Current status and future perspective of natural killer cell therapy for cancer

Introduction

Natural killer (NK) cells are a type of innate lymphoid cells (ILCs) and the third major subset of lymphocytes in the body in addition to T cells and B cells, accounting for approximately 10 %–15 % of the total peripheral blood (PB) lymphocytes. They are widely distributed in PB, liver, spleen, and other tissues, participating in forming the first line of defense in the human immune system. NK cells originate from CD34+ hematopoietic progenitor cells and undergo development into common lymphoid progenitor cells, eventually maturing into NK cells characterized by the upregulation of CD56 and downregulation of CD34 [1], [2], [3]. Unlike adaptive immune cells, NK cells possess the unique ability to rapidly respond to threats without requiring antigen stimulation or being restricted by major histocompatibility complex (MHC). Through the secretion of perforin and granzymes, as well as exerting antibody-dependent cell-mediated cytotoxicity (ADCC), NK cells have the ability to directly or indirectly eliminate target cells. Additionally, they can release a range of cytokines and chemokines, which play crucial roles in anti-tumor and anti-viral immune responses. In recent years, NK cell therapy has emerged as a promising approach in the field of anti-tumor treatment, with numerous preclinical trials and studies had been published or currently undergone [4, 5].

In this review, we will discuss the current status of biological research pertaining to NK cell anti-tumor activity, and address the challenges encountered by NK cells in the context of cancer treatment, particularly their susceptibility to dysfunction. Furthermore, we will provide a detailed overview of the application of NK cell-based therapies in the field of oncology, seen in Figure 1. These therapies encompass enhancing NK cell activity therapy and adoptive NK cell therapy, which includes purified NK cell therapy, expanded NK cell therapy, and chimeric antigen receptor (CAR)-NK cell therapy. By examining the existing literature and ongoing developments, we will shed light on the potential of NK cell-based approaches for combating cancer.

Figure 1:

Natural killer (NK) cell therapy: an overview. (A) The main NK cell sources for NK cell therapy. (B) NK cells can be expanded in vitro through various methods. (C) Multiple therapeutic approaches based on NK cell therapy for anti-tumor treatment. iPSC, induced pluripotent stem cell; ESC, embryonic stem cell; CAR, chimeric antigen receptor (created with BioRender.com).

Biological study of anti-tumor effect of NK cells

NK cells participate in immune responses to tumors by killing target cells and producing cytokines [6]. NK cells express a variety of activating and inhibitory receptors, and the balance of signals mediated by these receptors determines the outcome of NK-cell activation [7]. Roles of NK cells were important for the tumor control, and prognostic values of NK cells were explored in different kinds of tumors, including hematological malignancies and solid tumors. Impaired NK cell activity and the capability of secretion cytokines, such as interleukin (IL)-1β, interferon (IFN)-γ and IL-2, have been associated with early relapse of acute leukemia [8]. In acute myeloid leukemia (AML), the rapid recovery of NK cells post chemotherapy has been linked to improved leukemia-free survival (LFS). AML blasts exhibit heterogeneous expression of NK receptor ligands (NKRLs), including 6 activating (MHC class I chain-related protein A and B (MICA, MICB), CD155, CD112, UL16 binding protein (ULBP)1, and ULBP2/5/6) and 3 inhibitory (HLA class I, programmed cell death ligand 1 (PD-L1), and PD-L2) NKRLs. Notably, ULBP1 expression was significantly associated with improved 2-year overall survival (OS), relapse-free survival (RFS) and reduced relapse rates [9]. Moreover, cytomegalovirus (CMV) infection induced the expansion of memory-like NK cells (CD56dimCD57+NKG2C+ NK cells) at 6 months post-transplantation, which independently correlated with a lower 2-year relapse risk [10]. Moreover, Nersesian et al. conducted the first meta-analysis on NK cell infiltration in solid tumors to assess its prognostic value for OS, and discovered a strong association between NK cell infiltration and a decreased risk of death [11]. In lung cancer, NK cell infiltration has shown a significant and positive association with tumor high Scarff-Bloom-Richardson (SBR) grade. In resected pulmonary adenocarcinoma, count of NK cells was significantly related to the regulation of tumor progression, involving T classification, N classification, and stage [12]. NK cells have also been significantly associated with human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancer subtypes. At a median follow-up of 5.5 years, high CD56 expression (≥5 cells/10 high-power field) was associated significantly with improved OS and disease-free survival (DFS) [13]. In addition, NK cells in the blood were an independent predictor of survival in colorectal cancer patients. A higher percentage of NK cells indicated a longer survival time compared to a lower percentage [14]. In patients with HER2+ advanced gastric cancer treated with first-line fluoropyrimidine-platinum doublet plus trastuzumab, low baseline NK cell activity was associated with worse progression-free survival (PFS) and OS than high baseline NK cell activity [15].

