**5. Future directions**

#### **5.1. Genetic modification–Gene transfer to NK cells**

In order to genetically modify NK cells, efficient methods for gene transduction into NK cells are necessary. To date, such methods include viral transduction, electroporation, and nucleo‐ fection [145]. Gene transfer to not only NK cell lines such as NK‐92, but also primary NK cells has been conducted. Although various gene transfer efficiencies have been described, their transduction efficiency into NK cells is generally not high. Mainly retroviral vectors and lentiviral vectors are used for gene transfer. Since a retrovirus vector cannot transduce genes into non‐dividing cells, such a vector is suitable for use with NK cell lines such as NK‐92 cells. When a retrovirus vector is used for primary NK cells, it is necessary to amplify the NK cells, and the transduction timing is important.

In contrast, lentiviral vectors are capable of gene transfer into both dividing and non‐dividing cells. The lentiviral vectors RD114, 10A1, GALV, and VSV‐G are used for the envelope of the viral vector, and RD114 and VSV‐G seem to be suitable. However, it has been reported that viral vectors are recognized by antiviral mechanisms such as intracellular pattern recognition receptors, and apoptosis is induced [146]. The introduction efficiency was therefore not high.

Efforts are underway to improve the transduction efficiency. For example, by using a cytokine combination (e.g., IL‐2 + IL‐15 or IL‐2 + IL‐21), the transduction efficiency into NK cells by VSV‐G pseudotyped lentiviral vector was improved by approx. fivefold compared to single cytokine‐stimulated NK cells [146]. In addition, inhibitors of intracellular antiviral responses were evaluated, and the results indicated that BX795 (an inhibitor of the TBK1/IKKe com‐ plex) improved the transduction efficiency by approx. 10‐fold [146]. Guven et al. reported transduction into 75.4% of NK cells after 21 days of culture by a two‐round transduction with the GALV‐pseudotyped retroviral vector [147]. Further improvement of the transduction effi‐ ciency into NK cells by using viral vectors is desired.

In non‐viral gene transfer, mainly electroporation has been studied. Electroporation is a method of physically pulling a minute hole in a cell membrane by applying an electric pulse to a cell suspension and sending the nucleic acids into the cells. The transfer of genes into NK cells using this method has been performed. High transfection efficiency and a high survival rate have been reported in both NK cell lines and primary NK cells. Much higher transfection efficiency was achieved using mRNA compared to using DNA [148–152]. Introduction by nucleofection has also been tried, but the efficiency was not high [153, 154].

In light of these reports, the transfection of mRNA by electroporation is considered to be an efficient method from the viewpoint of gene transfer into NK cells. Shimasaki and colleagues reported that NK cells transfected with mRNA encoding CD19‐specific CAR (anti‐CD19‐4‐1BB‐CD3ζ) by electroporation showed enhanced cytotoxicity for tumors in animal models [154]. On the other hand, the transfection of mRNA can obtain transient gene expression. Further research is required to determine whether a treatment effect can be expected. When persistent gene expression is the goal, gene transfer by retroviral or lentiviral vectors is suitable.

It is necessary to select a suitable gene transfer method for the purpose of treatment. In any case, it is desirable to develop more efficient, simple, highly reproducible and clinically appli‐ cable gene transfer methods for NK cells.

#### **5.2. Improved persistence of NK cells** *in vivo*

*4.5.2. Combination with a checkpoint inhibitor: expansion of the therapeutic spectrum*

reactivity against this class of antigens.

synergistic effect with a checkpoint inhibitor.

and the transduction timing is important.

