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Open access peer-reviewed chapter

Natural Killer Cell-Based Cancer Immunotherapy: From Bench to Bedside

Written By

Li Zhang and Chang Liu

Submitted: 10 November 2022 Reviewed: 29 November 2022 Published: 22 December 2022

DOI: 10.5772/intechopen.109218

Natural Killer Cells IntechOpen
Natural Killer Cells Lessons and Challenges Edited by Leisheng Zhang

From the Edited Volume

Natural Killer Cells - Lessons and Challenges

Edited by Leisheng Zhang

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Abstract

Natural killer (NK) cells are innate cytotoxic lymphocytes involved in the surveillance and elimination of cancer. The increasing number of studies have identified novel methods for enhancing the anti-tumor immunity of NK cells and expanding NK cells ex vivo, which paved the way for a new generation of anticancer immunotherapies. In this chapter, we will review the following aspects regarding NK cells, including the inhibitory and activating receptors modulating NK cell activity, NK cell development, the cytotoxic mechanism of NK cells, isolation, expansion and characterization of NK cells, and the source for NK cells. Moreover, we will highlight the cutting-edge immunotherapeutic strategies in preclinical and clinical development such as chimeric antigen receptor (CAR)-NK cells, as well as the adoptive NK transfer to target cancer stem cells (CSCs). Last, we will discuss the challenges NK cells face which should be overcome to achieve cancer clearance.

Keywords

  • natural killer (NK) cells
  • expansion
  • cancer immunotherapy
  • cancer stem cell (CSC)
  • CAR-NK

1. Introduction

Activating the immune system by immunotherapy is an innovative way to target cancers. Immune checkpoints such as cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin, and ITIM domain (TIGIT) are the most studied ones. PD-1 and CLTA-4 have received great attention since the blockage of PD-1 or CTLA-4 signaling improved the tumor patient survival significantly. Immune checkpoints consist of pairs of receptors-ligands present in immune cells as well as tumor cells, and the interaction of the receptors in tumor cells with their ligands in immune cells inhibits the immune activity of immune cells. Immune checkpoint blockade has been regarded as the recovery of cytotoxic lymphocyte activity. However, partial or complete loss of HLA-I expression has been attributed to be one of the immune escape mechanisms in tumors, which evade T-cell surveillance [1]. Under these circumstances, the cytotoxic T cells and NK cells, which are capable of recognizing and killing tumor cells despite of HLA-I expression, seem to be vital [2, 3, 4]. Immune checkpoints have been mainly studied in T cells, but NK cells are also affected by these interactions.

NK cells, which are cytotoxic lymphocytes with high antitumor, antiviral, and antimicrobial activities in the innate immune system, were first identified as lymphoid subsets and were critical mediators of antitumor immunity [5]. NK cells were capable of responding to a variety of infections as well as transformed cells by directly killing and secreting pro-inflammation cytokines without prior antigen sensitization or recognition of specific tumor antigens [6, 7]. NK cells develop at numerous sites including bone marrow, lymph nodes, secondary lymphoid organs, thymus, liver, gut, and tonsils [8], and the effector functions are similar to CD8+ T cells. CD56dim and CD56bright cells are the two subsets of human NK cells, which have distinct immune phenotypes and functions. In peripheral blood, CD56dim cells constitute 90% of NK cells, and they have strong cytotoxic capability, while CD56bright subset is mostly involved in cytokine production. The NK cells in the secondary lymphoid tissue are different from the NK cells in the peripheral blood.

There have been many efforts to exploit these potent effector cells such as NK cells or T cells either by endogenous activation or by adoptive transfer. Unlike donor T cells, NK cells do not induce graft-versus-host disease (GVHD). It would be highly likely that NK cells would exploit an important position targeting tumors with deficient HLA-I molecules.

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2. NK cell regulation and NK cell-based cancer immunotherapy

2.1 NK cell activity regulation

It was hypothesized that NK cells were derived from CD34+CD45RA+ hematopoietic progenitor cells (HSC). NK precursors were identified in the hematopoietic population and differentiated into NK cells. Several transcription factors such as Ets-1, Id2, Ikaros, and PU as well as soluble and membrane factors were involved in the regulation of the NK cell phenotypic and functional maturation [9, 10, 11]. Moreover, IL-15-dependent signaling pathway plays an essential role in NK cell development, homeostasis, and survival.

In healthy individuals, 90% of NK cells in peripheral blood are mature with CD16bright and CD56dim expression. The rest of the NK cells are an immature subset, which is CD16dim or CD16−, CD56bright, and CD25+, and the main function of these NK cells is to produce cytokines [12].

It was found that lymphoma cells that lost MHC class I surface molecules could be killed by NK cells, but the original MHC class I+ cells were otherwise resistant. This phenomenon drives the hypothesis that NK cells are able to sense the absence of “self” MHC class-I molecules on target cells [13]. Years later, the hypothesis was verified by the discovery of inhibitory as well as activating NK receptors.

