An Alternative Cell Therapy for Cancers: Induced Pluripotent Stem Cell (iPSC)-Derived Natural Killer Cells

Cell therapy is usually defined as the treatment or prevention of human disease by supplementation with cells that have been selected, manipulated, and pharmacologically treated or altered outside the body (ex vivo). Induced pluripotent stem cells (iPSCs), with their unique characteristics of indefinite expansion in cultures and genetic modifications, represent an ideal cell source for differentiation into specialized cell types. Cell therapy has recently become one of the most promising therapeutic approaches for cancers, and different immune cell types are selected as therapeutic platforms. Natural killer (NK) cells are shown to be effective tumor cell killers and do not cause graft-vs-host disease (GVHD), making them excellent candidates for, and facilitating the development of, “off-the-shelf” cell therapies. In this review, we summarize the progress in the past decade in the advent of iPSC technology and review recent developments in gene-modified iPSC-NK cells as readily available “off-the-shelf” cellular therapies.


Introduction
Cell therapy (also called cellular therapy or cytotherapy) is defined as therapy in which cellular material is injected into a patient. It is a technology that relies on replacing diseased or dysfunctional cells with healthy, functioning ones. This idea was initialized in 1931 when Paul Niehans (1882-1971) attempted to cure a patient by injecting material from calf embryos or harvesting cells from young animals or the fetus to treat severely ill patients [1]. Therefore, he is regarded as the inventor of cell therapy even though his claims have never been validated. In fact, this major breakthrough in stem cell research is considered the discovery of hematopoietic stem cells (HSCs), which are broadly applied for treating hematological cancers and various disorders of the blood and immune system in the clinic [2]. Before 2006, people believed that the cells were divided from the "organspecific stem cells" of the tissue in which they resided, and that the cell types were also very limited. In addition, these cells may have irreversibly lost the capacity to generate other cell types. In 2006, Shinya Yamanaka and Kazutoshi Takahashi introduced induced pluripotent stem cells (iPSCs) [3], which can stably proliferate and serve as an unlimited Immunotherapy has become a cornerstone in cell therapy and is an innovative approach for the treatment of cancer. The first FDA-approved gene-edited T-cell products (chimeric antigen receptor-modified T, CAR-T) for lymphoma and leukemia came out on the market in 2017 [11]. Recently, CAR-T cell therapy has been successful and become a clinical hotspot in tumor immunotherapy. However, a key challenge for the wider implementation of cell therapy concerns the laborious procedures of identifying HLA-matched healthy related or unrelated donors and harvesting their cells for engineering and infusion into one patient. Additionally, this application is limited by inherent risks such as graftversus-host disease (GvHD), cytokine release syndrome (CRS), and immune effector cellassociated neurotoxicity syndrome (ICANS) [12]. The process is very lengthy and cumbersome [13,14]. Thus, the first clinical trial by Ruggeri et al., who proposed the potent antitumor efficacy of allogeneic NK cells, was performed in the context of hematopoietic stem cell transplantation (HSCT) [15], and the results revealed the potential realization of off-the-shelf products, making CAR NK cell therapies universal products, which might have a better safety profile than CAR-T cell therapy.
At present, it is known that NK cells not only detect and identify malignant cancer cells but also induce cancer cell death and even help trigger a broader adaptive immune response to fully engage and fight tumor cells. The safety of NK cell-based therapies is demonstrated in both autologous and allogeneic haploidentical settings [16][17][18][19]. Clinical studies show that NK cells are cytotoxic against a wide range of solid cancer tumor cells in vitro. The antitumor activities of adoptively transferred NK cells in vivo have also been Immunotherapy has become a cornerstone in cell therapy and is an innovative approach for the treatment of cancer. The first FDA-approved gene-edited T-cell products (chimeric antigen receptor-modified T, CAR-T) for lymphoma and leukemia came out on the market in 2017 [11]. Recently, CAR-T cell therapy has been successful and become a clinical hotspot in tumor immunotherapy. However, a key challenge for the wider implementation of cell therapy concerns the laborious procedures of identifying HLA-matched healthy related or unrelated donors and harvesting their cells for engineering and infusion into one patient. Additionally, this application is limited by inherent risks such as graft-versus-host disease (GvHD), cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS) [12]. The process is very lengthy and cumbersome [13,14]. Thus, the first clinical trial by Ruggeri et al., who proposed the potent antitumor efficacy of allogeneic NK cells, was performed in the context of hematopoietic stem cell transplantation (HSCT) [15], and the results revealed the potential realization of off-the-shelf products, making CAR NK cell therapies universal products, which might have a better safety profile than CAR-T cell therapy.
