Open Access, Peer-reviewed
pISSN 1598-2629 eISSN 2092-6685
Indexed in PubMed, Scopus and SCIE
    Immune Netw. 2020 Apr;20(2):e14. English.
    Published online Mar 09, 2020.
    https://doi.org/10.4110/in.2020.20.e14
    Copyright © 2020. The Korean Association of Immunologists
    Review

    NK Cell-Based Immunotherapies in Cancer

    Min Hwa Shin,1 Junghee Kim,1 Siyoung A. Lim,1 Jungwon Kim,1 Seong-Jin Kim,2 and Kyung-Mi Lee1
      • 1Department of Biochemistry and Molecular Biology, College of Medicine, Korea University, Seoul 02841, Korea.
      • 2Precision Medicine Research Center, Advanced Institutes of Convergence Technology, Seoul National University, Suwon 16229, Korea.
    • Correspondence to Kyung-Mi Lee. Department of Biochemistry and Molecular Biology, College of Medicine, Korea University, 73 Goryeodae-ro, Seongbuk-gu, Seoul 02841, Korea. Email: kyunglee@korea.ac.kr
      Correspondence to Seong-Jin Kim. Precision Medicine Research Center, Advanced Institutes of Convergence Technology, Seoul National University, 145 Gwanggyo-ro, Yeongtong-gu, Suwon 16229, Korea. Email: jasonsjkim@snu.ac.kr
    Received October 31, 2019; Revised March 01, 2020; Accepted March 01, 2020.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    With the development of technologies that can transform immune cells into therapeutic modalities, immunotherapy has remarkably changed the current paradigm of cancer treatment in recent years. NK cells are components of the innate immune system that act as key regulators and exhibit a potent tumor cytolytic function. Unlike T cells, NK cells exhibit tumor cytotoxicity by recognizing non-self, without deliberate immunization or activation. Currently, researchers have developed various approaches to improve the number and anti-tumor function of NK cells. These approaches include the use of cytokines and Abs to stimulate the efficacy of NK cell function, adoptive transfer of autologous or allogeneic ex vivo expanded NK cells, establishment of homogeneous NK cell lines using the NK cells of patients with cancer or healthy donors, derivation of NK cells from induced pluripotent stem cells (iPSCs), and modification of NK cells with cutting-edge genetic engineering technologies to generate chimeric Ag receptor (CAR)-NK cells. Such NK cell-based immunotherapies are currently reported as being promising anti-tumor strategies that have shown enhanced functional specificity in several clinical trials investigating malignant tumors. Here, we summarize the recent advances in NK cell-based cancer immunotherapies that have focused on providing improved function through the use of the latest genetic engineering technologies. We also discuss the different types of NK cells developed for cancer immunotherapy and present the clinical trials being conducted to test their safety and efficacy.

    Keywords
    NK cells; Cancer; Immunotherapy; Tumor microenvironment

    INTRODUCTION

    Due to the severe and/or obvious side effects of the available cancer drugs and radio therapeutic approaches, a much attention is being paid to cancer immunotherapy, which activates the immune system of patients with cancer. Among the different types of immune cells, NK cells represent innate immune cells that are CD3 negative and CD56 positive; they play important roles in cancer immune surveillance. Constituting approximately 5%–15% of the circulating lymphocytes in humans, NK cells can be classified into subpopulations based on their maturation status and functional characteristics. Unlike T cells or other adaptive immune cells, NK cells exhibit high cytotoxicity against tumors and virus-infected cells without prior sensitization. Activated NK cells can also act as regulatory cells by secreting various cytokines to trigger and expand the adaptive immune responses against targeted tumor cells (1). Various NK cell sources are currently being used for adoptive cancer immunotherapy; they include autologous NK cells, allogeneic NK cells, NK cell lines from peripheral blood and stem cells, and genetically engineered NK cells (2).

    The function of NK cells is tightly regulated by the balance between activating and inhibitory receptors. Inhibitory signals blocking NK cell activation generally result from the interaction between killer cell Ig-like receptors (KIRs) and MHC class I (3). Activating NK cell receptors, such as NK group protein 2 family member D (NKG2D), NKp30, NKp46, NKp44, and DNAM-1, allow for the recognition of the stress-induced ligands expressed on tumor cells, and prompt NK cells to kill tumor target cells through the release of cytotoxic granules containing perforin and granzyme B (4, 5). NK cells also mediate Ab-dependent cellular cytotoxicity (ADCC), an immune mechanism through which Fc receptor-bearing NK cells can recognize and kill Ab-coated target cells expressing tumor-derived Ags on their surface. NK cells express the low-affinity Fc activating receptor CD16, which is composed of FcγRIIIa (CD16a) and FcγRIIIb (CD16b) (6). Upon binding to the Fc portion of IgG, the cross-linking of Fc receptor with the surface of NK cells induces phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by cellular Src kinases, resulting in cytotoxic granule release and lysis of the target cell via the predominant perforin/granzyme cell death pathway.

    NK cells are also crucial to rebuffing undesirable cellular targets, which include tumors, virally infected cells, and allogeneic bone marrow through their ability to differentiate MHC-1 molecules between normal and abnormal non-self-cells (2). When MHC-I molecules that are present on target cells interact with NK cell inhibitory receptors, NK cells are unable to eliminate the target cells (7, 8). A lack of MHC-I on target cells, however, leads to NK cell activation through a mechanism called ‘missing-self recognition’, which allows NK cells to dispose of the target cells (9). For example, due to the lack or downregulated expression of MHC on bacteria, viruses, and impaired or infected cells, NK cell activity, which is usually suppressed by inhibitory receptors on target cells, is unhindered and NK cells are more prone to attack these target cells (10). Thus, the MHC-dependent inhibitory signal is essential during the effector phase for NK cell tolerance.

