**5. NK cell in cancer immunotherapy**

Cancer immunotherapy is the targeted therapy designed to induce antitumor response against malignancies by harnessing the power of the immune system [71]. The ability to recognize and lyse transformed cells without prior immunization, the ease of isolation and expansion *ex vivo,* and the shorter life span make NK cells a good alternate to immunotherapy. Furthermore, NK cell can kill cancer cells without damaging healthy tissues or risking the T cell–driven inflammatory cytokine storm that can accompany other immunotherapies. The NK cells derived from peripheral or umbilical cord cells, embryonic or induced pluripotent stem cells, and NK cell lines were being tested for treating various malignancies. Several promising clinical therapies have been used to exploit NK cell functions in treating cancer patients.

#### **5.1. Adoptive NK cell transfer therapy**

Adoptive NK cell transfer therapy is a strategy aimed at enhancing the biological function of the immune system by means of autologous or allogeneic NK cells. NK cells for adoptive NK cell transfer therapy (autologous or allogeneic) are usually obtained from the peripheral blood of the patient or from a donor. They can also be derived from the bone marrow, umbilical-cord blood, human embryonic stem cells, or induced pluripotent stem cells and are now considered as alternative sources of therapeutic NK cells [72].

Various approaches exist for the therapy with the adoptive transfer of NK cells. In autologous transfer, NK cells from the patient are activated and expanded in vitro in the presence of cytokines. IL-2 has been used for this purpose, but recently, the combination of IL-12, IL-15, and IL-18 might generate NK cells that are more functional and have memory properties. The expanded and activated NK cells are then transferred back into the patient. To sustain the expansion and function of the infused NK cells, patient receives IL2 cytokine administration. 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.

In allogenic transfer, NK cells can be obtained from HLA-matched or haploidentical (partially matched) donors. 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. NK cells are expanded through processes similar to those used for autologous transfer except that T cells should be removed.

#### *5.1.1. CAR-engineered NK cells*

NK cells can be transduced with activating chimeric antigen receptors (CARs) that specifically bind to antigens overexpressed by tumor cells. CARs are designed by the fusion of an antigen binding with a hinge region, a transmembrane domain and one or more stimulatory molecules. CARs can be engineered in autologous or allogeneic NK cells or in NK cell lines such as NK-92. Each CAR has the CD3ζ chain (or sometimes the FcRγ chain) as its main signaling domain. To increase persistence and superior functionality, co-stimulatory domains, usually from CD28 or CD137, can be added to the CAR construct. CARs from the first generation have no stimulatory domain, whereas CARs from the second generation and third generation have one costimulatory 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 the elimination of cancer cells. Several recent studies have documented a success using NK cells engineered to express activating chimeric antigen receptors (CARs) specific to tumor antigens [73]. Many B-cell acute and chronic leukemia can escape killing by natural killer cells. The introduction of chimeric antigen receptors (CAR) into T cells or NK cells could potentially overcome this resistance [74]. NK-92 leukemia cell lines were transduced to express CARs specific for CD19 [75] and CD20 [76] expressed on B cell malignancies and also for disialoganglioside GD2, a glycolipid expressed on neuroblastoma and various other cancer types [77].

In glioblastoma, the most aggressive primary brain malignancy, intracranial administration of NK-92-EGFR-CAR cells represents a promising therapy [78]. In human multiple myeloma (MM), CS1-specific (a surface protein highly expressed on MM cells) chimeric antigen receptor (CAR)-engineered natural killer cells [79] enhance responses to tumor cells in vitro and suppressed tumor growth when tested in vivo in xenograft models [65, 78, 80]. Autologous or allogeneic transplantation of CS1-specific CAR NK cells may be a promising strategy to treat multiple myeloma.

#### **5.2. Cytokine-induced NK cell activation**

**5. NK cell in cancer immunotherapy**

58 Natural Killer Cells

tions in treating cancer patients.

**5.1. Adoptive NK cell transfer therapy**

transmitted by self-HLA molecules.

*5.1.1. CAR-engineered NK cells*

be removed.

considered as alternative sources of therapeutic NK cells [72].