However, there are a lot of factors that influence the anti-tumor effect of NK cells, as depicted in Figure 2. Firstly, the maturation status of NK cells plays a crucial role, as mature NK cells exhibit a stronger anti-tumor ability [16, 17]. The development and functional maturation of NK cells are complex process involving multiple cytokines (e.g., IL-15, IL-3 and IL-7), transcription factor (e.g., Ras–MEK–MAPK, JAK–STAT5, and PI3K–AKT–mTOR) and so on [6, 18], [19], [20], [21]. For instance, Wang et al. performed conditional deletion of mechanistic/mammalian target of rapamycin complex 1 (mTORc1) and complex 2 (mTORc2) in mice, demonstrating that mTORc1 played a positive role in NK cell maturation and effector functions while mTORc2 negatively regulates NK cell cytotoxicity in a synergistic and non-redundant manner [20]. Meissl et al. disclosed the significance of signal transducer and activator of transcription 1 (STAT1) isoforms (STAT1α and STAT1β). In gene knockout mice, the absence of STAT1α rather than STAT1β was related to defects in IFN-γ signaling, leading to reduced surface levels of IL-15 receptor α in splenic dendritic cells, monocytes and macrophages and further impaired NK cell maturation [21]. Secondly, NK cell education correlated with enhanced anti-tumor effect. NK cell education refers to the interaction between inhibitory NK cell receptors (e.g., Killer Immunoglobulin-like receptors (KIR), NKG2A, and T cell immunoglobulin and ITIM domain [TIGIT]) and self-MHC-I molecules, allowing NK cells to obtain an immune response. Recently, some researches have shown that nonclassical MHC and non-MHC ligands also play an important role in NK cell education [22]. NK cell education is regulated by a combination of inhibitory signals, activating signals, and adhesion molecule signals, representing functional status of NK cells. Several models have been proposed to explain the mechanisms of this procedure [23, 24]. To investigate why educated NK cells have stronger function, our team prospectively enrolled 114 malignant hematological patients who underwent haploidentical hematopoietic stem cell transplantation (HSCT) to study the expression of activating receptors on the surface of reconstituted NK cells with the interaction of KIR with both donor HLA and recipient HLA [25]. We demonstrated that the expression of the activating receptor DNAX accessory molecule-1 (DNAM-1) was modulated by both donor and recipient KIR/MHC-I interaction. Additionally, in vitro studies have shown that compared to uneducated NK cells, educated NK cells are associated with high intensity expression of DNAM-1, NKP30, and NKG2D, resulting in stronger function. However, NK cell education is remolded by its environment factors, and phenotypic changes could not always consistently identify NK cell subsets and functional fate [26]. In addition, changes in cellular metabolism can distinguish NK cell education status in some way. A higher expression of glucose transporter and higher rate of glycolysis was observed in educated NK cells [27, 28]. Thirdly, the memory status of NK cells is another factor correlated with sustained anti-tumor effect of NK cells. Multiple studies have shown the existence of memory and memory-like functionality in NK cells after exposure to haptens, chronic viral infection, or cytokines [29, 30]. Cytokine-induced memory-like (CIML) NK cells can impart increased longevity and enhanced anticancer functionality through short priming of cytokine combination of IL-12, IL-15 and IL-18 in vitro [30]. Mature NK cells have a short lifespan of about 2 weeks post infusion, while memory NK cells induced by human CMV can survive for at least 7 months post-infusion [31]. By analyzing the phenotypic characteristics of CIML, it can be found that the expression of CD25 and semaphorin 7A are upregulated, while the expression of KIRs (e.g., KIR2DL2/L3, KIR2DL1 and KIR3DL1) and transforming growth factor (TGF)-β receptors are downregulated, which may help enhance anti-tumor response [32, 33]. Lastly, the dysfunction induced by tumor as well as immune suppressive microenvironment correlated with poor tumor control [34]. There are multiple factors contributing to NK cell dysfunction, and more detailed information will be elaborated in the following text. Therefore, in the future study, focused on the biological study of NK cells would be help to improve the curative effect of NK cell therapy.

