**3. NK cells for clinical use**

#### **3.1. Sources of NK cells**

**2.2. Death Ligands**

92 Natural Killer Cells

tumors [50].

Fas [45].

**2.3. Perforin and granzyme**

NK cell‐target cell interface[55] (**Figure 2**).

NK cells express members of the tumor necrosis factor (TNF) superfamily, the so‐called death ligands. The cognate of the receptor on target cells by these ligands on NK cells which include TNF‐related apoptosis‐inducing ligand (TRAIL), Fas ligand (FasL) and TNF‐ like weak inducer of apoptosis (TWEAK) — results in classical caspase‐dependent apoptosis [42, 43]. In the TRAIL/TRAIL receptor system, at least five receptors have been identified and two of them, TRAIL‐R1 (DR4) and TRAIL‐R2 (DR5), contain cytoplasmic death domains and are able to transduce an apoptotic signal [44]. Other TRAIL receptors — TRAIL‐R3 (DcR1) and TRAIL‐R4 (DcR2) and a soluble receptor called osteoprotegerin (OPG: TRAIL‐R5) lack a death domain, but they are dedicated as decoy receptors to regulate TRAIL‐mediated cell death [45]. In target cell expressing TRAIL receptors, the ligation of TRAIL can lead to the activation of caspase‐8 and subsequently caspase‐3 to induce apoptosis [46, 47]. TRAIL is up‐regulated after the stimulation of interferon‐gamma (IFN‐γ) *in vitro* [48, 49] and also *in vivo*, which demonstrates that TRAIL is required for the IFN‐γ‐mediated prevention of

FasL is expressed on activated NK cells and cytotoxic T lymphocytes (CTLs) [42]. Although it is known that FasL is expressed on NK cells at a low level, significant amounts are stored intracellularly [51]. An activating signal of rodent NK1.1 up‐regulated the expression of FasL [52]. After FasL has bound to Fas, the Fas associated with two specific proteins, Fas‐ associated death domain (FADD) and caspase‐8, to form the death‐inducing signal complex (DISC). Fas is expressed on various tissues, but the molecule is downregulated in cancers during its progression [53]. NK cells are capable of directly inducing Fas expression on tumor cells via IFN‐γ secretion, and NK cells show cytotoxicity to tumor cells expressing

Cytolytic killing using perforin and granzyme is a major mechanism in the elimination of infected cells and tumor cells by NK cells. NK cells contain cytoplasmic granules including perforin (a membrane‐disrupting protein) and granzyme (a family of serine proteases). Once NK cells recognize target cells, they form an immunological synapse, and the secretory gran‐ ules fuse with the presynaptic membrane and release perforin and granzyme into the syn‐ aptic cleft. Released perforin provides transmembrane pores on the target cell and enables granzyme to diffuse into the cell. Granzyme then initiates the apoptosis of the target cells, and the NK cells detach from the dying cells and can interact with other target cells to accom‐ plish serial killing [54]. The release of such granules stored in the NK cells is dependent on the polarization of both microtubules and actin filaments in the cytoskeleton. The increase of intracellular calcium concentration triggered by a positive balance of activating and inhibitory signals initiate the rapid move of microtubule‐organizing center (MTOC) in the cytoplasm towards the target cell, and then cytotoxic granules migrate along the MTOC [54]. Granules fuse with the presynaptic membrane, subsequently, the lytic granules can be released at the There are several options for sources of NK cell therapy, including peripheral blood mono‐ nuclear cells (PBMCs), umbilical cord blood (UCB), bone marrow (BM), cell lines, human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs).

#### *3.1.1. Peripheral blood mononuclear cells (PBMCs)*

PBMCs are the most common source of NK cells, and PBMCs can be collected by apheresis or a specific gravity centrifugal method (e.g., Ficoll separation). However, the percentage of NK cells in PBMCs is low (5–20%), and because there is a limit on the number of cells that can be recovered from a donor by lymphocyte apheresis, it is not possible that a sufficient number of NK cells for killing the target can remain in the recipient [7]. That is, in order to stay in the recipient's body until the target cells are killed, it is necessary to collect peripheral blood fre‐ quently, which is a heavy burden on the patient.