Cancer immunotherapy is the targeted therapy designed to induce antitumor response against malignancies by harnessing the power of the immune system [71]. The ability to recognize and lyse transformed cells without prior immunization, the ease of isolation and expansion *ex vivo,* and the shorter life span make NK cells a good alternate to immunotherapy. Furthermore, NK cell can kill cancer cells without damaging healthy tissues or risking the T cell–driven inflammatory cytokine storm that can accompany other immunotherapies. The NK cells derived from peripheral or umbilical cord cells, embryonic or induced pluripotent stem cells, and NK cell lines were being tested for treating various malignancies. Several promising clinical therapies have been used to exploit NK cell func-

Adoptive NK cell transfer therapy is a strategy aimed at enhancing the biological function of the immune system by means of autologous or allogeneic NK cells. NK cells for adoptive NK cell transfer therapy (autologous or allogeneic) are usually obtained from the peripheral blood of the patient or from a donor. They can also be derived from the bone marrow, umbilical-cord blood, human embryonic stem cells, or induced pluripotent stem cells and are now

Various approaches exist for the therapy with the adoptive transfer of NK cells. In autologous transfer, NK cells from the patient are activated and expanded in vitro in the presence of cytokines. IL-2 has been used for this purpose, but recently, the combination of IL-12, IL-15, and IL-18 might generate NK cells that are more functional and have memory properties. The expanded and activated NK cells are then transferred back into the patient. To sustain the expansion and function of the infused NK cells, patient receives IL2 cytokine administration. 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

In allogenic transfer, NK cells can be obtained from HLA-matched or haploidentical (partially matched) donors. 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. NK cells are expanded through processes similar to those used for autologous transfer except that T cells should

NK cells can be transduced with activating chimeric antigen receptors (CARs) that specifically bind to antigens overexpressed by tumor cells. CARs are designed by the fusion of an antigen binding with a hinge region, a transmembrane domain and one or more stimulatory molecules. To promote NK cell expansion, the use of IL-2 has demonstrated the effectiveness on NK cell activation and anti-tumor responses [81]. It was reported that NK cells from lung cancer patients could regain the cytotoxicity against targets after activation by IL-2 [82]. However, NK cells activation using high-dose IL-2 has some side effects because of severe capillary leaky syndrome. To improve the therapeutic efficacy and safety, a different strategy combining IL-2 with other NK cell activators was used. Hellstrand et al. [83] administered IL-2 together with histamine to 22 acute myeloid leukemia (AML) patients and showed a good clinical outcome. IL-2 diphtheria toxin (IL2DT), a recombinant cytotoxic fusion protein has been used in order to increase the depletion of regulatory T cells (Treg) and therefore improving in vivo donor NK cell expansion and remission induction [84].

#### **5.3. NK cells targeting cancer stem cells**

Tumor harbors a population of cancer cells with "stem-cell" like properties including selfrenewal and the ability to produce differentiated progeny [85]. These cells termed cancer stem cells (CSCs) can drive tumor progression and therapeutic resistance to standard cancer therapy. In fact, cancer stem cells have been proposed as an important mechanism of tumor initiation and/or repopulation after tumor debulking by chemotherapy and/or by radiotherapy.

In addition, CSCs have been associated with tumor relapse and metastasis, even in cases of apparent complete response to systemic therapy [86]. Then, targeting CSCs is a promising strategy for cancer therapy. Natural killer cells have the ability to reject allogeneic hematopoietic stem cells, and there are increasing data demonstrating that NK cells can selectively identify and lyse CSCs. Tallerico et al. [87], for example, demonstrated that metastatic colorectal cancer, which contains a high proportion of CSCs, showed increased susceptibility to NK cytotoxicity. Similarly, Castriconi et al. [88] reported that glioblastoma-derived CSCs were susceptible to NK cell cytotoxicity. Human cancer cells with stem cell-like phenotype exhibit enhanced sensitivity to the cytotoxicity of IL-2 and IL-15 activated natural killer cells [89]. IL-2- and IL-15-activated NK cells were found to be cytotoxic against human breast cancer stem cells and CD 133+ melanoma CSCs [90]. Recently, Ames et al. [91] showed that NK cells kill CSCs from different kinds of tumors, through the interaction of the NKG2D activating receptor with its ligand (MICA/B).
