorylation restrains NK cell cytotoxicity and tumor surveillance. Cell Rep. 4, 437444 (2013).
  135. Delconte, R.B. et al. CIS is a potent checkpoint in NK cell-mediated tumor immunity. Nat. Immunol. 17, 816824 (2016).
    This study identified CIS, the suppressor of IL-15 signaling in NK cells, as a potent checkpoint in NK cell activation. Deletion of CIS had a profound anti-metastastic effect in vivo, and adoptive therapy of CIS-null NK cells transferred resistance to melanoma metastasis.
  136. Maghazachi, A.A. Role of chemokines in the biology of natural killer cells. Curr. Top. Microbiol. Immunol. 341, 3758 (2010).
  137. Bernardini, G. & Santoni, A. The pathophysiological role of chemokines in the regulation of NK cell tissue homing. Crit. Rev. Oncog. 19, 7790 (2014).
  138. Young, A., Mittal, D., Stagg, J. & Smyth, M.J. Targeting cancer-derived adenosine: new therapeutic approaches. Cancer Discov. 4, 879888 (2014).
  139. Huntington, N.D. The unconventional expression of IL-15 and its role in NK cell homeostasis. Immunol. Cell Biol. 92, 210213 (2014).
  140. Delconte, R.B. et al. The helix-loop-helix protein ID2 governs NK cell fate by tuning their sensitivity to interleukin-15. Immunity 44, 103115 (2016).
  141. Stanietsky, N. et al. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity. Proc. Natl. Acad. Sci. USA 106, 1785817863 (2009).
  142. Stanietsky, N. et al. Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR. Eur. J. Immunol. 43, 21382150 (2013).
  143. Fuchs, A., Cella, M., Giurisato, E., Shaw, A.S. & Colonna, M. Cutting edge: CD96 (tactile) promotes NK cell-target cell adhesion by interacting with the poliovirus receptor (CD155). J. Immunol. 172, 39943998 (2004).
  144. Jones, B.S., Lamb, L.S., Goldman, F. & Di Stasi, A. Improving the safety of cell therapy products by suicide gene transfer. Front. Pharmacol. 5, 254 (2014).

Download references

Author information

  1. These authors contributed equally to this work.

    • Nicholas D Huntington &
    • Mark J Smyth


  1. Immunology of Cancer and Infection Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia.

    • Camille Guillerey &
    • Mark J Smyth
  2. School of Medicine, University of Queensland, Herston, Australia.

    • Camille Guillerey &
    • Mark J Smyth
  3. The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.

    • Nicholas D Huntington
  4. Department of Medical Biology, The University of Melbourne, Melbourne, Australia.

    • Nicholas D Huntington

Competing financial interests

M.J.S. has research agreements with Medimmune and Bristol Myers Squibb.

Corresponding author

Correspondence to:

Author details

Additional data