At present, it is known that NK cells not only detect and identify malignant cancer cells but also induce cancer cell death and even help trigger a broader adaptive immune response to fully engage and fight tumor cells. The safety of NK cell-based therapies is demonstrated in both autologous and allogeneic haploidentical settings [16][17][18][19]. Clinical studies show that NK cells are cytotoxic against a wide range of solid cancer tumor cells in vitro. The antitumor activities of adoptively transferred NK cells in vivo have also been demonstrated in preclinical xenograft mouse models of ovarian cancer, glioblastoma, and metastatic colorectal cancer [20]. NK cell-based immunotherapy has emerged as a promis-ing therapeutic approach for hematological malignancies and solid tumors. Currently, NK cells can be derived from autologous or allogeneic sources, such as peripheral blood (PB), and can also be differentiated from induced iPSCs and HSCs [21]. Similar to T cells, NK cells can be engineered to better recognize a specific tumor. However, they have some advantages: they can detect a greater number of chemical signals from tumors than T cells; they are less prone to attack healthy tissues than T cells are. Thus, NK cell therapy could complement, and in some scenarios substitute for, T cell-based adoptive therapies to maximize antitumor effects and reduce treatment toxicity [22].
Moreover, scientists are investigating the mechanisms of those transcription factors involved in the generation of iPSCs [27,31,33,40]. Currently, it is well known that Oct4, Sox2, and Nanog, when bound together, activate the promoters of both genes (Sox2 and Oct4) and subsequently enhance the stability of pluripotency gene expression [32,41]. In fact, Sox2 and Oct4 have attracted attention since the discovery that these genes play critical roles during embryogenesis [42]. Sox2, which is a high-mobility group DNA-binding domain transcription factor, is essential for early embryogenesis in mice [43]. The increased expression (~2-fold) of Sox2 in embryonic stem cells (ESCs) induces ESC differentiation into cells that express markers of ectoderm and mesoderm, but not endoderm [44]. Oct4 is highly expressed in pluripotent cells and becomes silenced upon differentiation [45], such as by Oct4-deficient embryos, which fail to form an inner cell mass [46]. However, if one of these transcription factors is utilized, no function is detected until they are grouped together in complexes composed of a wide array of other proteins [42].
It appears that reprogramming towards iPSCs also benefits the introduction of genes into iPSCs and enables the correction of disease-causing gene mutations in patient-derived iPSCs or the delivery of specific mutations into non-disease-affected wild-type iPSCs. This process helps to create iPSC-based disease cell models, which could be beneficial for new drug screenings [60]. iPSC-based drug screening platforms are especially helpful for complicated or unintelligible diseases such as Parkinson's disease [61,62], Alzheimer's disease [63,64] and spinal cord injuries [65,66]. Figure 2 summarizes the generation of iPSCs and applications.
plicity of design and ease of use. It is defined by a guide RNA that binds to the Cas nuclease and facilitates the design of new targeting constructs [72]. Unlike CRISPR-Cas9, ZFN and TALEN systems require the dimerization of the attached Fok1 endonuclease to induce their targeted double strand breaks (DSBs) [73]. However, the above technologies encounter several challenges. The major challenge is the "off-target effect", which may induce unintended responses, leading to the risk of disaster damage. The societal challenge is how to mitigate the sense of uncertainty and fear of catastrophic misuse [74]. The first clinical trials (NCT03655678 and CT03745287) involving ex vivo "CRISPR-Cas9 genome editing in HSCs" for the treatment of hemoglobinopathies were initiated in 2018 [75,76]. Since off-target effects remain a challenge for the clinical translation of CRISPR, it is important to develop ultrasensitive methods to identify them. "Prime editing" based on RNA uses a modified Cas9 coupled to an engineered reverse transcriptase to make precise modifications in genomic sequences without creating DSBs [77][78][79]. The precision and versatility of prime editing may allow it to rival the CRISPR system as the preferred genomic editing tool [67].  Gene editing technology is a next-generation stem cell therapy. Nuclease-based gene editing systems, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the CRISPR-Cas9 system, are among the most commonly used [67]. CRISPR-Cas9 technology in particular has attracted much attention and gained wide usage in the gene editing of human ESCs [68,69] and iPSCs [70,71], owing to its simplicity of design and ease of use. It is defined by a guide RNA that binds to the Cas nuclease and facilitates the design of new targeting constructs [72]. Unlike CRISPR-Cas9, ZFN and TALEN systems require the dimerization of the attached Fok1 endonuclease to induce their targeted double strand breaks (DSBs) [73]. However, the above technologies encounter several challenges. The major challenge is the "off-target effect", which may induce unintended responses, leading to the risk of disaster damage. The societal challenge is how to mitigate the sense of uncertainty and fear of catastrophic misuse [74]. The first clinical trials (NCT03655678 and CT03745287) involving ex vivo "CRISPR-Cas9 genome editing in HSCs" for the treatment of hemoglobinopathies were initiated in 2018 [75,76]. Since off-target effects remain a challenge for the clinical translation of CRISPR, it is important to develop ultrasensitive methods to identify them. "Prime editing" based on RNA uses a modified Cas9 coupled to an engineered reverse transcriptase to make precise modifications in genomic sequences without creating DSBs [77][78][79]. The precision and versatility of prime editing may allow it to rival the CRISPR system as the preferred genomic editing tool [67].