**5.1. Genetic modification–Gene transfer to NK cells**

**5. Future directions**

104 Natural Killer Cells

A loss or down‐regulation of HLA class I antigens in tumor cells has been frequently observed in a variety of human malignancies, and this represents an important cancer‐immune escape mechanism [130–134]. Using a panel of monoclonal antibodies on tumor tissue sections, these loss or down‐regulation has been found in 60–90% of tumors [135–140]. Early studies using immunohistological analyses of different tumors showed a very low frequency of allelic loss. However, with the arrival of other techniques, such as studies of microsatellites to detect the loss of heterozygosity (LOH) on chromosome 6, it has been shown that LOH (haplotype loss) is the most frequent alteration of HLA class I expression [139, 141–144]. This alteration is caused by various defects in the HLA genomic region (i.e., the short arm of chromosome 6, 6p21), including chromosomal dysfunction, mitotic recombination, and genetic conversion. The nature of the antigens that allow the immune system to distinguish cancer cells from non‐ cancer cells has long remained obscure. Recent technological innovations have made it possi‐ ble to dissect the immune response to patient‐specific neoantigens that arise as a consequence of tumor‐specific mutations, and emerging data suggest that the recognition of such neoanti‐ gens is a major factor in the activity of clinical immunotherapies. These observations indicate that the neoantigen load may form a biomarker in cancer immunotherapy and provide an incentive for the development of novel therapeutic approaches that selectively enhance T‐cell

If there is a neoantigen that can be a target of CTL and the patient has MHC on which the antigen is presented, and if the MHC is not lost from the tumor cells, treatments using CTL as an effector (e.g., checkpoint inhibitors) may be effective. NK cells that preferentially kill tumor cells whose expression of MHC has decreased by MHC non‐restriction are expected to have a

In order to genetically modify NK cells, efficient methods for gene transduction into NK cells are necessary. To date, such methods include viral transduction, electroporation, and nucleo‐ fection [145]. Gene transfer to not only NK cell lines such as NK‐92, but also primary NK cells has been conducted. Although various gene transfer efficiencies have been described, their transduction efficiency into NK cells is generally not high. Mainly retroviral vectors and lentiviral vectors are used for gene transfer. Since a retrovirus vector cannot transduce genes into non‐dividing cells, such a vector is suitable for use with NK cell lines such as NK‐92 cells. When a retrovirus vector is used for primary NK cells, it is necessary to amplify the NK cells,

In contrast, lentiviral vectors are capable of gene transfer into both dividing and non‐dividing cells. The lentiviral vectors RD114, 10A1, GALV, and VSV‐G are used for the envelope of the As a strategy for the genetic modification of NK cells to improve their survival *in vivo*, meth‐ ods for transducing cytokines such as IL‐2 and IL‐15 into NK cells have been reported, since it was demonstrated that a local administration of IL‐2 resulted in enhanced functioning of NK cells [155, 156]. IL‐15 has already been used in patients with metastatic melanoma and metastatic renal cell carcinoma, and it has been reported to induce the proliferation and clini‐ cal response of NK cells. Following these findings, attempts to introduce IL‐2 and IL‐15 genes into NK cells were reported. Nagashima et al. showed that the NK‐92 cell line transduced with IL‐2 genes with a retroviral vector propagated for >5 months irrespective of IL‐2 and showed higher antitumor activity than the parental cell line *in vitro* and *in vivo* [157]. Imamura et al. showed that the transfection of membrane‐bound IL‐15 into human PBMC‐derived NK cells using a retroviral vector resulted in the cells' survival for 2 months without the addition of cytokines *in vitro*, and that the cytotoxicity of the transduced NK cells was enhanced; more‐ over, the antitumor activity was observed in a mouse model [158].

Sahm et al. transduced IL‐15 gene into NK‐92 cells and observed cell proliferation in the absence of cytokines. They also showed that the co‐transduction of an EpCAM‐specific chi‐ meric antigen receptor and IL‐15 into NK‐92 cells enhanced the specific and efficient cytotoxic activity [159]. Jiang et al. reported high cytotoxic activity of a human NK cell line (NKL) transduced with IL‐15 *in vitro* against human hepatocellular carcinoma; the transduced NKL suppressed tumor growth and prolonged survival in human hepatocellular carcinoma‐trans‐ planted model mice [160]. These reports suggested that NK cells transduced IL‐2 or IL‐15 can proliferate sustainably *in vitro* and *in vivo*, which resulted in improved antitumor activity.