Numerous methods for amplification cultures of NK cells have been reported. Stem cell mobilization is a process whereby stem cells (CD34+ cells) are stimulated out of the bone marrow space (e.g., the hip bones and the chest bone) into the bloodstream, and granulo‐ cyte‐colony stimulating factor (G‐CSF) is widely used as a drug for harvesting peripheral blood stem cells from patients or healthy people. Another drug, Plerixafor [56], a CXCR4 antagonist approved in 2008, has an excellent mobilization effect in combination with G‐CSF [57, 58].

An increase in a number of CD34‐positive cells harvested by mobilization reduces the num‐ ber of apheresis sessions required for cell therapy, which may reduce the burden on patients. When allogeneic hematopoietic stem cells are used, GVHD in the acute phase, which is thought to be caused by T‐cell contamination, exists as a problem to be overcome. There is also a report that the induction of myeloid‐derived suppressor cells (MDSCs) by the adminis‐ tration of G‐CSF reduces the frequency of GVHD. It is important to note that MDSCs suppress T‐cell function through an arginine depletion by arginase‐1, the production of reactive oxygen species, and the induction of Treg cells [59, 60].

#### *3.1.2. Bone marrow (BM)*

Bone marrow aspiration removes a small amount of bone marrow fluid through a needle put into a bone under general anesthesia. Compared to apheresis, it is very rarely used as a start‐ ing material because of its high invasiveness to donors.

#### *3.1.3. Umbilical cord blood (CB)*

Hematopoietic reconstitution for Fanconi anemia treatment using cord blood was first per‐ formed in 1988 by Gluckman et al. [61]. Since then it has been widely accepted as a source of hematopoietic stem cells when autologous blood is not recommended or readily available. Cord blood (CB) can be donated to public CB banks for use by any patient worldwide for whom it is stored for potential autologous or family use. The majority of CB products used today are for hematopoietic stem cell transplantation and are accessed from public banks.

Cord blood presents no harm to the donor at the time of collection, and the frozen storage of collected samples is possible. In addition, cord blood of various blood types is classified and preserved in umbilical cord blood banks. When umbilical cord blood is used as the source of therapeutic NK cells, it is highly tissue‐compatible according to the patient's blood type and accompanying transplantation. It is easy to select blood type‐specific umbilical cord blood, with little side effects. In addition, it is possible to minimize the risk of GVHD from the absence of T cells [62–64]. As with PBMCs, a growth culture is essential because the amount of NK cells that can be obtained from a single CB sample is not an amount that can expect to provide a therapeutic effect.

#### *3.1.4. Embryonic stem cells and induced pluripotent stem cells (ES/iPS)*

Another possible source of NK cells is human embryonic stem cells (hESCs) and induced plu‐ ripotent stem cells (iPSCs). A differentiation/expansion culture from hESCs and iPSCs to NK cells is a regimen requiring more sophisticated technology compared to the PBMCs and UCB described above. The differentiation process of human iPSCs is divided into three stages of "maintenance amplification", "structure construction", and "differentiation induction". The culturing step is to generate CD34+ hematopoietic progenitor cells from hESCs and iPSCs and then differentiate these cells into NK cells.

A preclinical demonstration that NK cells can be separated from the sources of these plu‐ ripotent stem cells has been described [65, 66]. However, that demonstration enabled efficient proliferation by using mouse stromal cells as feeder cells, and the involvement of heterolo‐ gous cells may limit clinical application. As with UCB, advances in the development of safe, effective and standardized clinical‐grade manufacturing protocols will provide opportunities to develop ready‐made personalization and immunogenic cell therapy.