Clinical Application of iPSC-Derived Products
Cell therapy appears to be the best or most common alternative choice of available treatments for some diseases. However, immune rejection is the major challenge. Due to the autologous cell source, this is a privilege of iPSC-derived products in regenerative medicine. Autologous iPSCs are immunologically identical to the host and can be established from host somatic cells and differentiated into a variety of cell types for cell therapy, including immune cells such as natural killer (NK) cells and macrophages. The bank for storing iPSC cell lines was established according to donors with identified human leukocyte antigen (HLA) and has become an effective way to provide recipients with the maximum number of HLA-matched cell products derived from iPSC lines [80].
In 2014, the world's first clinical trial in Japan was performed based on allogeneic iPSCs, in which retinal pigment epithelial (RPE) cells derived from allogeneic iPSCs were transplanted into a patient with age-related macular degeneration (AMD) [81,82]. The 2-year follow-up trial demonstrated that the AMD patient's visual acuity improved and was stable due to the reprogrammed cells, which remained intact. In addition, no serious adverse effects on transplanted iPSC-derived RPE cells were observed [83]. In summary, HLA is a molecule that enables the immune system to distinguish between self and nonself entities, and HLA compatibility is positively correlated with graft survival rates after transplantation [84]. However, the establishment of autologous iPSCs from individual patients validated for clinical use is costly and time-consuming and hampers the standardization of the therapy. Therefore, Japan and the European Union are promoting the use of clinicalgrade allogeneic iPSCs, which are established from the peripheral blood mononuclear cells of HLA-matched healthy donors and are less susceptible to immune rejection. The iPSC bank in Japan reported that the stock of their iPSCs was able to cover approximately 32% of the Japanese population in view of HLA matching for clinical use and the targeting of various diseases in 2018 [85].
Global trends in clinical trials including pluripotent stem cells involve ophthalmic diseases, cardiovascular diseases, neurological disorders, metabolic diseases, genetic syndromes, reproductive and urogenital diseases, hematologic disorders, otorhinolaryngologic diseases and defects in the immune system [86]. Additionally, they can be applied for the treatment of spinal cord injury (SCI) [87][88][89], which is considered a refractory traumatic disease. However, with recent advances in stem cell transplantation, the field of regenerative medicine has gained hopeful momentum in developing a novel treatment for this challenging pathology. SCI usually results in permanent disability, and its symptoms include a loss of muscle function, sensation, or autonomic function in the parts of the body served by the spinal cord [87]. Hideyuki O et al. reported the first study to investigate the therapeutic efficacy and safety of human iPSC-neural precursor cell (NPC) transplantation for animal SCI models [90]. In 2018, the Keio University Certified Special Committee for Regenerative Medicine approved the unintegrated human iPSC product for clinically treating SCI patients with the ASIA impairment score A [91].