#### **5.3. Improvement of homing**

It was reported that NK cells expressed different chemokine receptors depending on the acti‐ vating state [161, 162]. Proper homing to the tumor tissue is an important factor in eliciting the antitumor activity of NK cells. Carlsten et al. showed that *ex vivo* expanded NK cells derived from PBMCs transfected with CCR7 mRNA using electroporation migrated significantly to CCL19, a ligand of CCR7 [163]. Sonamshi et al. reported that NK cells transferred CCR7 pro‐ tein from feeder cells using trogocytosis, but not genetic manipulation, promoted the migra‐ tion to CCL19 and CCL21 *in vitro*, and that the NK cells were transferred to lymph nodes in a mouse model [101]. There are few reports of genetic modification targeting homing receptors, but further progress in this field is expected.

#### **5.4. Improvement of tumor‐specific cytotoxic activity**

As an approach to enhance specificity to tumor cells, a technique using a chimeric antigen receptor (CAR) should be mentioned. CAR is a chimeric protein composed of a single‐chain antibody (the antigen‐specific binding site) in which a light chain and a heavy chain derived from a monoclonal antibody recognize a tumor cell surface antigen, a transmembrane domain, and an intracellular signal domain.

In the first‐generation CARs, the intracytoplasmic signal domain is composed of only the CD3ζchain. In addition to CD3ζ, the second‐generation CARs have another T cell costimula‐ tory signal domain (CD28, 4–1BB, OX40, etc.), and the activation signal is efficiently transmit‐ ted. In the third‐generation CARs, two or more costimulatory signal domains are inserted. CAR‐introduced T cells have already been used clinically, and excellent results have been obtained in some cases [164–167]. One of the problems with CAR‐T cell therapy is a serious side effect. CAR‐T cells attack not only target cells but also non‐target normal cells. For exam‐ ple, CD‐19 CAR‐T cells kill not only tumor cells but also normal B cells, which cause B‐cell defi‐ ciency [168]. In HER2‐specific CAR therapy for colorectal cancer with lung metastasis, death due to a pulmonary complication accompanied by high cytokinemia has been reported [169].

NK cells are an attractive alternative to T cells, as NK cells have the following advantages. HLA matching is not necessary, and NK cells are used as allogenic cells. Their lifespan is limited, and they can be expected to be excluded from the body before severe side effects occur, after they kill cancer cells. The reported CAR‐NK cells are summarized in **Table 3**. NK cells transduced with CD19‐CAR or CD20‐CAR have been used when targeting B‐cell malignancies. Bossel et al. showed that NK‐92 cells transfected with anti‐CD19‐CAR by using electroporation had high cytotoxic activity against a CD19+ cell line and primary chronic lym‐ phocytic leukemia (CLL) [150]. They also showed that tumor cells can be eliminated in xeno‐ graft mouse models by using NK‐92 cells transduced with anti‐CD19 CAR with lentiviral vectors [170]. Imai et al. also showed that primary NK cells transduced with anti‐CD19–CD3ζ with a retroviral vector killed CD19+ cells and that the cytotoxicity was improved by adding a signaling domain of 4–1BB to anti‐CD19–CD3ζ [171].

et al. showed that the transfection of membrane‐bound IL‐15 into human PBMC‐derived NK cells using a retroviral vector resulted in the cells' survival for 2 months without the addition of cytokines *in vitro*, and that the cytotoxicity of the transduced NK cells was enhanced; more‐

Sahm et al. transduced IL‐15 gene into NK‐92 cells and observed cell proliferation in the absence of cytokines. They also showed that the co‐transduction of an EpCAM‐specific chi‐ meric antigen receptor and IL‐15 into NK‐92 cells enhanced the specific and efficient cytotoxic activity [159]. Jiang et al. reported high cytotoxic activity of a human NK cell line (NKL) transduced with IL‐15 *in vitro* against human hepatocellular carcinoma; the transduced NKL suppressed tumor growth and prolonged survival in human hepatocellular carcinoma‐trans‐ planted model mice [160]. These reports suggested that NK cells transduced IL‐2 or IL‐15 can proliferate sustainably *in vitro* and *in vivo*, which resulted in improved antitumor activity.