    MHC-I molecules can be categorized as classical MHC-Ia or non-classical MHC-Ib. NK cell receptors that interact with MHC-Ia consist of the human KIR family receptors and the murine Ly49 family of inhibitory receptors. MHC-Ib molecules, such as HLA-E in humans and Qa1 in mice, are recognized by receptors such as NKG2/CD94 in both humans and mice (11, 12). KIRs are Ig superfamily receptors that are involved in NK cell inhibition and activation as well as missing-self recognition (10). Long-tailed KIR receptors have one or two immunoreceptor tyrosine-based inhibition motifs (ITIMs) that support inhibitory signaling. Since a considerable percentage of the human population does not have activating KIRs, these receptors are not as clinically critical for NK cell activation (13).

    C-type lectin receptors can substitute the lack of activating KIRs (13). Ly49 and NKG2 family receptors are both type II C-type lectin-like receptors. Murine Ly49-family receptors include both inhibitory and activating receptors that are characterized by the existence or lack of ITIM domains in the cytoplasmic tail (14, 15). Ly49 inhibitory receptors are capable of inhibiting a missing self-response by binding to MHC-I. NKG2 family members contain 2 ITIMs in its cytoplasmic tail (16, 17). NKG2A/CD94 is an inhibitory receptor that plays a significant role in hindering NK cell-mediated killing by ligating to HLA-E (18). Like inhibitory KIRs, NKG2A/CD94 also moderates missing self-recognition to aid NK cell self-tolerance. NKG2A/CD94 contributes to the migration of NK cells as well, which augments target cell binding and the probability of cell elimination (19). When Ly49 inhibitory receptors and NKG2A/CD94 receptors bind to MHC-I, the interaction leads to the phosphorylation of tyrosine residue in the ITIM domain, which then leads to recruitment of the Src-homology 2 domain-containing protein tyrosine phosphatases, such as SHP-1, which then generates inhibitory signals (20).

    Based on the actions and abilities of NK cells, these cells attract significant attention, and their use shows a great promise in the field of cancer immunotherapy. In this review, we focus on the current status and recent advances in NK cell-based cancer immunotherapy, including autologous and allogeneic NK cells, NK cell lines, and human induced pluripotent stem cell (iPSC)-derived and chimeric Ag receptor (CAR)-NK cells, along with the clinical trials being carried out in this field.

    CURRENT STATUS OF CANCER IMMUNOTHERAPY

    Traditional methods of cancer treatment include surgery, chemotherapy, and radiotherapy. Despite the significant efficacy of conventional therapies with respect to eliminating primary tumors, tumor recurrence remains a common issue. Therefore, alternative strategies are required to solve these problems.

    The field of cancer immunotherapy is rapidly growing and is becoming an attractive strategy for the use against severe malignant tumors. This includes mAbs such as immune checkpoint blockades, cancer vaccines, and adoptive cell therapies utilizing endogenous and engineered T cells, and NK cells (21, 22). Among these, Ab-targeted therapy becomes the standard treatment for several malignant cancers. To date, immune checkpoint blockade has proved to be the most successful approach for use in the clinic (23). Immune checkpoint therapy comprises blocking Abs that obstruct proteins that inhibit T cell activation, thus allowing cytotoxic T cells to target tumors (24). Clinical trials using Abs against PD-1/PD-L1 have demonstrated durable response in melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and Hodgkin's lymphoma (25). In addition, patients with melanoma have shown reliable clinical results with anti-CTLA-4 treatment (26). These convincing clinical results have enabled the Ab against CTLA-4 (ipilimumab) and 2 different Abs against PD-1 (pembrolizumab and nivolumab) to achieve Food and Drug Administration (FDA) approval in 2011 and 2014 for melanoma treatment (26).

    In case of cancer vaccines, although preclinical studies have shown promising results over the past years, therapeutic cancer vaccination in humans has demonstrated only moderate success (27); only one cancer vaccine, sipuleucel-T (Provenge, Dendreon) for the treatment of asymptomatic or minimally symptomatic metastatic, castration-resistant prostate cancer, has been approved by the FDA until now (28).

    The low-frequency endogenous T cells (ETCs) reactive to tumor Ags from the peripheral blood of patients can be isolated and expanded ex vivo for adoptive transfer for the treatment of melanoma and other solid tumors. Once tumor Ag has been ensured, ETC is immediately available for clinical trials. This characteristic of ETC is useful to develop personalized Ag-specific T-cell therapy for solid tumor patients, including colorectal, pancreatic, and ovarian cancers. Clinical trials with MART-1 and gp100-specific CD8+ T cells resulted in moderate clinical improvements in 8 of 10 metastatic melanoma patients (29).