#### *3.1.5. Cell lines*

antagonist approved in 2008, has an excellent mobilization effect in combination with G‐CSF

An increase in a number of CD34‐positive cells harvested by mobilization reduces the num‐ ber of apheresis sessions required for cell therapy, which may reduce the burden on patients. When allogeneic hematopoietic stem cells are used, GVHD in the acute phase, which is thought to be caused by T‐cell contamination, exists as a problem to be overcome. There is also a report that the induction of myeloid‐derived suppressor cells (MDSCs) by the adminis‐ tration of G‐CSF reduces the frequency of GVHD. It is important to note that MDSCs suppress T‐cell function through an arginine depletion by arginase‐1, the production of reactive oxygen

Bone marrow aspiration removes a small amount of bone marrow fluid through a needle put into a bone under general anesthesia. Compared to apheresis, it is very rarely used as a start‐

Hematopoietic reconstitution for Fanconi anemia treatment using cord blood was first per‐ formed in 1988 by Gluckman et al. [61]. Since then it has been widely accepted as a source of hematopoietic stem cells when autologous blood is not recommended or readily available. Cord blood (CB) can be donated to public CB banks for use by any patient worldwide for whom it is stored for potential autologous or family use. The majority of CB products used today are for hematopoietic stem cell transplantation and are accessed from public banks.

Cord blood presents no harm to the donor at the time of collection, and the frozen storage of collected samples is possible. In addition, cord blood of various blood types is classified and preserved in umbilical cord blood banks. When umbilical cord blood is used as the source of therapeutic NK cells, it is highly tissue‐compatible according to the patient's blood type and accompanying transplantation. It is easy to select blood type‐specific umbilical cord blood, with little side effects. In addition, it is possible to minimize the risk of GVHD from the absence of T cells [62–64]. As with PBMCs, a growth culture is essential because the amount of NK cells that can be obtained from a single CB sample is not an amount that can expect to

Another possible source of NK cells is human embryonic stem cells (hESCs) and induced plu‐ ripotent stem cells (iPSCs). A differentiation/expansion culture from hESCs and iPSCs to NK cells is a regimen requiring more sophisticated technology compared to the PBMCs and UCB described above. The differentiation process of human iPSCs is divided into three stages of "maintenance amplification", "structure construction", and "differentiation induction". The

hematopoietic progenitor cells from hESCs and iPSCs and

[57, 58].

94 Natural Killer Cells

species, and the induction of Treg cells [59, 60].

ing material because of its high invasiveness to donors.

*3.1.4. Embryonic stem cells and induced pluripotent stem cells (ES/iPS)*

*3.1.2. Bone marrow (BM)*

*3.1.3. Umbilical cord blood (CB)*

provide a therapeutic effect.

culturing step is to generate CD34+

then differentiate these cells into NK cells.

The cell lines that have been derived from NK cells are NK‐92, NK‐YS, KHYG‐1, NKL, NKG, SNK‐6, and IMC‐1 cells [67], and several research groups are exploring the possibilities of using these cell lines for therapeutic applications. The primary advantage of an NK cell line is that it is "ready to use", and it is possible to establish comprehensive standardization and characterization of the cell source by using the master cell bank. The cell therapy product is thus considered to be an attractive merit in manufacturing. Moreover, a more homogeneous population is obtained compared to that from peripheral blood, and its homogeneous charac‐ ter is another advantage when performing a genetic modification operation.

NK cell line has been applied to genetic modification technology for expressing intracellular IL‐2 for the forced expression of CD16, natural cytotoxicity receptor (NCR), chimeric antigen receptor (CAR) and NK cell activation [68, 69]. The most widely clinically used cell line is the NK‐92 cell line, which is cytotoxic to a wide range of malignant cells [70, 71]. NK‐92 cells express the receptor but hardly express KIR, NKp44 or CD16. NantKWest (Culver City, CA, USA) con‐ ducts clinical trials using NK‐92 cells (Neukoplast™) and has completed a Phase 1 study (U. S. National Clinical Trial [NCT] #00900809 and NCT #00990717). Moreover, the company has begun phase 1 and phase 2 trials of haNK (high‐affinity NK cells) [72] engineered to express CD16 (NCT #02465957 and NCT #03027128). In addition, as another attempt, the development of CAR‐TNK expressing CD7 or CD33 has been advanced (NCT #02944162 and NCT #02742727).