NK cell activity is modulated and controlled by a series of inhibitory and activating NK cell receptors. The inhibitory receptors in humans are mainly inhibitory killer Ig-like receptors (KIRs). KIRs consist of long cytoplasmic tails containing two ITIM domains, recruiting tyrosine phosphatases to transduce inhibitory signals, followed by two (KIR2D) or three (KIR3D) polymorphic extracellular Ig-like domains. The inhibitory KIRs recognize and directly interact with the human leukocyte antigen (HLA) class-I alleles and CD94/NKG2A heterodimer, especially the non-classical HLA-E molecule [14, 15, 16]. Different HLA class I allotypes contained the same specific epitopes, which are recognized by distinct KIRs. However, when a KIR-HLA-I mismatch or the loss of HLA-I occurs, inhibitory KIRs cannot interact with their ligands to activate NK cells [17].

The activating NK cell receptors play a fundamental role in the recognition and killing of transformed or virus-infected cells, which have normal HLA-I molecule expression, through direct interaction with ligands expressed [18, 19, 20]. These activating receptors contain the following families: activating KIRs, C-type lectin-like receptors, natural cytotoxicity receptors (NCR), and signaling lymphocyte activating molecule (SLAM) family of receptors [21].

A large number of activating NK cell receptors is responsible for the existence of 6000-30,000 different phenotypic NK populations, providing flexibility to respond to pathogens and tumor cells [22]. CD94/NKG2A in the C-type lectin-like receptor family was the first HLA-I-specific receptor expressed by the most immature CD56bright cells during NK cell differentiation. After a few steps of maturation, CD56dim NK cells replace the CD56bright cells; in addition, CD56dim NK cells carry KIR receptors but abandon NKG2A [23, 24, 25]. So most mature NK cells express KIR as well as the terminal differentiation marker CD57 [26]. HLA-I-specific inhibitory receptors normally prevent auto-reactive responses by the recognition of autologous cells. Nevertheless, for tumors, HLA-I-specific inhibitory receptors function as immune checkpoints to restrain the cytotoxic activity of NK cells [3, 27]. PD-1, a non-HLA-I specific inhibitory receptor, might be also expressed in NK cells. PD-1 was originally discovered in T cells and played a sharp inhibitory role in their tumor-killing activity. PD-1 is expressed on mature NK cells, which are KIR+CD57+NKG2A− cells, under normal conditions. However, for patients with tumors, the proportions of PD-1+ NK cells were increased significantly [28, 29]. Some other immune checkpoints are also expressed by NK cells to recognize additional ligands, including CTLA-4, T cell immunoglobulin and mucin-domain-containing molecule 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), T cell immune receptor with Ig and immune receptor tyrosine-based inhibition motif domains (TIGIT), and CD96 [4, 30, 31].

The inhibitory receptors recognize self-MHC class I molecule to inhibit NK cell activation, which enables the self-tolerance and prevents host cell killing. NK cells will be activated when they encounter the cells that are lack of MHC class I molecule, which is known as the “missing-self” hypothesis [32]. Through the recognition of MHC class I molecules, NK cells distinguish transformed or infected cells from normal host cells, and these abnormal cells with a lack of MHC class I expression or high expression of “stress ligands” could be lysed by NK cells. MHC class I molecules were usually downregulated in the tumor cells to escape the immune surveillance of cytotoxic T lymphocytes. Nevertheless, NK cells would still be activated and attack these tumor cells, since the activation receptors are no longer suppressed under these circumstances to induce potent stimulatory signals [33, 34].

2.2 NK cell development

NK cell development occurs predominantly within the bone marrow. At first, NK cell is referred to as a common lymphoid progenitor (CLP), which is identified by some markers including stem cells antigen-1 (Sca-1), c-kit (CD117), interleukin-7 receptor (IL-7Ra), and FMS-like tyrosine kinase-3 (Flt-3). Then, the CLP develops into a pre-NK precursor (pre-NKP) consisting of NK precursors and innate lymphoid cell precursors. Next, the pre-NKP becomes NKP and expresses IL-15 receptor complex (IL-15Rβ/γ) to maintain long-term NK cell development and survival. During this stage, symbolic NK cell marker CD56 is expressed, and NK cells are further subdivided into the immature CD56bright subset and mature CD56dim subset. CD56bright NK cells participated in immunomodulation through secreting cytokine interferon-gamma (IFNγ). While CD56dim NK cells which are the dominant cells in the peripheral blood and spleen have cytotoxic ability (Figure 1).

Figure 1.

The specific markers expression on CLP, NKP, immature NK (imNK) cells, and mature NK (mNK) cells during NK cell development.