Figure 2:

Schematic illustration of factors influencing the anti-tumor effect of natural killer (NK) cells. MHC, major histocompatibility complex; KIRs, killer inhibitory receptors; NKG2A, natural killer group 2, member A; TIGIT, T cell immunoreceptor with immunoglobulin and ITIM domains (created with BioRender.com).

NK cell dysfunction in the setting of cancer

NK cell dysfunction refers to a condition in which NK cells cannot function optimally or exhibit impaired activity. In new diagnosis AML patients, NK cell dysfunction was characterized by excessive maturation and downregulation of activating receptors (e.g., NKP30, NKP46, and NKG2D) [35], [36], [37], [38]. Effective therapeutic response following chemotherapy correlated with NK function restoration. Refractory or relapsed (R/R) patients demonstrated even worse immune impairments [39]. Compared with the normal control, NK cells from patients with B- and T-cell acute lymphoblastic leukemia (B/T-ALL) were also exhausted with less cytotoxic but exhibit an activated signature that was characterized by high CD56, high CD69, production of activated NK cell-origin cytokines, which was likely caused by their chronic activation. Increased frequencies of activated cytokine-producing NK cells were associated with increased disease severity and independently predicted poor clinical outcome in patients with ALL [40].

There are multiple mechanisms that contribute to NK cell dysfunction. Higher levels of active TGF-β1 have been linked to impaired effector function of bone marrow NK cells (BMNK) in AML patients. TGF-β1 activation was induced by the overexpression of glycoprotein A repetitions predominant (GARP), which was involved with a novel TGF-β processing pathway, on the surface of CD4+ T cells. Active TGF-β1 significantly suppressed mTORC1 activity, mitochondrial oxidative phosphorylation, the proliferation, and cytotoxicity of BMNK cells. Preclinical study has revealed that inhibition of the TGF-β1 pathway using galunisertib, a clinical inhibitor, would conversely restore mTORC1 activity, mitochondrial homeostasis, and cytotoxicity of NK cells in vitro, and improve the antitumor activity in a leukemia xenograft mouse model [41]. A similar relationship between TGF-β and NK cell dysfunction has been observed in breast cancer patients. There are also reduced IFN-γ production and cytotoxicity with clear metabolic deficits in PB NK cells and expression of GARP, and latency associated peptide (LAP) was increased on NK cells. Thus, blocking TGF-β and/or GARP can restore NK cell metabolism and function and is an important target for improving NK cell-based immunotherapies [42]. What’s more, in the setting of glioblastoma multiforme (GBM), the most aggressive brain cancer, the glioblastoma stem cells (GSCs) are sensitive to lysis by healthy allogeneic NK cells in vitro. However, primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched PB NK cells from patients with GBM or healthy donors. This immune evasion tactic to direct cell-to-cell contacts between GSCs and NK cells via αv integrin-mediated TGF-β activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-β signaling or with TGF-βR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth [43]. In addition, hypoxia is a common feature of solid tumors, resulting in upregulation of the transcription factor hypoxia-inducible factor (HIF)-1α in NK cells, which was also associated with NK cell dysfunction. Conditional deletion of HIF-1α in NK cells leaded to reduced tumor growth, elevated expression of activation markers, effector molecules, and an enriched NF-κB pathway in tumor-infiltrating NK cells. IL-18 from myeloid cells was required for NF-κB activation and the enhanced anti-tumor activity of HIF-1α (−/−) NK cells. Extended culture with an HIF-1α inhibitor increased human NK cell responses and could be exploited for cancer therapy [44].