    For patients with refractory or relapsed acute lymphoblastic leukemia (ALL) who were treated with engineered CD8 T cells retrovirally transduced with anti-CD19 CAR constructs, over 90% remission rate was achieved (30). Despite its success, the safety of CAR-T therapy is still in question due to the toxicity reported in some studies (31, 32). Other challenges to the use of CAR-T cell therapy in the mainstream include the exploration of target Ags that are not expressed in healthy tissues and overcome the tumor immunosuppressive microenvironment. In addition, adoptive immunotherapy with NK cells has shown great potential for treating malignant solid tumors (33). Unlike CAR-T cells, they do not need to be patient-specific, which makes them better applicable for use in cancer treatment. Several applications of NK cells in cancer immunotherapy will be discussed in this review.

    The recent development of cancer immunotherapy, such as CAR-T cells, NK cell adoptive immunotherapy, and checkpoint inhibitors, provides wide treatment options for individual patient. Therefore, improved complete response (CR) and overall survival in advanced cancer patients have become more conceivable. In addition to these immunotherapeutics, personalized combination therapy specifically tailored to match the genetic and epigenetic characteristics of each patient proved to be a promising approach to boost the effect of cancer therapy.

    NK CELL THERAPY: AN ALTERNATIVE TO CAR-T CELL THERAPY

    First described in the 1970s, NK cells have been a promising tool in the field of adoptive immunotherapy (34). They have the ability to target and destroy tumor cells without prior sensitization, via activation of NK cell-activating receptors against ligands present on tumor target cells. The function of NK cells is defined by the balance between the inhibitory receptors (killer inhibitory receptors and NK group protein 2 family member A [NKG2A] and killer cell lectin-like receptor subfamily G member 1) and the activating receptors (natural cytotoxicity receptors, NKp30, NKp44, NKp46, NKG2D) (34). Under the normal condition, inhibitory KIRs bind to the HLA-I and inhibit the tumor-killing activity of NK cells. However, upon encountering tumor cells, NK cell activation is triggered by binding NK activation receptors with their respective ligands expressed on target tumor cells (35). NK cells eliminate target cells by various mechanisms, such as releasing perforin and granzyme, ADCC, and mediating cytotoxicity by apoptotic pathways including TNF or FAS ligands (36, 37, 38).

    Several clinical studies have reported NK cell-based immunotherapy to be a promising treatment for cancer. In patients with cancer, NK cell function is generally inhibited due to the reduced expression of NK cell-activating receptors, thus impairing their tumor-killing activity. In this regard, adoptive immunotherapy with NK cells has emerged as a promising solution against a number of malignancies (39). One of the well-known methods of NK cell-based adoptive immunotherapy involves ex vivo expansion and activation. This method has been developed to increase both the number and antitumor activity of NK cells to overcome immunosuppression that is commonly observed in solid tumors.

    Several approaches have been developed to generate NK cells for adoptive immunotherapy. One of these approaches involves using cytokines, such as IL-2, IL-12, IL-15, IL-18, and IL-21, to culture and expand NK cells (40). These cytokines can upregulate the expression of activating receptors present on NK cells, thereby enhancing the anti-tumor activity of NK cells against the cells that express the respective receptor ligands. Co-culturing NK cells with growth-inactivated feeder cells may also be used to enhance NK cell proliferation and activation. Culturing NK cells ex vivo has shown to condition NK cells to target tumors that are resistant to the function of NK cells (41).

    LIMITATIONS OF CAR-T CELL THERAPY

    Currently, the most prominent form of immunotherapy uses CAR-T cells. Several pharmaceutical companies, including Gilead (Kite Pharma, Los Angeles, CA, USA), Novartis (Basel, Switzerland), and Juno Therapeutics (Seattle, WA, USA), have lined up several CAR-T cells in their pipelines. CAR-T cells are produced using autologous T cells from patients through genetic engineering to express a CAR for tumor-specific or tumor-associated Ags. Genetically expressed CAR sequences enable these modified T cells to kill tumor cells of corresponding patients via HLA-independent matter. Although CAR-T treatment represents a powerful and promising therapy for patients with cancer, CAR-T cells are associated with a high level of toxicity, potentially resulting in severe life-threatening conditions such as the cytokine release syndrome (CRS) (42).

    CD19 CAR-T therapies have shown high remission rates in patients with ALL and B-cell lymphomas, but durable remissions have not been reported. Approximately 40% of patients showing disease remission at 1 month with CD19 CAR-T eventually relapse within 1 year of treatment, possibly due to the low rate of in vivo persistence and Ag loss (43). In addition, the use of CAR-T cells for the treatment of solid tumor has the limitation due to the complex structures of solid tumors such as extracellular matrix and inhibitory tumor microenvironment (43). These structures limit the contact of CAR-T cells to solid tumors (44).

    Compared to the disadvantages of CAR-T cells, the significant advantages of NK cells for patients with cancer include their ability to be used as “off-the-shelf” treatments, as NK cells are not required to be specific to a particular patient. This advantage enables their use in the clinic, in combination with other drugs for cancer treatment. In this review, we discuss the key types of NK cell-based cancer immunotherapy (Fig. 1) considering the characteristics and advantages of NK cells.

    Figure 1
    Methods of NK cell-based immunotherapy for cancer. Types of NK cells used for cancer immunotherapy. Allogeneic NK cells and autologous NK cells, manipulated with cytokines (IL-12, IL-15, and IL-18) and CAR, resulted in various types of NK cells for cancer immunotherapy. Somatic cells from the donor could be used as functional iPSC-based NK cells. Irradiated NK-92 cell line also served as an important resource for NK cell-based cancer therapy.