During immune response, NK cells can receive signals from other immune cells as well as tumor cells. For example, dendritic cells regulate the proliferation and immune function development of NK cells through the secretion of IL-12, type I IFN, trans-presenting IL-15, and secretory exosomes [35, 36]. And CD4+ cells could regulate NK cell proliferation and survival by IL-2. On the contrary, the regulatory T (Treg) cells can suppress NK cell proliferation and function through transforming growth factor beta (TGF-β) [37, 38].

2.3 The cytotoxic mechanism of NK cells

The death receptor pathway and the granule-dependent pathway are classic NK cell cytotoxic mechanisms. The two predominant pathways jointly induce the apoptosis of the target cells. The former one is a caspase-dependent apoptosis pathway, activated by the tumor necrosis factor (e.g., Fas/CD95) on target cells with their equivalent ligands such as FasL and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on NK cells [23, 39]. TRAIL and FASL interact with their receptors on the targeting cells. Interaction between FASL and FAS as well as TRAIL and TRAIL receptors promotes death-inducing signaling complex formation, which includes Fas-associated death domain protein (FADD), caspase-8, along with caspase-10 [40]. Caspase-8 activation leads to the activation of the other caspases or Bid proteolysis, resulting in the subsequent caspase activation as well as cytochrome C release [23].

After identifying and bounding to the target cells, NK cells initiate the granule-dependent pathway to deliver the granzymes, which are cytotoxic granules containing perforin and proteases to kill the target cells [41]. Distinct granzymes (A, B, H, K, and M) activate different apoptotic or non-apoptotic cell death pathways. Granzyme A activates non-apoptotic cell death, and Granzyme B activates apoptosis by activating caspases as well as by cytochrome C release. Granzymes H, K, and M were less studied, and it was reported that CD56bright cells released Granzymes K to mediate the non-apoptotic tumor cell death [42]. Granzyme H was shown to help remove infected and transformed cells. And Granzyme M functioned as an anti-tumor effector after adoptive NK cell transfer [43]. The death receptor pathway and the granule-dependent pathway endow NK cells with the different killing abilities to eliminate different tumor cells.

B7-H3 was reported to be correlated with the poor prognosis of cancer patients, through the inhibition of NK cell activity; on the contrary, the inhibition of B7-H3 restrains tumor growth and increases the cytotoxic activity of NK cells [44]. Moreover, the tumor cells can also be killed by antibody-dependent cellular cytotoxicity (ADCC), as they express a low-affinity Fc receptor for IgG, FccRIII.

2.4 NK cell isolation, expansion, and characterization

The commercial NK isolation kits were available nowadays. The commonly used peripheral blood NK cell isolation kit was Miltenyi Biotec. NK cells could be either isolated from peripheral blood directly as well as from the peripheral blood mononuclear cells (PBMCs). In this regard, PBMCs should be isolated from the peripheral blood first. Layer 35 mL of peripheral blood on 15 mL of Ficoll-Paque, and then centrifuge at 400 g for 20 minutes without brake. Harvest the PBMCs from the interface of Ficoll-Paque and plasma, and wash the three times with PBS (centrifuge at 400g for 10 minutes). NK cell isolation protocol is as follows: Pass PBMCs through a 30-μm filter to remove cell clump, and then centrifuge cell suspension at 500 g for 7 minutes to count cells. Use precooled solutions to keep cells cold, resuspend 107 cells in 40 μl solution, and add 10 μl of biotin-conjugated antibodies cocktail to remove unwanted cells. Then, add 30 μl of separation buffer and 20 μl of MicroBeads, mix well, and incubate for 10 min at 4 °C. Wash once in separation buffer (centrifuge at 500 g for 7 minutes) and resuspend labeled cells in 500-μl separation buffer. Place an MS column in the magnetic field of the MACS separator and pre-rinse with separation buffer. Apply cell suspension to the column. Collect the flow-through fraction containing enriched untouched NK cells. Wash the column three times with separation buffer, and also collect the NK cells.

As to isolate the NK cells from the peripheral blood instead of PBMCs, use the RosetteSep™ Human NK Cell Enrichment Cocktail from STEM CELL Technologies. Transfer 10 ml whole blood in a 50-mL tube, and add 500 μl RosetteSep Human NK Cell Enrichment Cocktail, incubate for 20 min at room temperature and mix gently. Dilute the sample with RPMI with 2% FBS (1:1), mix gently, and lay on Lympholyte-H cell separation media. Centrifuge for 20 min at room temperature at 800 g without brake. Collect enriched NK cells from the interface of Lympholyte-H and plasma, and wash the NK cells twice with a complete medium. Either way, NK cell number and viability should be determined by Trypan Blue dye exclusion, in addition to assess NK cell purity by flow cytometry.