Besides dysfunction, NK cells can transform to regulatory cell and inhibit the anti-tumor effect of T cells through expression of immune checkpoint receptor CD73. These CD73-positive NK cells undergo transcriptional reprogramming and upregulate IL-10 production via STAT3 transcriptional activity, suppressing CD4-positive T cell proliferation and IFN-γ production [45]. IL-22, a cytokine produced by T helper (Th) 17 cells, gamma delta (γδ) T cells, invariant natural killer T (iNKT) cells, and ILCs, is known to promote cancer cell growth, enhance migration, protect from apoptosis, induce epithelial-to-mesenchymal transition, and sustain stemness of malignant cells [46], [47], [48]. Meanwhile, IL-22 mediated immunosuppression in the metastatic niche by promoting the expression of CD155 on cancer cells, which was associated with decreased expression of CD226 on NK cells and reduced IFN-γ production. Therefore, an IL-22-CD155 axis triggers decreased expression of CD226 in NK cells and renders them inert in the tumor microenvironment, and blocking CD155 would be help for cancer control [49]. In hepatocellular carcinoma (HCC), dysfunction of both tumor-infiltrating NK cells and liver resident NK cells was mediated by T cell immunoglobulin and mucin domain 3 (Tim-3)-mediated PI3K/mTORC1 interference. Blockade of Tim-3 has shown to retard HCC growth in an NK cell-dependent manner, suggesting a potential strategy for immune checkpoint-based targeting [50].

In conclusion, how to avoid tumor induced NK cell dysfunction during NK cell therapy would be important to better improve the therapeutic effect of NK cell therapy in the future study.

Enhanced NK cell activity therapy for cancer

Enhancing the activity of NK cells can be achieved through several primary methods, including the use of exogenous activating cytokines, monoclonal antibodies targeting NK cell surface receptors, and NK cell engagers. Notably, exogenous cytokines such as IL-2, IL-15, and TGF-β have been extensively studied and proven to effectively induce NK cell proliferation and enhance their cytotoxic activity. In the realm of clinical research, exogenous cytokines are primarily employed through three distinct approaches. Firstly, they can be utilized to stimulate the ex vivo expansion of NK cells, which serves as the foundation for high-dose NK cell infusion therapy [51]. Secondly, cytokines can be administered intravenously to trigger the activation of NK cell activity in vivo. In a phase I multicenter trial led by Romee et al., the IL-15 superagonist complex ALT-803 was investigated in patients who experienced relapse after undergoing allogeneic HSCT for more than 60 days. The trial involved the administration of ALT-803 once a week to 33 patients, either intravenously or subcutaneously, for a total of 4 doses. The results of the study demonstrated that ALT-803 effectively stimulated the activation, proliferation, and expansion of NK cells and CD8+ T cells, while not increasing the levels of regulatory T cells. Additionally, a positive treatment response was observed in 19 % of the patients who were evaluated [52]. Lastly, cytokines can be expressed in NK cells through genetic modification. This approach involves introducing specific genes into NK cells to enable them to produce cytokines internally [53]. In addition, targeting the inhibitory and activating receptors on the surface of NK cells is a crucial component of tumor immunotherapy. Currently, monoclonal antibody therapies targeting PD-1/PD-L1 and NKG2A have been widely utilized in clinical practice [54, 55]. It is worth mentioning that in clinical research, monoclonal antibodies are often used in combination with cytokine therapy or adoptive NK cell transfer therapy.

NK cell engagers refer to engineered antibodies that are derived from the structure of monoclonal antibodies and have been modified to target multiple tumor antigens and NK cell receptors. These agents are composed of two essential components: a targeting moiety that binds to specific antigen on cancer cells, and an activating moiety that binds to NK cells. Some pre-clinical studies have demonstrated that NK cell engagers facilitate the selective destruction of cancer cells by stimulating the cytotoxic activity of NK cells by acting as a bridge between cancer cells and NK cells. For instance, in a study conducted by Arvindam et al. [56], a tri-specific killer engager (TriKE) molecule called CLEC12A TriKE was developed. This molecule consists of a humanized anti-CD16 heavy chain camelid single-domain antibody (sdAb) to activate NK cells, an IL-15 molecule to promote NK cell proliferation, activation, and survival, and a single-chain variable fragment (scFv) that specifically targets human CLEC12A, a myeloid antigen highly expressed on AML cells. The researchers demonstrated through in vitro experiments that CLEC12A TriKE effectively stimulated NK cell proliferation, enhanced NK cell activation, and induced the death of AML cell lines, while preserving the viability of healthy bone marrow cells. Moreover, CLEC12A TriKE demonstrated its potential in reducing tumor burden in preclinical mouse xenograft models [56]. Gauthier et al. reported the efficacy of TriKE in targeting NK cell activation receptors NKp46, CD16, and tumor antigens. The preclinical study involved in vivo experiments, which demonstrated that when used to treat solid tumors or invasive tumors, TriKE exhibited a remarkable capability to reduce tumor size. Notably, the anti-tumor effects of these engagers were found to be superior to those of tumor antigen-specific monoclonal antibodies [57]. In addition, a phase 2, open-label, multicenter study (NCT04101331) conducted in 2019 evaluated the efficacy of AFM13, a bispecific CD30/CD16 antibody, in patients with R/R peripheral T cell lymphoma. A total of 108 patients were enrolled in the study and received weekly intravenous infusions of AFM13 at a dose of 200 mg. The median number of infusions administered was 9.0, ranging from 1 to 116. The study reported an overall response (OR) rate of 32.4 %, with a complete response (CR) rate of 10.2 %. The median duration of response, PFS, and OS were found to be 2.3, 3.5, and 13.8 months, respectively [58]. Furthermore, NK cell engagers are also commonly combined with NK cell adoptive transfer or other therapies to enhance their anti-tumor activity [59], [60], [61].