    AUTOLOGOUS AND ALLOGENEIC NK CELL THERAPY

    NK cell therapy can be split into autologous and allogeneic cell therapy. Autologous cell therapy implies collection of the cells from a patient before being expanded, processed, and activated. These cells are then refused back into the initial patient. Cells in allogeneic NK cell therapy are not collected from the patient who undergoes treatment, but from various donors before being processed and infused into patients. Although autologous NK cell expansion and activation for adoptive cancer immunotherapy has been reproducibly shown in vitro, they have limited function against the autologous tumors in the clinic. The Rosenberg group has shown that the adoptive transfer of autologous NK cells does not demonstrate significant clinical results in patients with melanoma and renal cell carcinoma (RCC) (45). Although the adoptively transferred ex vivo IL-2 activated autologous NK cells survived for a long period of time, their clinical activity was minimal as well (45). This limitation may be explained partly by the inhibition of NK cell-mediated cell killing by the recognition of the “self” class I MHC on tumor cells by the inhibitory KIR.

    Another reason underlying this limitation could be the difficulty of ensuring the pure and enhanced expansion of autologous NK cells from patients with cancer. In addition, autologous NK cells are vulnerable to the presence of resistant tumor cells, and show a tumor-targeting issue; therefore, tumor cells resistant to autologous NK cells can survive and grow (46). These significant disadvantages limit the use of autologous NK cells for cancer immunotherapy. Selected clinical trials with autologous NK cells, based on these features, are presented in Table 1 (47).

    Table 1
    Selected clinical trials with autologous NK cells

    With the discovery of inhibitory KIR that decrease NK cell-mediated killing of self-MHC-I expressing tumor cells, researchers have begun to explore the possibility of using allogeneic NK cells for cancer treatment, considering their enhanced anti-tumor activity (48). The functional activity of autologous NK cells is limited in patients with cancer, primarily due to the KIR ligand match (49). However, the alloreactivity of NK cells promoted by the mismatch between KIR receptors and their ligands expressed on the surface of target tumor cells in hematopoietic stem cell transplants (HSCT) has been demonstrated to induce potent anti-tumor activity and to limit graft-versus-host disease (GvHD) (50, 51). Unlike T cells, NK cells are not related to GvHD since activated NK cells are not able to attack host Ag-presenting cells, and they are not able to proliferate in patients with cancer. Therefore, toxic effects resulting from the infusion of allogeneic donor NK cells are minimized.

    Alloreactive NK cells can be used either as a type of HSCT or as adoptive immunotherapy. In an allogeneic HSCT settings, T cells from the donor are the main cause of GvHD. In comparison to T-cells, NK cells result in limited incidence of GvHD and have thus been used in T cell-depleted HSCT (52). HSCT is a well-known method for the treatment of hematological cancers, and NK cells are the first lymphocytes to be reconstituted after allogeneic HSCT (53).

    In allogeneic NK cell adoptive therapy, NK cells can be obtained from umbilical cord blood, clonal cell lines (including NK92), and products of lymphapheresis from allogeneic donor PBMCs (54, B55, 56). While treatments with autologous NK cells have not shown persistent anti-tumor activity, researchers have demonstrated the improved efficacy of allogeneic NK cells co-cultured with feeder cells and cytokines, such as IL-12, IL-15, and IL-18, for treating hematological and solid cancers (57). Several methods have now been established to choose NK cell donors based on the genotype of their KIR and KIR ligands (58).

    Despite the efficacy of allogeneic NK cells in hematological malignancies, the function of NK cells is often impaired in solid tumors. The high degree of heterogeneity of solid tumors causes complications in the progression of these NK cells, and conditions and components of the tumor microenvironment such as hypoxia and TGF-β promote the downregulation of activating NK cell receptors (59). Various approaches have been established for improving the effect of adoptively transferred NK cells into solid tumors, such as genetic engineering to confer trafficking to solid tumors as well as combination therapy with other targeted drugs, including checkpoint inhibitors, cytokines, Abs, and immunomodulatory agents (60, 61). Selected clinical trials that have used allogeneic NK cells are presented in Table 2 (47).

    Table 2
    Selected clinical trials with allogeneic NK cells

    NK CELL LINES

    NK cell lines that are sequestered from patients are important in understanding the underlying mechanisms and advancing immunotherapy. NK-92, YT, NK-YS, NKL, and NK3.3 are the most commonly used NK cell lines. These NK cell lines were established from expanded clones of patients with malignant leukemia/lymphoma, with the exception of NK3.3 that originated from the blood of a healthy donor (62). Each cell line has unique characteristics, which must be accounted for when creating an ideal environment to maintain and grow them. The proliferation of these cells can be easily compared to the expansion of PBMCs or stem cell-derived NK cells ex vivo. Due to this observation, these patient-isolated NK cell lines are excellent models for research on human NK cell immunotherapy (63, 64).