NK cell expansion requires multiple signals for survival, activation, and proliferation. In the early years, in vivo expand NK cells were the main purpose of the clinical trials. Administrating systemic cytokines, mainly IL-2, did not improve the clinical outcome of cancer patients due to the serious side effects of cytokines [45]. Additionally, IL-2 also stimulates the regulatory T cell, which plays the immunosuppression effect. Thus, ex vivo NK cell stimulation with other cytokines can also help to increase the survivability of the NK cells. NK cell expansion can be initiated from both PBMCs and purified NK cells using the following protocol. The initiating number of PBMCs or NK cells can be varied based on the amount of NK cells desired after expansion. For each 5×106 cell to be expanded, count and irradiate 107 K562 Cl9 mIL21 using a gamma irradiator at 100 Gy. Wash the K562 cells with PBS and resuspend in NK cell expansion media (NKEM) after the irradiation. Seed 5×106 PBMCs with 107 irradiated K562 Cl9 mIL21 in 40 mL of NKEM in a T75 flask and place it upright in an incubator at 37°C and 5% CO2. After 5 days, collect NK cells by centrifugation and replace half of the media with fresh NKEM. Count the number of cells after a week. For every 5×106 cell, count and irradiate 5×106 K562 Cl9 mIL21 as previously described. Add an equal number of irradiated K562 Cl9 mIL21 and resuspend in NKEM with the density of 2.5×105 cells/ml, then seed cells in T75 flasks. After 10 days, change the entire media with fresh NKEM based on the cell numbers. Resuspend NK cells with the irradiated same amount of K562 Cl9 mIL21 in NKEM and change the whole media with fresh NKEM after 17 days. At the end of the expansion, count the cell number and analyze the NK cell phenotyping.

It was reported that in vitro pre-activation of NK cells with novel cytokines such as IL-12, IL-15, and IL-18 induces CD25 expression in NK cells [46]. Thus, expansion strategies have been focused either to substitute these factors using autologous feeder cells or to use genetically modified allogeneic feeder cells. Autologous PBMC as feeder cells to expand NK cells in vitro has been shown to generate enough functional active NK cells [47]. The number of purified NK cells increased 2500 folds after 17 days by using autologous PBMCs as feeder cells. Combined feeder cells with OKT3 and IL-2 endowed NK cells with cytolytic activity against tumor cells [48]. Genetically modified K562 cells or Epstein-Barr virus-transformed lymphoblastoid cell lines were used as feeder cells for NK cell expansion. The leukemia cell line K562 was genetically altered to express a membrane-bound form of IL-15 and the 4-1BB (CD137L) [49]. By using this feeder cell, NK cells were expanded by 277-fold after 3 weeks [49]. K562 feeder cells expressing CD137L, MICA, and soluble IL-15 were also produced, and they promoted the NK cell expansion of 550 folds [50]. Denman et al. have constructed K562-based feeder cells with the expression of membrane-bound chimeras of IL-21 (mbIL21) and IL-15 (mbIL15), and investigated NK cell expansion, phenotype, and function. It was found that IL-21 was superior to IL-15 in terms of promoting NK cell growth, but as to NK phenotype or function, the function of IL-21 and IL-15 expressing k562 feeder cells had no significant differences [51].

NK cells are characterized by a lack of CD3/TCR molecules and expression of CD16 and CD56 surface antigens. CD56dim NK cells express high levels of CD16, while CD56bright NK cells are CD16dim or negative [52]. Cytotoxicity of NK cells against various malignant cell lines, and the expression patterns of NK cell receptors including cluster of differentiation (CD)-16, CD69, CD158b, natural killer group-2 member D (NKG2D), NKp30, NKp44, NKp46 define NK cell activity [53]. The cytotoxic assay of NK cells was performed as follows, using 6×105 NK cells and 3×105 target cells to perform the experiment. Resuspend 106 target cells in 1 ml of NKEM composed of Calcein-AM, incubated for 1 hour at 37 °C, with occasional shaking. Prepare NK cells at 1×106 cells/ ml, add 200 ul of NK cell suspension to 3 wells of a 96-well plate corresponding To 10:1 (E:T) ratio. Dilute the NK cells for the five subsequent E:T ratios. After 1 hour of calcein loading, wash target cells in NKEM twice, centrifuging for 5 minutes at 1200 rpm. Re-count the target cells and resuspend at 1×105 cells/ml. Add 104 target cells to NK cells, and centrifuge for 1 minute at 100 g to enable cell contact. Then, incubate the cell mixture at 37 °C and 5% CO2 for 4 hours. Mix them gently, centrifuge at 100g for 5 minutes to pellet the cells, and transfer 100 μl of the supernatant to a new plate. Read the absorbance (excitation filter 485 nm, emission filter 530 nm). Calculate Percent Specific Lysis according to the formula [(test release-spontaneous release)/(maximum release - spontaneous release)] × 100 [54].

2.5 Source of NK cells

It was agreed that NK cell transfer is safe and well-tolerated by patients. Interleukin (IL) 2 was administered to activate NK cells in vitro or in vivo after immunosuppressive treatment by fludarabine and cyclophosphamide.