It was reported that NK cells expressed different chemokine receptors depending on the acti‐ vating state [161, 162]. Proper homing to the tumor tissue is an important factor in eliciting the antitumor activity of NK cells. Carlsten et al. showed that *ex vivo* expanded NK cells derived from PBMCs transfected with CCR7 mRNA using electroporation migrated significantly to CCL19, a ligand of CCR7 [163]. Sonamshi et al. reported that NK cells transferred CCR7 pro‐ tein from feeder cells using trogocytosis, but not genetic manipulation, promoted the migra‐ tion to CCL19 and CCL21 *in vitro*, and that the NK cells were transferred to lymph nodes in a mouse model [101]. There are few reports of genetic modification targeting homing receptors,

As an approach to enhance specificity to tumor cells, a technique using a chimeric antigen receptor (CAR) should be mentioned. CAR is a chimeric protein composed of a single‐chain antibody (the antigen‐specific binding site) in which a light chain and a heavy chain derived from a monoclonal antibody recognize a tumor cell surface antigen, a transmembrane domain,

In the first‐generation CARs, the intracytoplasmic signal domain is composed of only the CD3ζchain. In addition to CD3ζ, the second‐generation CARs have another T cell costimula‐ tory signal domain (CD28, 4–1BB, OX40, etc.), and the activation signal is efficiently transmit‐ ted. In the third‐generation CARs, two or more costimulatory signal domains are inserted. CAR‐introduced T cells have already been used clinically, and excellent results have been obtained in some cases [164–167]. One of the problems with CAR‐T cell therapy is a serious side effect. CAR‐T cells attack not only target cells but also non‐target normal cells. For exam‐ ple, CD‐19 CAR‐T cells kill not only tumor cells but also normal B cells, which cause B‐cell defi‐ ciency [168]. In HER2‐specific CAR therapy for colorectal cancer with lung metastasis, death due to a pulmonary complication accompanied by high cytokinemia has been reported [169].

over, the antitumor activity was observed in a mouse model [158].

**5.3. Improvement of homing**

106 Natural Killer Cells

but further progress in this field is expected.

and an intracellular signal domain.

**5.4. Improvement of tumor‐specific cytotoxic activity**



**Table 3.** CAR‐NK cells.

The gene human epidermal growth factor receptor 2 (HER2) is overexpressed in many breast cancers and is correlated with disease progression [172, 173]. It is therefore considered one of the suitable targets of CAR. Kruschinski et al. transduced anti‐HER2–CD28‐CD3ζ into human primary NK cells using a retroviral vector, and the results demonstrated cytotoxicity to an HER2+ cell line. This cytotoxicity was correlated with the HER2 expression level on target cells [174]. Uherek et al. reported that anti‐HER2–CD3ζ‐CAR retrovirally transduced NK‐92 cells efficiently killed cell lines derived from ErbB2‐positive breast carcinoma, ovarian carcinoma, and squamous cell carcinoma *in vitro* and *in vivo* [175]. In a study by Liu et al., the plasmid coding anti‐HER2–CD28‐CD3ζCAR was transfected into NK‐92 cells by electroporation, and the cells specifically killed the ErbB2‐expressing human breast cancer cell lines MDA‐MB‐453 and SKBr3. The adoptive transfer of NK‐92 cells specifically reduced the tumor size and lung metastasis of nude mice transplanted with MDA‐MB‐453 cells and significantly prolonged the survival of these mice [176].

As shown in **Table 3**, CAR against various tumor‐associated antigens (TAA) has also been evaluated in NK cells. Chang et al. reported a modification by CAR using NKG2D, one of the human NK cell activating receptors, instead of the antigen‐binding site of the antibody. Since NKG2D can bind to eight types of ligands expressed in solid tumors and blood tumors, it can be applied to a broader range of tumor cells. Primary human NK cells were transduced with a retroviral vector, using constructs designed to combine the extracellular domain of NKG2D with CD3ζ and further to express DAP10 simultaneously. This approach showed strong cyto‐ toxic activity against various tumor cell lines and showed no damage to normal cells. It also showed strong tumor growth suppression in a mouse model of osteosarcoma [177].