Purified NK cell therapy for cancer

Purified NK cells therapy for cancer comes from autologous or allogeneic NK cells. In an early clinical trial, 8 patients with metastatic melanoma or renal cell carcinoma were treated with adoptively transferred in vitro activated autologous NK cells with an average of 4.7 × 10¹⁰ (±2.1 × 10¹⁰) NK cells after the patients received a lymphodepleting but nonmyeloablative chemotherapy regimen. The infused NK cells exhibited high levels of lytic activity in vitro. Although the adoptively transferred NK cells seemed to persist in the peripheral circulation of patients for at least one week post transfer and, in some patients, for several months, no clinical responses were observed [62]. This lack of efficacy may be attributed to the absence of NK cell inhibitory receptor mismatching with autologous tumor cells. To overcome this limitation, allogeneic NK cell infusions have become a promising approach. By 2005, Miller JS et al. reported the outcomes of patients receiving haploidentical, related-donor NK cell infusions after lower intensity regimens in a nontransplantation setting. This resulted in transient persistence but no in vivo expansion of donor NK cells. In contrast, when a more intense regimen of Hi-cyclophosphamide/fludarabine (Hi-Cy/Flu) was used, there was a marked rise in endogenous IL-15, leading to the expansion of donor NK cells and induction of complete hematologic remission in 5 of 19 poor-prognosis patients with AML. These findings suggest that haploidentical NK cells can persist and expand in vivo and may have a role in the treatment of selected malignancies used alone or as an adjunct to HSCT [63].

In order to enhance the efficacy of allogeneic NK cells infusions, in vivo expansion of haploidentical NK cell infusions with IL-2 can induce remission of refractory AML, but efficacy may be hampered by concurrent stimulation of host regulatory T cells [64, 65]. To overcome this limitation, 42 patients received either intravenous or subcutaneous recombinant human IL-15 (rhIL-15) after lymphodepleting chemotherapy and haploidentical NK cells. Escalating doses of rhIL-15 (0.3–1.0 μg/kg) were given on 12 consecutive days in a phase 1 trial. Of 26 patients, 36 % had robust in vivo NK-cell expansion at day 14, and 32 % achieved CR. Hypothesizing that subcutaneous dosing of rhIL-15 would be safer and better tolerated, 16 patients received 10 once per day doses of subcutaneous rhIL-15 at 2.0 μg/kg on a phase 2 trial. NK cell expansion at day 14 was seen in 27 % of the patients, and 40 % achieved remission. RhIL-15 induced better rates of in vivo NK cell expansion and remission compared with previous trials with IL-2, but it was associated with previously unreported cytokine release syndrome (CRS) after subcutaneous but not intravenous dosing. CRS was observed in 56 % of patients given stem cell (SC) rhIL-15 (with concurrent neurologic toxicity in 5 of 9 patients) and was responsive to steroids and tocilizumab. SC administration was associated with slower pharmacokinetic clearance and higher levels of IL-6 than IV dosing [66]. Maintenance of an exhausted T cell state at day +14 has been shown to permit haploidentical NK cell expansion, suggesting that selectively depleting recipient T cells or modulate their dysfunction would be help for NK cells expansion [67].