Nevertheless, adoptive transfer of NK cells for cancer immunotherapy has been hindered by the inability to obtain sufficient NK cells, as these cells represent a small fraction (about 10% comprise the third largest population of lymphocytes following B and T cells) of blood mononuclear cells, and long-term expansion and persistence of NK cells ex vivo was still a challenge. Moreover, the alloreactive T cells would eliminate NK cells after transfer [55, 56].

Various protocols have been used to isolate and preferentially expand primary NK cells from PBMC [57]. The combination of cell selection and depletion using immunomagnetic beads was the common protocol [58]. Leukapheresis products were used for the clinical-grade purification of NK cells by depleting CD3+ cells followed by the selection of CD56+ cells [59] or in combination with subsequent a 14-day of stimulation with IL-2 [60]. It was reported that autologous adoptive NK-cell therapy had drawbacks, which were mainly that self-MHC I molecules on the tumor cells inhibited NK cells. So the autologous adoptive transfer of NK cells may not be efficient, and healthy allogeneic NK cells were an optimal option.

Since a large number of activating and inhibitory receptors, cooperative receptor pairs, and overlapping signaling pathways involved in NK cell maturation, activation, and proliferation, it is difficult to identify signaling molecules to expand NK cells in vitro [61]. NK cell propagation in vitro needs feeder cells [62], various cytokines such as IL-2, IL-15, IL-21 [63, 64], as well as fusion proteins [65, 66].

In umbilical cord blood, NK cells account for about 30% of lymphocytes. In contrast, NK cells account for 10% of lymphocytes in peripheral blood. Furthermore, the immunophenotype of NK cells from umbilical cord blood is CD3-CD56+, which is roughly classified as the less differentiated CD56bright and mature CD56dim NK cells in a broad sense. CD34+ hematopoietic progenitors from umbilical cord blood or bone marrow are considered as an excellent source for cell therapeutic applications [67]. Previously, it was a challenge to obtain efficient numbers of NK cells for the low number of NK cells in cord blood. In recent years, different protocols have been developed for the generation of NK cells from CD34+ cells from bone marrow as well as cord blood through co-cultured with stromal cell lines and a combination of cytokines [68, 69, 70]. More recently, ex vivo expansion protocol of NK cells, derived from cord blood CD34+ cells, has been established. This method uses a clinical-grade serum-free culture medium and a mixture of cytokines as a substitute for the extracellular microenvironment of bone marrow in static cell culture bags and an automated bioreactor without feeder cells [71]. Up to 1010 NK cells derived from CD34+ cells were possible by adding high levels of several activating receptors such as NKG2D and NC [72].

The healthy allogeneic NK cells can be sourced from UCB, adult donor lymphopoiesis products, or even from NK-cell lines such as NK-92. Moreover, human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) were verified to be the new source of functional NK cells.

The hESC and iPS-derived NK cells with a sophisticated approach are relatively new compared with the NK cell from PBMC and or CD34+ cells [73]. Currently, research focused on the optimization of experimental design and culture conditions to generate hESC as well as iPS-derived NK cells [74, 75]. These cells could lyse malignant cells by both direct cell-mediated cytotoxicity and antibody-dependent cytotoxic cell lysis. Kaufmann and co-workers produced mature and functional NK cells from hESC and iPS though IL-21 expressing antigen-presenting cells, it took at least 2 months. And the harvested NK cells were on clinical scale [76].

CARs were developed to equip immune effector cells with the ability to recognize antigens on the surface of tumors and kill their targets in an HLA-unrestricted fashion [77]. CAR is short for chimeric antigen receptor, which is composed of three regions, an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain in CAR-T cells guaranteed the specificity of T cells toward a specific target expressed in tumor cells without antigen presentation. It was derived from the single chain variable fragment (scFV) of a monoclonal antibody (mAb). The intracellular domain would activate the lytic pathway of T cells, which is derived from the T cell receptor (TCR)/CD3 complex. In addition, the co-stimulatory signaling endo-domains (CD28, 4-1BB, or OX40) activate T-cell proliferation after the encounter with the target cell. The number of co-stimulatory domains can differ between the different CARs [78].

CAR-T cell therapy has appeared as a revolutionary immunotherapy option for the treatment of hematological malignancies nowadays, whereas CAR-NK cell is still under development. A large number of activating receptors would initiate NK cell cytotoxic activity, based on this one would hypothesize that NK cells do not need a CAR. However, many negative clinical results regarding NK cells transferred into refractory tumor patients indicated otherwise. NK cell activation can be promoted by augmenting activating signals and down-regulating inhibitory signals through genetic engineering techniques. Nevertheless, the addition of a CAR into NK cells might add another option to recognize tumor cells. Specifically, for patients with down-regulation of the ligands required for activation of NK receptors, CAR was necessary. Furthermore, after recognition of tumor cells, the CAR would induce NK cell expansion and increase NK cell persistence. Recently, a few preclinical studies using CAR-NK cells have been published [79, 80, 81, 82]. These studies produced CAR-NK cells targeting CD19 and CD20 for B cell malignancies, targeting CD5 for T cell malignancies, and targeting CD138 and CS1 for multiple myeloma.