    The NK-92 cell line is generated from the peripheral blood of a patient with large granular lymphocyte (LGL) non-Hodgkin's lymphoma (65). As NK-92 is derived from a human tumor, it must be irradiated prior to infusion for safety reasons; this treatment eventually results in limiting its therapeutic efficacy (66). However, this cell line still displays a high degree of toxicity against various malignant cells (67). This cell line is dependent on recombinant IL-2 and the cells die within 72 hours due to the lack of the cytokine (65). NK-92 is positive for CD56, CD2, CD7, CD11a, CD28, and CD45, and negative for CD16, NKp44, and NKp46 (47). The lack of CD16, however, results in an impaired ADCC (65, 68, 69). The NK-92 cell line is the only U.S. FDA-approved cell line for use in clinical trials (70). Researchers are trying to enhance the specificity and efficacy of NK-92 cells through genetic manipulations, such as those used for creating CAR-NK92 cells. CAR-engineered NK-92 cells have several advantages over primary NK cells (71). Overall, the NK-92 cell line is unlimited, uniform, well-characterized, reproducible, and homogeneous. This homogeneity of NK-92 cells results in a more consistent and effective transduction efficiency as compared to those in primary NK cells.

    The YT cell line is derived from the pericardial effusion of a patient with acute lymphoma and thymoma with an inducible receptor for T cell growth factor (TCGF) and IL-2. These cells do not require conditioned media to be maintained and can be grown in RPMI-1640 media supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Unlike many NK cell lines, YT cells proliferate continuously in vitro in an IL-2–independent manner (72). Pre-treatment of these cells with IL-2 did not generate an increased cytolytic activity. This could be beneficial in NK cell adoptive transfer treatments where, oftentimes, high doses of IL-2 are needed to activate the cells, whether they are autologous or allogeneic NK cells (73). YT cells are negative for CD2, CD3, and CD16, and are IL-2-independent. They exhibit irregular cell size and nuclei and exert cytolytic effect against K562, MOLT-4, and HPB-ALL.

    The NK-YS cell line is established from a patient with leukemic-state nasal angiocentric NK cell lymphoma and systemic skin infiltration (74). This cell line is generated through co-culturing leukemic cells from the patient with a mouse stromal cell line (SPY3-2) with recombinant human interleukin 2 (rhIL-2). NK-YS cells express CD2, CD5, CD7, and CD56, but are negative for CD3, CD16, and CD57. The NK-YS cells preserved toxicity against K562 and Jurkat cells and show a type-II latent infection of Epstein-Barr virus (EBV) (74).

    The NKL cell line is established from the peripheral blood of a patient with LGL-leukemia (75). This cell line is IL-2 dependent and expresses CD2, CD6, CD11a, CD27, CD29, CD38, CD43, CD58, CD94, and CD95. In conditions of prolonged in vitro culture, the cell surface expression of CD16, CD56, and CD57 is rapidly decreased (75, 76). These cells have diverse tumor-killing activities and exhibit high and low specific killing activity against NK-sensitive target cells, K562 and 721.221, respectively (75, 76, 77, 78).

    NK3.3 is a normal NK-derived cell line that originates from a mixed lymphocyte culture isolated from the peripheral blood of a healthy donor (79). This cell line is generated by mixing the peripheral blood mononuclear lymphocytes (PBL) from heparinized blood from a responder cell donor and a stimulator cell donor. The responder PBL is incubated with an equal amount of irradiated stimulator PBL in upright flasks for 6 days to generate a mixed lymphocyte culture. To maintain this cell line, the cells must be grown with IL-2 conditioned media to continue proliferation (62). These cells are IL-2-dependent, and express CD2, CD11a, CD38, CD45, CD16, and CD56 (62, 80). NK3.3 cells exhibit strong cytolytic activity against NK-susceptible target cells, such as K562 and MOLT-4 (62, 79).

    Other than NK cell lines described above, we summarized patents introducing the methods of generating the NK cell lines in Table 3 (81-84). EP3138905A1 provides a method for expanding human donor-derived NK cells with IL-21, IL-2, IL-15, and B cell-derived EBV-transformed lymphoblastoid cell lines (EBV-LCL) (81). This method resulted in a 1×1011-fold expansion of NK cells in 7 wk (81). EP2539442A1 introduces a method for the generation and expansion of cytokine-induced killer (CIK) cells and NK cells from human peripheral blood cells in the presence of IL-15, IL-7 in combination with IL-2, stem cell factor (SCF), and Fms-related receptor tyrosine kinase 3 (FLT3) (82). This method showed 15- to 48-fold NK cell expansion within 3 weeks and significantly high toxicity against K562 target cells with the effector:target ratio 20:1 (82). WO2017017184 showed the methods of NK cell modification to produce increased cytotoxic phenotype (83). CD96, CD328, and TNF-related apoptosis-inducing ligand (TRAIL) ligand of KHYG-1 and NK92 cells were modified and showed increased toxicity against K562 or MM1.2 target cells (83). ZNK® cells have been developed by Tella Inc. (Tokyo, Japan) and Kyushu University from human PBMC and cord blood cells (84). ZNK® cells show several hundred to 10,000-fold expansion, 10 times more perforin and Granzyme B release than that before culturing, and elimination of almost all cancer cells within 2 h (84).

    Table 3
    Generation of NK cell line from patents for future therapeutic aspect

    Although these NK cell lines are relatively easy to culture ex vivo, they need to be irradiated prior to clinical use owing to the risk of generating chromosomal abnormalities from the malignant transformation of the cell line . For this reason, in vivo persistence is limited, which leads to frequent injecion of NK cell lines into the patients to achieve promising clinical results. Furthermore, lack of NK activating receptors on some of the NK cell lines make them less cytolytic to tumor cells, hence require genetic incorportation of proper NK activating receptors, such as NKG2D and DNAM-1. However, NK cell lines retain characteristics similar to those of normal human NK cells and serve as an important tool in NK cell research, despite having different origins and phenotypes.