In a study involving 16 myelodysplastic syndrome (MDS) patients, fludarabine/cyclophosphamide (Flu/Cy) conditioning combined with total lymphoid irradiation was followed by adoptive immunotherapy with IL-2-activated haploidentical NK cells. The NK cell infusions were well-tolerated, with only transient adverse events observed in 16 patients. six patients achieved objective responses with CR, marrow CR, or partial remission (PR). Five of these patients proceeded to allogeneic HSCT. Three patients remain DFS over 3 years after treatment. All evaluable patients with objective responses (5/5 evaluable) had detectable donor NK cells at days 7/14 following infusion and displayed reduction of tumor cell clones, some of which carried poor prognosis mutations. This suggested that high-risk MDS was responsive to NK cell therapy and supported the use of haploidentical NK cell infusions as a bridge to HSCT in refractory patients. Responding patients displayed less pronounced activation of CD8+ T cells and lower levels of inflammatory cytokines following NK cell infusions, further highlighting the potential benefits of depleting recipient T cells or modulating their dysfunction to enhance the efficacy of NK cell therapy [68]. In another study involving 17 AML patient in first CR (median age 64 years, range 53–73), NK cells from haploidentical KIR-ligand mismatched donors were administered after Flu/Cy chemotherapy, followed by IL-2. All patients with molecular disease achieved molecular CR. A significantly higher number of donors alloreactive NK cell clone was observed in responders over non-responders. The infusion of a higher number of alloreactive NK cells was associated with prolonged DFS [69].

As previous mentioned, NK cells exhibit memory like properties with an enhanced recall response after cytokine pre-activation [70]. CIML NK cellular therapy has been shown to be safe and induces remissions in AML patients, with the potential for long-lasting effects [71]. Mass cytometry revealed an in vivo memory-like NK-cell phenotype, where NKG2A served as a dominant checkpoint, and CD8α was associated with treatment failure after CIML NK cell therapy [72]. Meanwhile, PB NK cells from allogeneic healthy donor or patients with advanced melanoma can be differentiated into ML NK cells for use as a novel immunotherapeutic treatment for advanced melanoma, which warrants testing in early-phase clinical trials [73].

Expanded NK cell therapy for cancer

Besides purified NK cell therapy, the rapid development of ex vivo NK cells expansion has facilitated the clinical study of clinical grade expanded NK cells for cancer treatment. Expanded NK cells can be derived from a wide range of sources, including PB, umbilical cord blood (UCB), NK cell lines, as well as stem cells, such as human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC). Each source of NK cells has its own unique advantages and limitations, as summarized in Table 1. And there are several methods that can be used to expand NK cells derived from various cellular sources, including cytokine stimulation, co-culture with feeder cells, magnetic bead selection and genetic modification [74].

The most efficient way to expand NK cell is based on feeder cells. In our previous study, we demonstrated that expansion for 2 and 3 weeks produced approximately 4 and 8 × 10⁹ NK cells from 2 × 10⁷ PB mononuclear cells with by co-culture with K562 cells transfected with 4-1BBL and membrane-bound IL-21 (mbIL-21/4-1BBL NK). The NK cell products generated through this expansion method exhibited higher levels of CD107a and TNF-α production in response to AML cell lines and primary blasts compared to resting NK cells. When the 2-week expanded NK cell products were xenografted into immunodeficient mice with leukemia, they were persistently detected in the BM, spleen, liver, lung, and PB for at least 13 days. Furthermore, these expanded products reduced AML burden in vivo. Compared with matched AML patients with persistent or relapsed minimal residual disease positive (MRD (+) who underwent regular consolidation therapy, MRD (+) patients who underwent NK treatment had better OS and showed no major adverse events. Meanwhile, repeated infusions of mbIL-21/4-1BBL NK cells every 7 days showed a stronger anti-leukemia effect, providing valuable insights for the design of further clinical studies [75]. NK cells from healthy donors and myeloma patients could expand a median of 804- and 351-fold, respectively by co-culture with K562 cells transfected with 4-1BBL and membrane-bound IL-15. These expanded NK cells could kill both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D, natural cytotoxicity receptors, and DNAM1 played a central role. Furthermore, when transferred into the body, these expanded NK cells continued to proliferate in vivo in an IL-2 dose-dependent manner and could persist for up to 4 weeks. They were readily detectable in the human BM and had been shown to inhibit myeloma growth while also protecting the bone from myeloma-induced osteolysis [76].

