**6. Conclusions**

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

**NK cell type Target antigen Cancer Co‐stimulatory** 

PBMC‐NK CD19/GD2 CD3ζ or 2B4

PBMC‐NK NKG2D ligands wide range CD3ζ (with

PBMC‐NK ErbB2 (HER‐2) Breast, ovarian and

YT cell line CEA Colon carcinoma CD3ζ Electroporation

renal cell carcinoma

PBMC‐NK CD19 B‐ALL, CLL CD3ζ Retrovirus [171]

PBMC‐NK CD19 B‐ALL, CLL CD137/CD3ζ Electroporation(mRNA) [152] PBMC‐NK CD20 B‐ALL, CLL CD137/CD3ζ Electroporation(mRNA) [186]

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].

 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

**domain**

DAP10 CD137/CD3ζ

alone

2B4/CD3ζ CD137/CD3ζ

DAP10)

**Gene transfer method Ref**

Retrovirus [185]

Retrovirus [177] Electroporation(mRNA)

[148]

(plasmid DNA)

CD28/CD3ζ Retrovirus [174]

HER2+

**Table 3.** CAR‐NK cells.

108 Natural Killer Cells

the survival of these mice [176].

Because NK cells are difficult to culture and it is a challenge to transduce foreign genes into NK cells, research concerning NK cells has been delayed compared to research involving T cells. However, culture and gene transfer technologies for NK cells are now developing. As introduced here, genetically modified NK cells acquired enhanced antitumor functions. These NK cells are very intriguing and are expected to be revolutionary cellular medicines for the treatment of malignancies.

NK cells are heterogeneous populations that exhibit various maturation stages and different KIR expression patterns. Since this heterogeneity has not been completely elucidated, it is not easy to choose the appropriate subset of NK cells for cancer treatment. Although alloge‐ neic NK cell therapy using a KIR mismatch shows a strong antitumor effect against several blood cancers, the mechanisms underlying the activation and maintenance of NK cells in cancer patients are not completely understood. Cancer patients usually undergo a variety


**Table 4.** Products of NK cells for clinical use.

of standard treatments before receiving immunotherapy, and it is important to understand the factors that influence NK cell activity in order to select the correct clinical setting for NK cell therapy. In this review, we have explained the combination with antibodies, the genetic modification technique, and the KIR mismatch pattern (which is the basis of patient selection) that has been tested to maximize the use of NK cells as a treatment for cancer. Products of NK cells for clinical use that have been developed worldwide are shown in **Table 4**.

Advances in the understanding of NK cells may also lead to the development of small‐mol‐ ecule inhibitors targeting intracellular signals. Because NK cells are difficult to handle, their development is delayed compared to several other immunotherapies, but it is highly likely that they will be established as innovative cell therapy in the near future.