iPSCs are established from patients across a panoply of diseases, leading to the development of a wide range of cell-based disease models, which are beneficial for understanding the pathogeneses of diseases and facilitating drug discovery. For example, histone deacetylase 4 (HDAC4) is shown to be mislocalized in patient iPSC-derived dopaminergic neurons, which model Parkinson's disease and cause the downregulation of some critical genes [92]. Alternatively, adoptive immunotherapy with iPSC-derived immune effector cells (dendritic cells (also called DCs), tumor-specific T cells and NK cells) is also applied to cancer treatments [93][94][95][96][97], some examples of which are provided as follows: iPSC-T: Adoptive immunotherapy with antigen-specific cytotoxic T lymphocytes (CTLs) represents a potential therapeutic strategy that can reduce tumor development and provide a survival advantage for patients undergoing cancer therapies. Maeda T et al. developed a simple method to generate antigen-specific CD8αβ T cells from the iPSCs of healthy volunteers and demonstrated their therapeutic potential against leukemia [95]. Wilms' tumor antigen 1 (WT1)-specific CTLs regenerated by this method demonstrated antigen-specific cytotoxic activity in vitro and showed comparable potential to primary CTLs in producing IFNγ and TNFα [94]. When applied in vivo in a xenograft model, these CTLs prolonged the survival of mice bearing WT1-expressing leukemic cells [94].  [98]. iPSC-NK cells were applied to treat NOD/SCID/γc −/− (NSG) mice, which were inoculated with ovarian cancer cells (MA148), and the median survival improved from 73 to 98 days. Moreover, such iPSC-NK cells were found in the peritoneal cavity of mice and were able to markedly inhibit tumor growth [97], indicating the therapeutic potential of iPSC-NK cells for treating solid tumors.
Although iPSC-Ts provide an alternative cell source for allogeneic T-cell immunotherapy, they appear to have poor outcomes and severe side effects [99,100]. Experimental mice receiving iPSC-T treatment died of tumor relapse and/or graft-versus-host disease (GvHD) [99]. In fact, it is reported that patients may develop cytokine release syndrome (CRS) and/or immune effector cell-associated neurotoxicity syndrome (ICANS) [100]. In addition, although more effective than traditional chemotherapy, T cell therapies are usually costly (approximately USD 373-475,000 per dose), require longer preparation times, and partly depend on the quality of the cell source after leukapheresis [101]. However, NK cells function as allogeneic effectors and do not need to be collected from a patient or a specific HLA-matched donor to reduce GvHD [102]; therefore, it is not necessary to spend much effort preparing autologous NK cells, and off-the-shelf sources of allogeneic iPSC-NK cells may become available. Several trials have demonstrated that 30-50% of patients with refractory or relapsed acute myelogenous leukemia (AML) can achieve complete remission after receiving allogenic NK cells [16] stimulated with cytokines (typically IL-2 or IL-15) [103].

Generation of iPSC-Derived NK Cells
Recently, several generation methods have been described to mass-produce iPSC-NK cells to provide unlimited NK cells for research or clinical applications, and the representative NK cells are summarized in Recently, biotechnology has advanced to the point where NK cells can be generated directly from CD34+ HSCs and iPSCs. However, the different culture methods might give rise to different subsets of NK cells. Therefore, there is an urgent need to standardize the phenotyping protocol because the specific phenotypes may be associated with the function of NK cells, which is critical for the therapeutic applications of NK cells [107].

Natural Killer Cells (NK cells)
NK cells are a type of lymphocyte (a white blood cell) that was first described in the mid 1970s as an innate immune cell and recently reclassified as a member of the group 1 innate lymphoid cells (ILCs) [108]. Five major groups of ILCs have been defined on the basis of their cytokine production patterns and developmental transcription factor requirements: natural killer (NK) cells, group 1 ILCs (ILC1s), ILC2s, ILC3s and lymphoid tissue-inducer (LTi) cells. ILC1s, ILC2s and ILC3s resemble the corresponding T helper cell subsets (T helper 1 (TH1), TH2 and TH17 cells, respectively) and produce cytokines that shape both innate and adaptive immune responses [109]. They are able to recognize stressed cells in the absence of antibodies and the major histocompatibility complex (MHC), allowing for a much faster immune reaction. NK cells are typically activated by missing MHC class I marker-harmful cells, which play a very important role. Missing MHC class I cells cannot be detected and destroyed by other immune cells, such as T lymphocytes [109][110][111][112].