In addition to feeder cells, another commonly used method for expanding NK cells is pure cytokine culture. Tschan-Plessl et al. conducted a study where they demonstrated that NK cells, expanded with IL-2 and IL-15 under Good Manufacturing Practice (GMP) conditions, exhibited a remarkable 56.0-fold increase in proliferation (ranging from 37.4- to 75.5-fold). In their study, 10 myeloma patients were treated with induction therapy followed by high-dose melphalan (200 mg/m²) at day 1, autologous stem cell transplantation (ASCT) at day 0 and escalating doses of NK cells (1.5 × 10⁶, 1.5 × 10⁷, and multiple doses of 1.0 × 10⁸ cells/kg body weight) from day +1 to day +30 after ASCT. Donor NK cells were detectable in the PB, peaking 1 h after each dose, with up to 90 % of the NK cells being of donor origin. Importantly, this treatment was found to be safe and well tolerated, with no evidence of graft-versus-host disease (GVHD). And comparison with a control group of patients who received ASCT without NK cells infusions showed no significant difference in relapse, PFS and OS [77]. Similarly, Heinze et al. investigated the cytotoxic capabilities of IL-activated NK cells compared to cytokine-induced killer (CIK) cells for treating neuroblastoma (NB) [78]. The separated NK cells from PB mononuclear cells (PBMCs) were expanded with various cytokines (e.g., IL-2, IL-15, and/or IL-21) in feeder-cell free conditions. CIK cells were generated by stimulating PBMCs with IFN-γ, IL-2, OKT-3, and IL-15. After 10–12 days of expansion, NK cell preparations exhibited significantly higher median cytotoxicity against NB cells compared to CIK cells [78].

In contrast to obtaining NK cells from PB, there are two distinct approaches for acquiring a significant quantity of NK cells from UCB. The first approach involves directly isolating and expanding NK cells from UCB. The second approach involves inducing the differentiation of CD34 hematopoietic stem and progenitor cells (HSPCs) derived from cord blood into NK cells, followed by subsequent expansion of the NK cells [79]. Additionally, when working with stem cells, it is necessary to utilize a differentiation system to generate NK cells, which may involve the use of a feeder system or a feeder-free system [80]. However, the primary method for expanding NK cell lines still relies on cytokine-dependent culture methods, particularly IL-2 [81]. Importantly, studies have shown that expanded NK cells derived from UCB, stem cells and NK cell lines were effective to kill tumor cells [82, 83]. More research is imperative to elucidate the variances in cytotoxic functions and the underlying factors among NK cells derived from different sources within diverse tumor environments, which will provide a solid groundwork for improved implementation of NK cell therapy in clinical settings.

CAR-NK cell therapy for cancer

CAR-NK cell therapy is a promising approach in which NK cells are artificially constructed NK cell activation signaling pathways through gene modification to specifically enhance their anti-tumor capabilities. Compared to CAR-T cell therapy, CAR-NK cell therapy offers superior safety, a broader range of cell sources, multiple killing pathways, and wider indications. It also holds significant potential for development as off-the-self product.

iPSC-derived CAR-NK cells therapy for cancer

Although CAR-NK cells can derive from a wide range of sources, iPSC has the advantages both from primary NK cells and NK cell lines, and has become a research hotspot. Similar to primary NK cells, iPSC-NK cells maintain high cytotoxicity and can expand well in vivo after cryopreservation [106]. Meanwhile, iPSC-NK cells exhibit clonal growth, high in vitro expansion, and differentiation capabilities, enabling the production of a large number of homogeneous NK cells with reduced tumorigenic risk compared to NK cell lines [107]. More importantly, the efficiency of transduction or transfection for iPSC-NK cells is high, allowing them to be better genetically engineered through viral vectors, transposons, and CRISPR-Cas9 to create more lethal NK cells [108], [109], [110]. Several laboratories are devoted to developed the differentiation process of NK cells from iPSC, and they commonly follow the differentiation route of hematopoietic precursor cells [93, 111].