As for solid malignancies, many researches have been done to use CAR-NK cells against the tumor. The ErbB2/HER2-specific chimeric antigen receptor was linked to NK cells to target breast cancer and glioblastoma [83, 84]. Disialoganglioside GD2-specific NK cells were engineered to target against drug-resistant neuroblastoma [85]. Genssler et al. have used NK-92 cells, which expressed CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding, to recognize EGFR, EGFRvIII, or an epitope common to both antigens, to kill heterogeneous glioblastoma cells [86]. Functional NK cells for adoptive therapy can be derived from several different sources [87]. It was verified that autologous NK cells had limited activity against the patient’s own tumor cells since self-HLA molecules inhibited the efficacy of autologous NK cells [88, 89, 90]. Liu and co-workers have genetically modified cord blood NK cells with CARs that carry the CD19 gene to redirect specificity to CD19 [91]. These CAR-NK cells ectopically produced IL-15 to promote proliferation and survival. In addition, this CAR carried a suicidal gene, inducible caspase-9 (iC9), in order to eliminate transduced cells whenever needed [92].

The most widely studied NK cell line by far is NK-92 cells, which was originally established from a patient with non-Hodgkin’s Lymphoma. The lack of almost all inhibitory KIRs made NK-92 cells have higher anti-tumor activity. NK-92 cells have been engineered to express various CARs such as CD19, CD20, CD38, and CS1 for hematologic malignance, and Her2 for solid tumors [83, 93, 94, 95]. CAR-NK-92 cells have also been intratumorally injected, allowing them to traffic to tumor sites and exert their effect via a vaccine-like mechanism [96].

The promising results in some studies indicated that the addition of IL15 into the CAR construct would enhance the persistence of NK cells in vivo [97]. Moreover, Muller et al. have added the C-X-X motif chemokine receptor 4 (CXCR4) into the CAR to enable NK cells to traffic to tumor sites [98]. Nevertheless, NK-92 cells have inherent drawbacks that must be taken into account, which were the potential tumorigenicity, multiple cytogenetic abnormalities, and latent infection with the Epstein-Barr virus. So it should be irradiated prior to clinical use [99].

Both hESCs and iPSCs-derived NK cells were also suitable for CAR expression, which could be maintained indefinitely to provide an almost limitless supply of NK cells. It was reported that NK cells from cord blood CD34+ cells were modified to express CD19-CAR [100].

2.6 NK cell-based cancer immunotherapy

It was demonstrated that in multiple malignant tumors including acute myeloid leukemia and multiple myeloma, NK cell number, and surface-activating receptors NKp30, NKG2D were downregulated, while the inhibitory receptors were overexpressed [101, 102]. Any activating and inhibitory receptor expression disorders would render NK cells unable to activate normally, the ability to secrete cytokines and chemokines would be inhibited, and the cytotoxicity would be affected. Hence, many efforts have been made to produce “off-the-shelf” NK cells to treat cancers [103, 104, 105].

NK cells lead immune surveillance against cancer and early elimination of small tumors. The first clinical use of NK cells involved infusing IL-2-activated lymphokine-activated killer cells (LAK cells)) into cancer patients in the 1980s [106]. And recently, many in vitro as well as in vivo studies had documented the ability of activated NK cells to kill both hematologic malignancies and solid tumors.