The Physiological Conditions in NK Cells
NK cells differentiate from and mature in the bone marrow (BM), lymph nodes (LNs), spleen, tonsils, and thymus and then enter the circulation. In humans, they are typically characterized as CD56+CD3 − lymphocytes and can be broadly categorized into two subpopulations based on the level of CD56 and CD16 (Fc receptor FcR III) expression: CD56 bright /CD16 neg cells and CD56 dim /CD16 pos cells (Figure 3). Previous studies have suggested that CD56 bright /CD16 neg cells are immature precursors of mature CD56 dim /CD16 pos cells [113][114][115]. The majority of PB-NK cells are CD56 dim /CD16 pos cells, which are highly cytotoxic against target cells. In contrast, approximately 2-10% of PB-NK cells are CD56 bright /CD16 neg cells, which have a low cytotoxic activity while displaying a high capacity to produce immune regulatory cytokines (such as IFNγ, TNF, and GM-CSF) [116], interacting with dendritic cells and T-cell polarization to participate directly in adaptive immune responses [117]. The unique characteristic of the metabolism of NK cells is that they do not use glutamine as a fuel to drive oxidative phosphorylation (OXPHOS). In fact, ATP production is primarily fueled by glucose. The inhibition of OXPHOS or glycolysis in NK cells significantly impairs IFNγ production in a short time [111].
Mature NK cells are known as CD56 dim /CD16 pos cells that can trigger antibodydependent cellular cytotoxicity (ADCC), which is known as one of the important functions of NKs. In fact, several different mechanisms are involved in the NK-mediated lysis of target cells [118,119], including (1) the release of cytoplasmic granules containing perforin and granzymes, (2) the production of IFN-γ, (3) the expression of FasL and TRAIL, and (4) the expression of ADCC. For example, in tumor cell therapy, the type III Fc-gamma receptor (FcγR), also known as CD16, on NK cells recognizes the Fc portion of antibodies bound to tumor cells and triggers the cell death of tumor cells through ADCC. This antibody may be a monoclonal antibody (mAb), preferentially of class IgG1 or IgG3 since these two antibodies are able to link different FcγRs [120]. Over 100 monoclonal antibodies (mAbs) on the market involving several mechanisms of action are used as tumor cell therapeutics [121], in addition to NK cell-mediated ADCC, including checkpoint inhibitors, targeting radiation, blocking cell growth, inhibiting neovascularization and inducing leukocyte effector functions [120,122,123].

NK Cell-Based Therapy in Tumors
Many trials of adoptive NK cell-based immunotherapy have been performed over the past decade and growing clinical and experimental evidence highlights the clear and direct role of NK cells in controlling human cancer development and/or progression. Epidemiologic studies indicate that a reduced function of NK cells is related to cancer incidence [107]. Endogenous NK cells in cancer patients usually have impaired function because of the alteration of the receptor repertoire in the cells. Therefore, the primary method in immunotherapy treatments is to "push" for immune activation by including additives such as cytokines (IL-2 therapy [138], IL-15 therapy [139] and TGFβ inhibitors [140]) and antibodies that help to modulate the mechanisms that improve the quantity and/or quality of the antitumor immune response. For example, some immunomodulatory drugs targeting NK cells, such as lenalidomide and pomalidomide, are approved for the treatment of multiple myeloma, mantle cell lymphoma and a subset of myeloid-derived suppressor cells (MDSCs). These drugs induce cell cycle arrest and apoptosis in tumor cells. Moreover, they increase NCR expression, expand NK cell populations and increase the immune cell recognition of tumor cells in various models. Lenalidomide, for instance, is shown to decrease the immunosuppressive activity of MDSCs and regulatory T (Treg) cells and to increase NK cell cytotoxicity and IFNγ production. NK cell dysfunction in patients with chronic myeloid leukemia (CML) is associated with immune evasion and disease progression, but tyrosine kinase inhibitor (TKI) treatment can restore NK cell numbers and functions [141].
However, NK cell-mediated control of large solid tumors is usually inefficient, although tumors often express large amounts of activating ligands and low levels of inhibitory ligands, presumably due to tumor escape through the alteration of NK cell function and resistance. Currently, the augmentation of the receptor affinity and activation of ADCC and accessibility to the tumor site, in combination with other molecules and immune-modulatory strategies, such as radiotherapy and checkpoint blockade, might be good targets for NK cell-mediated attack. A new type II glycoengineered anti-CD20 mAb with increased FcγR III binding and ADCC is represented by obinutuzumab (GA101).