#### **3.2. Manufacturing method**

The number of NK cells contained in a collectable amount of UCB or peripheral blood is not enough to achieve a clinical therapeutic effect. A long‐term culture method is necessary to overcome this problem. For starting material, T cells and/or B cells are removed with mag‐ netic beads to increase the purity of NK cells. This is also to prevent the proliferation of T cells caused by IL‐2 during the culture period of NK cells and to avoid lower purity of the final product [8, 73].

In addition to the important cytokine IL‐2, there are IL‐15, which is necessary for both the maturation and survival of NK cells [21]. IL‐2 and IL‐15 share the same receptor component IL‐2/15Rβ and a common γ chain, and they are used in a culture method without the use of feeder cells [74, 75]. IL‐21 [76], a member of the IL‐2 cytokine family, is a potent immunostim‐ ulatory cytokine that shows diverse regulatory effects on NK cells, T cells and B cells [77, 78] and also has the effect of enhancing rituximab‐mediated antibody‐dependent cell mediated cytotoxicity (ADCC) of mantle cell lymphoma [79]. In addition, there is a culture method using feeder cells to efficiently expand NK cells *ex vivo*. As feeder cells, monocytes, irradiated PBMCs, the K562 cell line and a genetically modified cell line are used.

For example, there are systems using a co‐culture with NK cells and monocytes [80], with CB CD34+ cells and bone marrow stromal cells [81], or K562 cells transfected with IL‐21 [82]. There is also a completely closed culture using Epstein‐Barr virus‐transformed lymphoblas‐ toid cell lines [83]. Thus, the use of feeder cells is an important method for securing a number of cells that can be expected to have a therapeutic effect. However, because of the problem of infectious disease risk presented by the use of an allogeneic feeder, the regulatory hurdles in the manufacture of pharmaceutical products are high [84].

To overcome this problem, Yonemitsu et al. reported a method of culturing highly acti‐ vated NK cells with ≥90% purity from PBMCs in a completely closed and feeder‐free system under the good manufacturing practices (GMP) [13]. As another feeder‐free culture method, Spanholtz and colleagues reported a culture method that achieved amplification efficiency and high purity of 10,000 times or more in 6 weeks from UCB CD34<sup>+</sup> hematopoietic stem cell (HSCs), using a closed system process based on GMP [85]. Knorr and colleagues reported dif‐ ferentiation induction from CD34+ hematopoietic progenitor cells produced under feeder‐free conditions to cytotoxic NK cells [86].

#### *3.2.1. NK cell‐based immunotherapy: autologous cells*

Clinical trials using autologous NK cells have been performed targeting solid tumors such as colorectal cancer, non‐small cell lung cancer, melanoma, kidney cancer and esophageal can‐ cer [87–89]. In general, autologous NK cell therapy is safe without side effects such as GvHD [87], but its therapeutic effect is limited to some cancer types [74]. An activation culture with IL‐2 and OKT3 or Hsp70 has been reported to be able to efficiently induce the proliferation of NK cells [74], in particular, the retronectin culture method of Sakamoto et al. showed a high amplification efficiency (about 4720‐fold) [89].

#### *3.2.2. NK cell‐based immunotherapy: allogeneic cells*

Allogeneic NK cell products are used for the treatment of malignant tumors such as leukemia, renal cell carcinoma, colorectal cancer, and lymphoma. The major risk of allogeneic NK cell transplantation is the onset of GvHD. Measures against GvHD include the use of immunosup‐ pressive agents, injection of high‐purity NK cells by CD3 depletion, and the selection of donors consistent with the host HLA [74, 75]. In the case of haploidentical donors and recipients, to avoid GvHD, it is necessary to strictly perform T‐cell depletion. In many studies, CD56<sup>+</sup> is enriched after the removal of CD3+ T cells [17, 90, 91]. In cases of an allogeneic type, HLA typ‐ ing and confirmation of KIR by flow cytometry are carried out, particularly in order to select the optimum donor. For details on the selection criteria, please refer to a later section of this article.