    HUMAN IPSC-DERIVED NK CELLS

    Human pluripotent stem cells, particularly the iPSCs, are considered to be the standard starter tool for the generation of immune cells, such as NK cells (85, 86, 87). NK cells generated from iPSCs are regarded as effective potentiators of tumor lysis, both in vitro and in vivo, as the receptor and gene expression profiles of these cells are similar to those of NK cells purified from peripheral blood or human umbilical cord blood (88, 89). Moreover, human NK cells derived from iPSCs can be generated at a scale appropriate for clinical studies on cancer immunotherapy (Fig. 2) (90). Compared to T cells, primary NK cells are difficult to isolate, purify, and genetically modify, as they represent a heterogeneous mixture of cells that expand hardly (91, 92, 93). However, human iPSCs can be effectively differentiated into NK cells, without the risk of generating chromosomal abnormalities (89, 94, 95); this is in addition to other advantages arising from their being homogeneous and excluded from the donor variation (96). Another advantage of human iPSC-NK cells is that they can be genetically engineered with relative ease using viral vectors and CRISPR technologies (95).

    Figure 2
    Schematic representation of human iPSC-based NK cell therapy. iPSCs are generated from somatic cells of the donor by reprogramming them using crucial various transcription factors, including Oct4, Sox2, c-Myc, and Klf4. The iPSCs are further differentiated into CD34+ cells and NK cells and can be expanded for clinical use.

    A number of studies have reported the development of mature NK cells from human iPSCs (86, 97). Methods for the generation of NK cells from human iPSCs involve the creation and proliferation of CD34+ hematopoietic precursor cells while retaining NK cell cytokine production (IL-15, IL-3, IL-7, SCF, and FLT3 ligand) and the use of a cell line expressing a membrane-bound IL-21 to boost their development, thereby scaling-up NK cell production to a clinical-level (40, 85). Human iPSC-NK cells are reported to efficiently kill hematological malignancies, including acute myeloid leukemia (AML) and multiple myeloma, as well as solid cancer such as ovarian cancer by direct ADCC, and IFN-γ production (86, 89). Furthermore, human iPSC-NK cells can kill leukemia and ovarian cancer cells efficiently, without further generating teratomas (89). Studies have demonstrated the role of KIRs in human iPSCs, and have provided a way to produce NK cells with customized KIR expression in patients with different HLA types (85). In 2018, Li et al. (98) reported the modification of iPSC-derived NK cells with CAR to increase the killing activity of NK cells against mesothelin-expressing tumors, both in vitro and in vivo. In this regard, CAR-iPSC-NK cells may provide an attractive option for CAR therapy, although the safety concerns and clinical effectiveness need to be resolved.

    In February 2019, Fate Therapeutics and the University of California, San Diego (UCSD) undertook the first clinical trial for evaluating the effect of FT500 cell therapy (99). FT500 is an “off-the-shelf” and one master iPSC line-derived NK cell product. In this ongoing trial, FT500 is being tested for safety, along with patient responses to its different doses for the treatment of various tumors (Table 4) (47). If this trial is successful, a new era of “off-the-shelf” cancer immunotherapy might be introduced.

    Table 4
    Clinical Trial with human iPSC-derived NK cells

    CAR-NK CELLS

    In 2017, the U.S. FDA had approved the first CAR-T cell therapy (Tisagenlecleucel, marketed as Kymriah; Novartis) for the treatment of B-cell ALL in children and young adults (100). After this, the second CAR-T cell therapy (Axicabtagene ciloleucel, marketed as Yescarta; Kite Pharma) was approved for the treatment of certain non-Hodgkin's lymphomas such as diffuse large B-cell lymphoma (101, 102). Despite these outstanding clinical results, CAR-T cell therapy still has disadvantages that need to be overcome, such as CRS and neurotoxicity. CAR-T cells are generally composed of three domains, extracellular domain, transmembrane domain, and intracellular signaling domain (Fig. 3). Extracellular domain has a single-chain variable fragment (scFv) that recognizes tumor surface Ag. Various tumor Ag-binding domains have currently been designed and tested as CAR extracellular domains. Between the extracellular and transmembrane domains, there is a hinge region that is generally derived from CD8 or IgG4. The intracellular signaling domain is most important, as this region determines the functionality of CAR. Currently, CAR is in the fourth generation of its technical development (103). The first generation of CARs had a domain with a scFv that recognized cancer Ags, along with an immunoreceptor tyrosine-based activation motif (ITAM, generally CD3ζ) (104). However, the structure of these first generation CARs could not produce long-term cell proliferation signals to retain anti-cancer activity. In second and third generations of CARs co-stimulatory molecules, including CD28, CD134 (OX40), and CD137 (4-1BB) were introduced to increase tumor cytotoxicity and T cell proliferation (105). The fourth generation CAR cells consist of two co-stimulatory molecules (CD28, CD134, or CD137) and secrete cytokines to fully enhance the anti-cancer activity by activating the innate immune system (106, 107).

    Figure 3
    Structure of CAR-NK cells. Design of CAR-NK cells. Three domains constitute the central part of the CAR structure (extracellular domain[s], a transmembrane domain, and intracellular domain[s]). The scFv region of the extracellular domain is composed of heavy and light chains. Components of the intracellular domain include CD3 signaling domain and co-stimulatory domains, such as CD28, 2B4, and CD137.