2.6.1 Hematologic malignancy

The therapeutic potential of NK cells has been extensively explored in hematological malignancies. Miller et al. have tested haploidentical, related-donor NK-cell infusions in patients with poor-prognosis acute myeloid leukemia resulting in a marked rise in endogenous IL-15, expansion of donor NK cells, and induction of complete hematologic remission in 5 of 19 poor-prognosis patients with AML [107]. Another study also used haploidentical NK cell transplantation, which was killer immunoglobulin-like receptor-human leukocyte antigen (KIR-HLA)-mismatched NK cells, to treat acute myeloid leukemia in children. It also verified the safety of the NK cells with limited hematologic toxicity and no GVHD. And the 2-year event-free survival estimate was 100% [108]. Liu and co-workers have administered HLA-mismatched anti-CD19 CAR-NK cells derived from cord blood to 11 patients with relapsed or refractory CD19-positive non-Hodgkin’s lymphoma or chronic lymphocytic leukemia (CLL). These cells were safe and were not associated with the development of cytokine release syndrome, neurotoxicity, or GVHD. Of the 11 patients who were treated, 8 (73%) had a response, 7 had a complete remission, and 1 had remission of the Richter’s transformation component but had persistent CLL. Responses were rapid and seen within 30 days after infusion at all dose levels. The infused CAR-NK cells expanded and persisted at low levels for at least 12 months [109]. Torelli’s group has enrolled 103 newly diagnosed acute lymphoblastic leukemia patients with 46 adults and 57 children. Significantly higher expression of Nec-2, ULBP-1, and ULBP-3 was found in the pediatric blasts compared to adult cells. In addition, higher surface expression of NKG2D and DNAM1 ligands was found in BCR-ABL gene fusion group. Accordingly, the BCR-ABL fusion gene group was proved to be significantly more susceptible to NK cell-dependent lysis than the B-lineage group [110]. Moreover, NK cells were used to treat various malignancies, and mixed results were obtained. Shi et al infused haploidentical KIR-mismatched NK cells into 10 patients with relapsed multiple myeloma, followed 14 days later with an autologous stem cell graft. Five patients achieved near complete remission [111]. However, when six non-Hodgkin lymphoma patients were transplanted with infused haploidentical NK cells, the NK cells expanded poorly in vivo. Besides, the host regulatory T cells were significantly increased after NK cell infusion and IL-2 administration [112]. Pretreatment with ontak (denileukin diftitox) to deplete host regulatory T cells before NK cell transplantation would get a better result. Bachanova et al. have applied the combined Ontak treatment with infused haploidentical NK cells to AML patients, the NK cell expansion was increased, and the AML clearance was enhanced [113].

2.6.2 Solid malignancy

Tumor cells have developed several mechanisms to overcome the constant immune surveillance, prevent NK cell-induced apoptosis, and diminish the efficacy of NK cell-mediated tumor clearance. And the efficacy of NK cells in solid tumors remains undetermined since the preclinical and clinical data are not enough.

NAGAI and co-workers have enrolled nine patients with metastatic pancreatic, ovarian, colonic, renal, and adenocystic carcinoma to receive intravenously NK cell therapy. The NK cells were obtained from HLA/KIR mismatched healthy donors. The dose was starting from 106 to 108 cells for each patient at 2-week dosing intervals. The results showed that neither grade 2 or higher toxicities, nor adverse events causing discontinuation of protocol treatment were found after NK cell therapy. When the number of administered NK cells was increased to 108 cells in four cases, no serious dose-limiting toxicity was found. The overall response rate was 40%, one with partial response and three with stable disease, and the patient with the partial response is still alive after 4 year’s observation [114]. Janneke et al. have summarized the pre-clinical and clinical trials on NK cell immunotherapy in ovarian cancer [115]. In six clinical trials with intravenous infusion of NK cells, only 31 patients have been reported that received NK cell adoptive transfer. The majority of patients reached stable disease after NK cell therapy, with a mild pattern of side effects. More complete responses were found in patients with repeated NK cell infusions.

Some ex vivo studies also verified the NK cell efficacy against hepatocellular carcinoma (HCC) cell lines. Kamiya and co-workers have obtained activated NK cells from the peripheral blood of healthy donors with stimulation by K562-mb15-41BBL cell line. The viability of three HCC cell lines was reduced after sorafenib treatment, and after 4 hours of culture with NK cells at 1:1 ET ratio, the viability of HCC cells further decreased twofolds. In addition, they used immune-deficient NOD/SCID IL2RGnull mice engrafted with Hep3B to test the efficacy of NK cells, and it was found that NK cells markedly reduced tumor growth and improved the overall survival of the mice [116]. Bugide et al. have found that HCC cells downregulated NKG2D ligands and were resistant to NK cell-mediated eradication. Thirty-two chemical inhibitors of epigenetic regulators, which were able to re-express NKG2D ligands, were tested to investigate if the HCC cell eradication by NK cells was improved. It was found that the inhibition of EZH2, a transcriptional repressor of NKG2D ligand, by small-molecule inhibitors or genetic means enhanced HCC cell eradication by NK cells in an NKG2D ligand-dependent manner [117].

Xiao et al. have constructed NKG2D RNA CAR-NK cells to enhance the cytolytic activity against several solid tumor cell lines as well as in xenografts. In addition, local infusion of the CAR-NK cells was used to treat three patients with metastatic colorectal cancer. The results showed that the ascites generation of two patients, who were intraperitoneal infusion of low doses of the CAR-NK cells, reduced and tumor cell number in ascites samples was significantly decreased. The other patient with a metastatic tumor site in the liver was treated with intraperitoneal infusion of the CAR-NK cells. Rapid tumor regression in the liver region was observed with Doppler ultrasound imaging and complete metabolic response in the treated liver lesions was confirmed by positron emission tomography (PET)-computed tomographic (CT) scanning [118].