#### *3.2.3. Synergistic effect: antibody drugs*

An antibody drug is, in short, a medicine that functions based on the specificity with which an antibody recognizes an antigen. The characteristics of antibody drugs are high specificity (low toxicity) and high stability *in vivo*. The antibody binds only to the target antigen, and not to any other, which leads to the intended medicinal effect with only rare unexpected side effects. Antibodies are present at a stable level in the blood, and antibody medicines can also be detected at a stable level in the blood for a long period after administration, and they can exert their medicinal effects over a long term. More than 50 antibody drugs have been approved in Japan, the U.S. and Europe. Target diseases include cancer, rheumatoid arthritis and psoriasis, but most of the targets are cancers. One of the action mechanisms of antibody drugs used to treat cancer is ADCC, in which NK cell plays a central role. **Table 2** shows FDA‐approved antibodies for ADCC to treat cancer.

using feeder cells to efficiently expand NK cells *ex vivo*. As feeder cells, monocytes, irradiated

For example, there are systems using a co‐culture with NK cells and monocytes [80], with

There is also a completely closed culture using Epstein‐Barr virus‐transformed lymphoblas‐ toid cell lines [83]. Thus, the use of feeder cells is an important method for securing a number of cells that can be expected to have a therapeutic effect. However, because of the problem of infectious disease risk presented by the use of an allogeneic feeder, the regulatory hurdles in

To overcome this problem, Yonemitsu et al. reported a method of culturing highly acti‐ vated NK cells with ≥90% purity from PBMCs in a completely closed and feeder‐free system under the good manufacturing practices (GMP) [13]. As another feeder‐free culture method, Spanholtz and colleagues reported a culture method that achieved amplification efficiency

(HSCs), using a closed system process based on GMP [85]. Knorr and colleagues reported dif‐

Clinical trials using autologous NK cells have been performed targeting solid tumors such as colorectal cancer, non‐small cell lung cancer, melanoma, kidney cancer and esophageal can‐ cer [87–89]. In general, autologous NK cell therapy is safe without side effects such as GvHD [87], but its therapeutic effect is limited to some cancer types [74]. An activation culture with IL‐2 and OKT3 or Hsp70 has been reported to be able to efficiently induce the proliferation of NK cells [74], in particular, the retronectin culture method of Sakamoto et al. showed a high

Allogeneic NK cell products are used for the treatment of malignant tumors such as leukemia, renal cell carcinoma, colorectal cancer, and lymphoma. The major risk of allogeneic NK cell transplantation is the onset of GvHD. Measures against GvHD include the use of immunosup‐ pressive agents, injection of high‐purity NK cells by CD3 depletion, and the selection of donors consistent with the host HLA [74, 75]. In the case of haploidentical donors and recipients, to avoid GvHD, it is necessary to strictly perform T‐cell depletion. In many studies, CD56<sup>+</sup>

ing and confirmation of KIR by flow cytometry are carried out, particularly in order to select the optimum donor. For details on the selection criteria, please refer to a later section of this article.

An antibody drug is, in short, a medicine that functions based on the specificity with which an antibody recognizes an antigen. The characteristics of antibody drugs are high specificity (low

hematopoietic progenitor cells produced under feeder‐free

T cells [17, 90, 91]. In cases of an allogeneic type, HLA typ‐

cells and bone marrow stromal cells [81], or K562 cells transfected with IL‐21 [82].

hematopoietic stem cell

is

PBMCs, the K562 cell line and a genetically modified cell line are used.

the manufacture of pharmaceutical products are high [84].

ferentiation induction from CD34+

conditions to cytotoxic NK cells [86].

*3.2.1. NK cell‐based immunotherapy: autologous cells*

amplification efficiency (about 4720‐fold) [89].