    The clinical success of CAR-T cells is being used to drive the development of CAR-NK cells. Similar to CAR-T cells, CAR-NK cells have a basic structure, including the extracellular, transmembrane, and intracellular signaling domains. To develop an intracellular signaling motif, CAR-NK cells generally use CD3ζ as the first signal domain, followed by a co-stimulatory domain, such as CD28 or CD137 (4-1BB). Other co-stimulatory molecules, including NKG2D and CD244 (also known as 2B4), are also used by NK cells to promote cytotoxicity and secretion of cytokines by NK cells (108, 109, 110, 111, 112). Compared to CAR-T cells, CAR-NK cell therapy has advantages such as significantly reduced safety issues like on-target/off-tumor effects, GvHD, CRS and tumor lysis syndrome along with additional tumor-killing activity, such as ADCC (113, 114). NK cells produce low amounts of IFN-γ and GM-CSF and do not secrete the central cytokines that promote CRS, such as IL-1 and IL-6. In addition, CAR-NK cells maintain their activating receptors, such as NKp30, NKp44, NKp46, NKG2D, and DNAM-1. Therefore, relapses may be reduced due to the loss of CAR-targeting Ag.

    While CAR-NK cells cause reduced side effects, including the low occurrence of cytokine storms, further studies, including clinical trials, are required to thoroughly evaluate their safety. At present, there are on-going clinical trials registered on clinicaltrials.gov to test the safety and efficacy of CAR-NK cell therapy in both hematological and solid cancers (Table 5) (47).

    Table 5
    Clinical trials with CAR-NK cells

    IMMUNE CHECKPOINT THERAPY IN COMBINATION WITH NK CELLS

    Immune checkpoint inhibitors are Abs blocking the PD-1:PD-L1 and CTLA-4. These molecules have shown significant effects for the treatment of NSCLC, BRAF wild-type melanoma, and metastatic RCC (115, 116, 117). In 2011, ipilimumab, a CTLA-4 blocking Ab, was approved by the U.S. FDA for the treatment of melanoma (118). These checkpoint inhibitors have been reported to improve overall survival and the survival rate of many cancer patients and promote immune responses and tumor regression by reducing the immune suppressive mechanisms in many cancer patients.

    Although checkpoint blockade has shown significant responses, side effects including gastrointestinal and pulmonary toxicities and endocrine failure still remain to be a hurdle to overcome. Other limitations of immune checkpoint inhibitors are a low efficacy in cancers with the low mutational burden (119, 120, 121).

    To improve the response rate to checkpoint inhibitors, clinical trials of checkpoint inhibitors in combination with NK cells, are now on-going. We have summarized these clinical studies in Table 6 (47). NCT03853317 is a phase 2 study of combination therapy with an IL-15 superagonist (N-803), off-the-shelf CD16-targeted natural killer cells (haNK), and avelumab without cytotoxic chemotherapy in patients with Merkel cell carcinoma that has progressed on or after treatment with a checkpoint inhibitor. NCT03937895 is a phase 1/2a clinical trial to test the efficacy of allogeneic NK cells ("SMT-NK") in combination with pembrolizumab (Keytruda) for patients with gemcitabine-refractory biliary tract cancer. NCT04143711 is a phase I/II study to investigate the safety, tolerability, pharmacokinetics, biological, and clinical activity of DF1001 (new molecule that targets NK cell activation signals to specific receptors on cancer cells) in combination with pembrolizumab (Keytruda) in patients with locally advanced or metastatic solid tumors.

    Table 6
    Clinical trials of checkpoint therapy in combination with NK cells

    TUMOR MICROENVIRONMENT AND NK CELLS

    Tumor microenvironment (TME), which is created when a tumor progresses and invades surrounding tissues, provides a protective environment around the tumor cells, immune cells, stromal cells, and extracellular matrix. General conditions of the TME include low oxygen concentration, modified metabolic status, acidic pH, Tregs, myeloid-derived suppressor cells (MDSC), and a series of immunosuppressive cytokines, including TGF-β, IL-10, and IL-6 produced by the tumor cells, Tregs, and MDSCs (122). These harsh immunosuppressive conditions are able to cause differentiation of regulatory immune cells and inhibition of immune cell activation and proliferation, and act as negative regulators of NK cell infiltration into solid tumors (123, 124).

    Microenvironmental hypoxia is a well-known TME of solid tumors and is involved in the up-regulation of hypoxia-inducible factor (HIF)-1α, altered gene transcriptional profiles (125), a metabolic shift to glycolysis (126), and loss of immune reactivity by inducing immunosuppressive mechanisms (127). HIF-1α, a master regulator of hypoxic conditions, regulates key genes involved in cell proliferation, apoptosis, and metabolic pathways. HIF-1α, which is ubiquitinated or degraded in normoxic conditions, is generally expressed and stabilized in highly-glycolytic and hypoxic TME by interacting with HIF-1β in the nucleus (128). HIF-1α downregulates the expression of NK cell-activating receptors, such as NKp30, NKp44, and NKp46, NKG2D, granzyme B, and perforin (129). Hypoxic conditions in TME upregulates HIF-1α-dependent pro-angiogenic genes, such as VEGF and TGF-β, in NK cells (130). In addition, tumor-infiltrating NK cells present a CD56bright phenotype when they bind to PD-L1 in hypoxic TME (131).