Cancer stem cells (CSCs) are a subpopulation within the tumor, which is capable of self-renewal by asymmetrical cell division [119]. CSCs were considered as the basis of tumor’s cellular heterogeneity as well as the main culprit of tumorigenesis, tumor progression, and metastasis. CSCs are resistant to conventional cancer therapies, and their persistence following treatment is strongly believed to drive tumor recurrence [120, 121]. The lack/downregulation of consistent surface markers was found in CSCs including MHC-1 [122]. Nevertheless, CSCs were highly susceptible to NK cell-mediated cytotoxicity [123, 124]. By using multiple preclinical models, including autologous and allogeneic NK coculture with cancer cell lines and dissociated primary cancer specimens, as well as pancreatic cancer xenografts, Ames et al. have demonstrated that activated NK cells were capable of preferentially killing CSCs identified by multiple CSC markers including CD24+/CD44+, CD133+, and aldehyde dehydrogenase (ALDH) bright [125]. Similar work involving CSCs has been reported in osteosarcoma [126]. Fernández and co-workers have found osteosarcoma cells were susceptible to NK cells’ lysis both in vivo and in vitro, which relied on the interaction between NKG2D receptor and NKG2D ligands (NKG2DL). Moreover, spironolactone increased the susceptibility of osteosarcoma cells to NK cells and could shrink the osteosarcoma CSCs. Tallerico et al. demonstrated that colorectal CSCs showed increased susceptibility to NK cells, which was associated with the upregulation of the activating natural cytotoxicity receptors Kp30 and NKp44 [127]. Castriconi et al. reported that glioblastoma-derived CSCs were susceptible to NK cell cytotoxicity. Fresh tumor specimens from glioblastoma patients were used. They have found these neural-CSCs were resistant to unactivated NK cells, but were highly susceptible to both allogeneic and autologous NK cells in co-culture models after pre-treatment with IL-2 and IL-15 [128]. It was reported that when melanoma cell lines were exposed to IL-2-activated allogeneic NK cells, both CD133- non-CSCs and CD133+ CSCs showed sensitivity to NK cell cytotoxicity, which was possibly mediated by the DNAM-1 ligands Nestin-2 and PVR [129]. In breast cancer, CSCs were CD44+CD24- subpopulation. It was found that IL-2 and IL-15-activated NK cells could kill breast cancer CSCs mediated by increased expression of the NKG2D ligands such as ULBP1, ULBP2, and MICA on breast CSCs [130].

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3. Challenge and future perspective

Allogeneic NK cell treatment against cancer has seen rapid development. Clinical trials of NK cell-based adoptive transfer to treat relapsed or refractory malignancies have used peripheral blood, umbilical cord blood, hESC- and iPSC-derived NK cells, as well as NK cell lines. Although tumors may develop several mechanisms to resist attacks from endogenous NK cells, ex vivo activation, expansion, and genetic modification of NK cells can greatly increase their anti-tumor activity and equip them to overcome resistance. Some of these methods have been translated into clinical-grade platforms and support clinical trials of NK cell infusions in patients with hematological malignancies or solid tumors [131, 132]. Surprisingly, many of the clinical studies involving NK cells against tumors did not show optimal results [133]. It was reported that after in vitro expansion of NK cells, the KIR-HLA-I mismatch effect in some occasions can be bypassed, and the expression of NK receptors becomes homogenous, which inhibited the killing ability [134].

Effective adoptive NK cell-based immunotherapy requires NK cells to be activated, sufficient in number, and considerably persistent in the body, to enter tumor sites and effectively kill tumor cells. These factors can be co-administered with adoptive cell infusions or the NK cells themselves can be modified to secrete or present membrane-bound factors. IL-15, for example, would increase NK cell persistence in vivo [135]. hESC and iPSC-derived NK cells are phenotypically similar to the NK cells in peripheral blood, and they express more KIR compared to umbilical cord blood-derived NK cells, which made them unlimited source for the adoptive transfer of NK cells [73, 136]. On the other hand, the safety of hESC and iPSC-derived NK cells in terms of potential tumorigenicity needs to be determined before they can be utilized in the clinical setup.

Recently, gene-modified NK cells have been successfully developed to possess specific tumor antigens or secreting certain immunosuppressive cytokines to enhance the cytotoxicity of NK cells. Nevertheless, different transduction methods, both viral and non-viral, have been used to modify NK cells, and different expansion strategies were used. So it was difficult to obtain NK cells with homogenous functions. Additionally, lentiviral and retroviral vectors are designed to ensure persistent transgene expression. However, safety remains controversial [137, 138]. Alternatively, non-viral transduction methods, including electroporation, are being explored. Electroporation introduces CAR-encoding mRNA through pores in the cell membrane, resulting in the immediate expression of the CAR molecule. However, mRNA electroporation was reported to result in markedly lower efficiencies [139]. In future studies, more efficient and safe transduction methods should be developed to improve the activity and persistence of NK cells. Moreover, the functional homogenously NK cells should be produced as the “off-the-shell” products.

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Conflict of interest

The authors declare no conflict of interest.

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Written By

Li Zhang and Chang Liu

Submitted: 10 November 2022 Reviewed: 29 November 2022 Published: 22 December 2022

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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