NK Cell Education
NK cell activity is tightly regulated by a complex interplay between activating and inhibitory receptors that prevent the killing of normal autologous cells expressing an appropriate level of all self-HLA alleles and low/negative levels of ligands for non-HLAspecific activating receptors (aNKRs) [124]. The most frequently described activating receptor is natural killer group 2D (NKG2D, a transmembrane protein), which belongs to the natural cytotoxic receptor (NCR) family. The other family members include NKp46, NKp30, and NKp44, and the leukocyte adhesion molecule DNAX accessory molecule-1 (DNAM1, also called CD226). These receptors, expressed largely on NK cells, are potent inducers of NK cell cytotoxicity, and are crucial for NK cell-mediated tumor apoptosis. It is known that the expression of NKp44 is induced upon NK cell activation, while NKp46 and NKp30 are expressed on both resting and activated NK cells [125].
There are two main inhibitory receptors, the killer immunoglobulin-like receptor (KIR) family, which can bind HLA-class I, and the heterodimeric receptors CD94-NKG2A/B, which recognize HLA-E [124,126,127]. In malignancies, activating killer cell immunoglobulin-like receptors (KARs) are often decreased, while the expression of the most prominent inhibitory NK cell receptors, KIRs and CD94/NKG2A, may occasionally increase [128,129]. Remarkably, NK cell activation is determined by the balance of inhibitory and activating receptor stimulation [129,130]. Indeed, in the absence of inhibitory interactions, NK cells kill target cells and produce cytokines in great quantities [128]. By secreting large amounts of cytokines and chemokines, mature NK cells can not only directly kill target cells but can also elicit other immune cells, including monocytes, DCs, and T cells. Similarly, naïve NK cells can also be activated by different proinflammatory cytokines, such as IL-2, IL-15, IL-18 or IL-21, which stimulate NK cell survival and proliferation, as well as upregulating the expression of activating receptors and enhance NK cell cytotoxicity [109,110].
NK cells can recognize tumor cells and have antitumor and antimetastatic potential in cancerous cells [131]. The chemoattractant/receptor axes appear to control tumorinfiltrating NK cell migration, activation, survival, and persistence in the tumor microenvironment [132]. In the context of NK cells, chemokine receptors of note include CCR2, CCR5, CCR7, CXCR3, and CX3CR1 [133]. CCR2 and CCR5 regulate the migration of tumorassociated monocytes and macrophages. The cytolytic activity of NK cells is simultaneously augmented by CCL2 [134]. High levels of CCL2 correlate with increased monocyte and macrophage recruitment and appear to be an indicator of an adverse prognosis in patients with breast, ovarian, gastric, and esophageal carcinomas [135,136]. The presence of tumorinfiltrating NK cells confers a favorable outcome in many tumors. However, due to nutrient and oxygen deprivation, a higher concentration of tumor-derived metabolism causes the metabolic impairment of NK cells, which ultimately limits their effector functions [137].

NK Cell-Based Therapy in Tumors
Many trials of adoptive NK cell-based immunotherapy have been performed over the past decade and growing clinical and experimental evidence highlights the clear and direct role of NK cells in controlling human cancer development and/or progression. Epidemiologic studies indicate that a reduced function of NK cells is related to cancer incidence [107]. Endogenous NK cells in cancer patients usually have impaired function because of the alteration of the receptor repertoire in the cells. Therefore, the primary method in immunotherapy treatments is to "push" for immune activation by including additives such as cytokines (IL-2 therapy [138], IL-15 therapy [139] and TGFβ inhibitors [140]) and antibodies that help to modulate the mechanisms that improve the quantity and/or quality of the antitumor immune response. For example, some immunomodulatory drugs targeting NK cells, such as lenalidomide and pomalidomide, are approved for the treatment of multiple myeloma, mantle cell lymphoma and a subset of myeloid-derived suppressor cells (MDSCs). These drugs induce cell cycle arrest and apoptosis in tumor cells. Moreover, they increase NCR expression, expand NK cell populations and increase the immune cell recognition of tumor cells in various models. Lenalidomide, for instance, is shown to decrease the immunosuppressive activity of MDSCs and regulatory T (Treg) cells and to increase NK cell cytotoxicity and IFNγ production. NK cell dysfunction in patients with chronic myeloid leukemia (CML) is associated with immune evasion and disease progression, but tyrosine kinase inhibitor (TKI) treatment can restore NK cell numbers and functions [141].