    Accumulated glucose metabolic products in TME affect the tumor-infiltrating NK cells by modifying the expression of PMK2, PGK1, GLUT1, and FAS in a HIF-1α-dependent manner (132). Metabolic end products, such as lactate, adenosine, and tryptophan, accumulated in TME, promote the immunoregulatory functions of tumor-infiltrated NK cells (122). Various pathways, including HIF-1α, myc, p53, PI3K/Akt, and mTOR, have been reported to be involved in these dynamic metabolic changes (133).

    To improve persistence within a solid tumor microenvironment, adoptively transferred NK cells would need to avoid immunosuppressive factors to realize effective NK-cell-based therapies for solid tumors.

    CONCLUSION

    Despite the promising results of preclinical and clinical trials, cancer immunotherapy using NK cells still has several hurdles to overcome. Some of these obstacles include a lack of tumor trafficking, in vivo persistence, tumor cytotoxicity, and tumor immune escape. The lifespan of NK cells is also relatively short, and this causes a reduction in in vivo persistence and therapeutic efficacy of adoptively transferred NK cells. iPSC-derived or genetically engineered CAR-NK cells show promising pre-clinical results but are still in the early stages of development compared to CAR-T-based immunotherapy. The approaches to produce engineered NK cells, including optimal gene construct design and delivery methods, have not been fully established as well. Overcoming these challenges for clinical use in the next decades would greatly advance NK cell-based cancer therapeutics.

    Another important limitation of NK cell immunotherapy is its low efficacy against solid tumors. This may due to the low ability of NK cells to traffic through the solid tumor tissues. The tumor microenvironment is another obstacle to the adoptive therapy of NK cells since tumor microenvironment interferes with NK cell activation by secreting immunosuppressive cytokines and facilitating differentiation into MDSC, Treg, M2 regulatory immune subtypes. Further in-depth understanding of NK cells within tumor microenviroment will greatly facilitate NK cell therapy to be effective in various solid tumor types.

    As NK cells have natural anti-tumor toxicity, NK cell-based cancer immunotherapy can be used in a complementary or combinatory method with other anti-cancer agents. Although more research must still be conducted to establish the NK cell therapy as a legitimate form of therapy, NK cells possess the potential to become an ‘off-the-shelf’ product that may shift the current paradigm of cancer treatment modalities in the near future.

    Notes

    Conflict of interest:The authors have no financial conflicts of interests.

    Author Contributions:

    • Conceptualization: Shin MH, Kim JH, Lim SA, Kim JW, Kim SJ, Lee KM.

    • Investigation: Shin MH, Kim JH, Lim SA, Kim JW, Kim SJ, Lee KM.

    • Methodology: Shin MH, Kim JH, Lim SA, Kim JW, Kim SJ, Lee KM.

    • Supervision: Kim SJ, Lee KM.

    • Writing - original draft: Shin MH, Kim JH, Lim SA, Kim JW, Kim SJ, Lee KM.

    • Writing - review & editing: Shin MH, Kim JH, Lim SA, Kim JW, Kim SJ, Lee KM.

    Abbreviations

    ADCC Ab-dependent cellular cytotoxicity
    ALL acute lymphocytic leukemia
    AML acute myeloid leukemia
    CAR chimeric Ag receptor
    CIK cytokine-induced killer
    CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
    CRS cytokine release syndrome
    EBV Epstein-Barr virus
    EBV-LCL Epstein-Barr virus-transformed lymphoblastoid cell lines
    EGFR epidermal growth factor receptor
    ETC endogenous T cell
    FDA Food and Drug Administration
    FLT3 Fms-related receptor tyrosine kinase 3
    GvHD graft-versus-host disease
    haNK off-the-shelf CD16-targeted natural killer cells
    HIF hypoxia-inducible factor
    HSCT hematopoietic stem cell transplantation
    iPSC induced pluripotent stem cell
    ITAM immunoreceptor tyrosine-based activation motif
    ITIM immunoreceptor tyrosine-based inhibition motif
    KIR killer-cell Ig-like receptor
    LGL large granular lymphocyte
    MDSC myeloid-derived suppressor cell
    NCT National Clinical Trial
    NKG2A NK group protein 2 family member A
    NKG2D NK group protein 2 family member D
    NSCLC non-small cell lung cancer
    PBL peripheral blood mononuclear lymphocyte
    RCC renal cell carcinoma
    SCF stem cell factor
    scFv single-chain variable fragment
    TCGF T cell growth factor
    TME tumor microenvironment
    TRAIL TNF-related apoptosis-inducing ligand
    UCSD University of California, San Diego

    ACKNOWLEDGEMENTS

    Lee KM was supported by grants from the National Research Foundation of Korea (NRF) (NRF-2017R1A2B3004828, NRF-2016M3A9B6948342, NRF-2018M3A9D3079288, and NRF-2018M3A9D3079285). Shin MH was also supported by a grant from NRF (NRF-2018R1D1A1B07041442) and a Korea University Grant. This work was supported by the Korea Health Industry Development Institute (KHIDI-HI14C2640) grant funded by the Korean Government.

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    MeSH Terms
    Adoptive Transfer
    Cytokines
    Exhibition
    Genetic Engineering
    Humans
    Immune System
    Immunization
    Immunotherapy*
    Induced Pluripotent Stem Cells
    Killer Cells, Natural
    Sensitivity and Specificity
    T-Lymphocytes
    Tissue Donors
    Tumor Microenvironment
    Metrics
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