*3.2.2. NK cell‐based immunotherapy: allogeneic cells*

enriched after the removal of CD3+

*3.2.3. Synergistic effect: antibody drugs*

and high purity of 10,000 times or more in 6 weeks from UCB CD34<sup>+</sup>

CB CD34+

96 Natural Killer Cells

ADCC mediated by NK cells begins with the recognition of antibodies bound to target cells. NK cells express two Fc receptors, CD16a (FcγRIIIa) [92] and CD32c (FcγRIIc) [93]. These Fc receptors recognize and bind to IgG1 and IgG3 and have a high affinity for IgG3 [94]. NK cells that recognize the antibody on the target cells transmit their signals intracellularly and kill the cells.

The signaling of human CD16 is mediated via FcεRIγ, CD3ζ, or FcεRIγ‐CD3ζ heterodimer. These molecules contain an ITAM and are phosphorylated when the antibody binds to CD16 [42]. CD32c (FcγRIIc) contains an ITAM‐like sequence in the cytoplasmic domain (which is


**Table 2.** FDA‐approved antibodies for ADCC to treat cancer.

similar to that of FcγRIIa), suggesting that it transmits a signal via SRC‐SYK (the SRC family of kinases and spleen tyrosine kinase [SYK]) signaling pathways [95]. However, the expres‐ sion of CD32c was less than half of that of NK cells [96, 97]. Many studies focusing on CD16 have thus been conducted.

Several genetic polymorphisms of CD16 exist. Among them, the amino acid at position 158 has been shown to be important for the strength of the affinity for antibodies. The affinity depends on whether the amino acid at position 158 is phenylalanine or valine, and the valine type (158 V) has a higher affinity for the Fc of IgG. A number of reports have indicated that differences in the affinity for antibodies are correlated with therapeutic effects, and many studies have analyzed the clinical responsiveness of this gene polymorphism and antibody therapy. Cartron et al. examined the effects of rituximab treatment for non‐Hodgkin's lym‐ phoma, and they reported a higher objective response rate in CD16 (158 V) homozygous patients compared to CD16 (158F) carrier patients [98]. Wang et al. analyzed the outcomes of rituximab treatment for Follicular Lymphoma and reported significantly more likely progres‐ sion‐free survival at 2 years in CD16 (158 V) homozygous patients compared to CD16 (158F) carrier patients, at 45 and 14%, respectively [99].

These results suggest that the affinity of CD16 for antibodies correlates with the therapeutic effect. Thus, focusing on CD16, the modification of NK cells has been attempted. Binyamin et al. reported that introducing CD16 (158 V) into NK‐92 cells not expressing CD16 improved the cytotoxicity against B‐cell lymphoma with rituximab [100]. Carlsten et al. reported high ADCC activity of cultured NK cells from healthy donors with CD16 (158F/F) and transduced CD16 (158 V) mRNA by electroporation against rituximab‐coated CD20<sup>+</sup> B‐cell lymphoma cells [101].

As described above, since NK cells mediate antibody‐dependent cytotoxic activity via Fc receptors, compatibility with antibody drugs targeting ADCC is desirable. As a strategy to further augment the antitumor effect, a plausible strategy is to enhance the affinity between the Fc receptor and the antibody. Low‐molecular‐weight compounds that are able to inhibit the shedding of CD16 have been reported. It is known that CD16 is cleaved by a protease such as a disintegrin and a metalloprotease 17 (ADAM17) when cells are activated [102], and thus in order to exert more sustained and enhanced ADCC activity, a method of inhibiting the cleavage of CD16 on NK cells may be important. In fact, inhibitors of ADAM17 enhanced the activity of NK cells [102, 103]. Another method to inhibit the cleavage of CD16 is a genetic modification. The substitution of the serine residue at position 197 in CD16 by a proline pre‐ vents the cleavage of CD16 on NK cells [104]. It may be possible to promote the antitumor effect by using a CD16 mutant.