One notable study by the Kaufman group involved the engineering of iPSC with a CAR targeting the tumor-associated antigen mesothelin, named meso-CAR-iPSC-NK cells. Mesothelin, a membrane surface glycoprotein, is highly expressed in ovarian cancer but less expressed in normal mesothelial tissue. They revealed that meso-CAR-iPSC-NK cells were able to more effectively inhibit tumor growth and prolong survival in an ovarian cancer xenograft model, compared to conventional PB NK cell therapy, iPSC-NK cell therapy and CAR-T cell therapy. Furthermore, they tested multiple CAR constructs to identify the optimal combination, and found that CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain maximized the anti-tumor ability [112]. Apart from CAR design, researchers have also employed various modification to iPSC in many ways, including gene knockout and gene knockin, to improve in vivo persistence and efficacy of CAR-iPSC-NK cells [113]. Wang et al. developed CD33-CAR-iPSC-iNK cells (QN-023a) that showed superior antitumor activity among the candidates both in vitro and in NOG mouse model [113]. QN-023a exhibited 4 anti-tumor modalities: (1) a proprietary CD33-targeting CAR; (2) a high-affinity, non-cleavable CD16 (hnCD16) to enhance antibody-dependent cellular cytotoxicity (ADCC) and mitigate antigen escape; (3) an IL-15 receptor fusion (IL-15RF) to enable NK cell persistence without the need for exogenous cytokine support; and (4) a CD38 knockout to effectively avoid fratricide of CAR-NK cells.

At present, the number of clinical studies on iPSC-NK cells is increasing year by year, and some clinical data on iPSC-CAR-NK cell therapy has been disclosed, including data on FT596 and FT576. FT596 is an off-the-shelf, CD19-directed CAR-NK cell derived from iPSC, and is generated with a hnCD16Fc receptor and IL-15RF. Phase I clinical trial of FT596 in patients with R/R B-cell lymphomas and chronic lymphocytic leukemia was reported at the 2021 American society of hematology (ASH) annual meeting (NCT 04245722). Researchers evaluated the safety and efficacy of FT596 as a monotherapy or in combination with anti-CD20 monoclonal antibody at 3 different dose levels. The effect of FT596 was dose-dependent, and 5 of the 6 patients (83 %) reached OR at single-dose levels of 300 million cells [114]. Further, the clinical-stage biopharmaceutical company Fate Therapeutics, Inc. presented the outcomes of phase I trial of FT576 in patients with R/R MM at the 2022 ASH annual meeting to assess safety and tolerability (NCT05182073) [102]. FT576 is a first-of-kind, multi-engineered BCMA CAR-NK cell therapy derived from a clonal master engineered iPSC. Besides the construct of hnCD16 Fc receptor and IL-15RF, FT576 is engineered to knock out CD38 gene. Nine R/R MM patients have been treated and evaluated, and it is encouraging that nobody developed CRS and neurotoxicity. Three patients had a decrease in their myeloma disease burden (38 %–97 % decrease), with 2 subjects with a confirmed objective response.

Future perspective

NK cells, as crucial effector cells in innate immunity, possess potent anti-tumor functions, making them promising candidates for cancer treatment. The continuous development of biotechnology has unveiled the tremendous therapeutic potential of NK cell-based therapies in the field of anti-cancer treatment. However, it is important to acknowledge the current challenges and difficulties associated with NK cell therapy. On one hand, there is a limited sample size in preclinical research and clinical trials conducted so far, along with the relatively short follow-up periods. These limitations hinder the comprehensive validation of the safety and efficacy of NK cell therapy. On the other hand, it is necessary to be mindful of the numerous technical issues in the field of NK cell therapy. Determining the optimal sources of NK cells, developing effective NK cell expansion methods, and determining the duration of NK cell therapy are crucial considerations. Moreover, reducing immune evasion to prevent immune exhaustion in the tumor microenvironment is of utmost importance. For CAR-NK cell therapy, the design of target antigens and signaling domains, as well as strategies to enhance NK cell activity and persistence, such as combination therapy with other drugs, are anticipated to be research hotspots in the future of cellular therapy.

Furthermore, despite the promising anti-tumor effects demonstrated in preclinical and clinical research, particularly with CAR-NK cell therapy, NK cell therapy still faces significant ethical challenges in its clinical translation. Firstly, there is a lack of clear benefit assessment criteria for NK cell therapy, especially the genetic editing techniques used to enhance its anti-tumor efficacy requiring further research to investigate its long-term effects and safety. Additionally, with more clinical trials of iPSC-NK therapy, more attention should be pay to the safety issues. Moreover, with the growing number of clinical trials for NK cell therapy, there is a need for further refinement of admission criteria for clinical research platforms and relevant regulatory frameworks.

In summary, future basic research should focus on elucidating the key factors that determine the efficacy and persistence of NK cells, which enable to optimize and design the next generation of NK-based therapeutic product. By addressing these issues and conducting further research, we can unlock the full potential of NK cell-based therapies and pave the way for innovative and effective cancer treatments.