However, NK cell-mediated control of large solid tumors is usually inefficient, although tumors often express large amounts of activating ligands and low levels of inhibitory ligands, presumably due to tumor escape through the alteration of NK cell function and resistance. Currently, the augmentation of the receptor affinity and activation of ADCC and accessibility to the tumor site, in combination with other molecules and immunemodulatory strategies, such as radiotherapy and checkpoint blockade, might be good targets for NK cell-mediated attack. A new type II glycoengineered anti-CD20 mAb with increased FcγR III binding and ADCC is represented by obinutuzumab (GA101). GA101 induces NK cell activation regardless of the inhibitory immunoglobulin-like receptor (KIR) expression. Furthermore, its activity is not adversely modulated by KIR/HLA [120]. Monalizumab (IPH2201), an anti-NKG2A checkpoint inhibitor, has been evaluated in clinical trials in ovarian cancers, head and neck cancers, advanced malignancies and chronic lymphocytic leukemia (CLL). Its use might represent a novel approach in NK-based immunotherapy, not only by enhancing the cytotoxic potential of the cells but also by potentially proposing a role for them in ADCC augmentation [120].
Conversely, many studies have described the engineering of NK cells with CARs to improve the killing of solid tumors, and clinical trials utilizing CAR-expressing NK cells for the treatment of both hematological malignancies and refractory solid tumors have been initiated (ClinicalTrials.gov: NCT00995137, NCT01974479, NCT02839954, NCT02892695, NCT02742727, and NCT02944162, https://clinicaltrials.gov/, accessed on 29 July 2021). Most of these studies use CARs designed for T cells that are expressed in NK cells. However, due to the limitations of CAR-T cell therapy and the patient's specific clinical and diseaserelated features, a decision was made to take an alternative approach to using CAR natural killer (NK) cells that might circumvent these problems [141,142]. Even though allogeneic iPSC-NK cell therapy requires multiple doses for the treatment of solid tumors, genetic modifications, CAR-NK can further improve the specificity, strength, and efficacy of iPSC-CAR NK cell therapies [141].
Indeed, there is an increasing interest in iPSC-NK therapies due to their ability to address the supply chain bottlenecks associated with primary and cell line NK therapies ( Table 2). The advantages of iPSCs include their ease of generation from accessible sources such as fibroblasts or peripheral blood, the retention of pluripotency during expansion, and the capacity for long-term storage. The FDA has already approved a phase I clinical trial to investigate Fate Therapeutics' off-the-shelf iPS-NK product, FT500, representing the first FDA-approved clinical investigation of an iPS-derived cell product in the USA. To generate this product, NK cells were developed from a clonal master iPSC line cell bank. FT500 in combination with checkpoint blockade therapy was utilized to treat adults with advanced solid tumors (ClinicalTrials.gov: NCT03841110). FT516, engineered to include a high affinity, noncleavable CD16 (hnCD16) Fc receptor, was designed against hematological malignancies (ClinicalTrials.gov: NCT04023071). In the preclinical FT538 study, NK cells were engineered by the knockout of the CD38 receptor and knock-in of the high-affinity, noncleavable CD16 receptor and the fused IL-15 receptor. The cells were used in combination with daratumumab (anti-CD38) monoclonal antibody to treat multiple myeloma [101]. Cell line (K562, SKOV3, SW480, HCT-8, MCF7, SCC-25)

IL-2 stimulated
Efficiently killed all tested cancer cell lines (p < 0.5) [105] Ovarian cancer Targeting Mesothelin, engineered with chimeric, NKG2D-CAR-iPSC-NK NKG2D-CAR-iPSC-NK cells displayed in vivo function similar to NKG2D-CAR-iPSC-T cells [141] Hematological cancers, Hepatocellular carcinomas, Ovarian cancer Tetravalent bispecific trifunctional antibody targeting GPC3, NKp46-CAR-iPSC-NK-EGFR Effectively suppressed GPC3-expressing tumor growth in vitro and in vivo and confirmed the therapeutic quality and safety of the final product [142] Clinical trial As of the data cutoff date of April 16, 2021, the encouraging outcome for pateints in both trials of FT516 and FT538 was observed. Four patients in FT516 (n = 9) and one in FT538 (n = 3), respectively, achieved an objective response with complete leukemic blast clearance in the bone marrow. None of them observed dose-limiting toxicities and no events were of any grade of CRS, ICANS, or GvHD. Recently, a third-generation anti-GPC3-CAR 28bbz has demonstrated safety and efficacy in disseminating ovarian tumors [143]. Being engineered with chimeric antigen receptors, the iPSC-NK cells appears to be more effective