Natural Killer Cells for Cancer Immunotherapy

#### **Chapter 2**

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

*Li Zhang and Chang Liu*

#### **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 [2–4]. 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.

#### **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<sup>+</sup> 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 [9–11]. 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 [14–16]. 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].

#### *Natural Killer Cell-Based Cancer Immunotherapy: From Bench to Bedside DOI: http://dx.doi.org/10.5772/intechopen.109218*

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 [18–20]. 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 [23–25]. 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

**Figure 1.**

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

interferon-gamma (IFNγ). While CD56dim NK cells which are the dominant cells in the peripheral blood and spleen have cytotoxic ability (**Figure 1**).

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, transpresenting IL-15, and secretory exosomes [35, 36]. And CD4<sup>+</sup> 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 granuledependent 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 releasespontaneous 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<sup>+</sup> cells followed by the selection of CD56<sup>+</sup> 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<sup>+</sup> 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 [68–70]. 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 [79–82]. 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 [88–90]. 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, 93–95]. 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 [103–105].

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

#### *Natural Killer Cell-Based Cancer Immunotherapy: From Bench to Bedside DOI: http://dx.doi.org/10.5772/intechopen.109218*

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].

#### **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 membranebound 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.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Li Zhang\* and Chang Liu Dalian Municipal Central Hospital Affiliated to Dalian University of Technology, Dalian, China

\*Address all correspondence to: tyouri19652004@hotmail.com

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

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#### **Chapter 3**

### Advances in Natural Killer Cells and Immunotherapy for Gastric Cancer

*Shixun Ma, Li Li, Jintang Yin, Xiaohu Wang, Chongya Yang, Leisheng Zhang, Tiankang Guo and Hui Cai*

#### **Abstract**

Gastric cancer is one of the common malignant tumors in the gastrointestinal tract, and the treatment of gastric cancer includes the main ways such as radical resection, adjuvant chemotherapy, palliative care, and drug therapy; however, patients often have defects such as high recurrence rate, high treatment burden, and serious side effects, which impose a heavy burden on the economic and social construction and patients' families. In recent years, novel gastric cancer treatment methods featuring tumor immunotherapy have provided new treatment strategies to improve the above-mentioned defects and increase the cure rate of patients. Natural killer cells (NK cells) are key components of the body's intrinsic immune response and can participate in both the intrinsic and adaptive immune responses, exercising the functions of tumor killing, removing pathogenic microorganisms or abnormal cells and enhancing immunity, and thus have broad prospects for new drug development and clinical treatment. This article reviews the biological properties and functions of NK cells and their interrelationship with gastric cancer treatment, and provides a reference for clinical research.

**Keywords:** NK cells, gastric cancer, immunotherapy, progress

#### **1. Introduction**

Globally, the mortality rate of gastric cancer is high, with approximately 14.3/100,000 in men and 6.9/100,000 in women, with men being higher than women [1, 2]. There are significant geographical differences in the incidence of gastric cancer: it is highest in East Asia, Eastern Europe and South America and lowest in northern and southern Africa. Advanced gastric cancer can metastasise to the liver, pancreas, intestinal ducts, peritoneum, mesentery, greater omentum, esophagus, bile ducts, pelvis and lymph nodes, making treatment outcomes poor. Early diagnosis and early intervention are therefore crucial. The usual treatment for gastric cancer includes the main routes of surgery, chemotherapy and radiotherapy.

However, it often has drawbacks such as high recurrence rate, heavy treatment burden and side effects, which impose a heavy burden on society, economy and patients' families. In recent years, novel gastric cancer treatments featuring tumor immunotherapy have provided new treatment strategies to improve the abovementioned defects and increase the cure rate of patients [3, 4]. The natural killer cell (NK) is a key cell in the body's innate immune response. It is composed of a unique group of innate lymphocytes that can participate in both the innate and adaptive immune responses and has the intrinsic ability to recognize and eliminate virally infected and tumor cells [5–9]. NK cells play a key role in anti-cancer immunity due to their cytotoxic mechanisms and immune response modulating ability. 20 years ago, NK cells showed good safety and efficacy in the treatment of patients with advanced leukemia [10–13]. Studies suggest that NK cell content and phenotype are significantly altered in patients with a variety of malignant hematological tumors (AML, MDS) and metastatic solid tumors (gastric cancer, lung cancer). This evidence suggests that NK cells have important research value in the management of a wide range of tumors. In recent years, with the paradigm shift and technological advances in chimeric antigen receptor (CAR) engineered passaged T cell therapy, there is confidence in NK cells as a new tool for immunotherapy. Strategies to develop NK cell-based therapies focus on enhancing the potency and persistence of NK cells through co-stimulation of signaling, checkpoint inhibition, and cytokine armor, and aim to specifically redirect NK cells to tumors through CAR expression or using splice molecules. Notably, clinical studies have shown that the proportion of NK cells in the peripheral blood of patients with stage III and IV gastric cancer is significantly lower (P < 0.05). In the clinical setting, the promising results of the first generation of NK cell therapies, which showed encouraging efficacy and a remarkable safety profile, have inspired great enthusiasm for continued innovation [14–19]. In this paper, we review the biological properties and functions of NK cells in gastric cancer patients and their interrelationship with gastric cancer therapy, as well as the latest research progress in gastric cancer immunotherapy, to provide a reference for clinical research.

#### **2. Tumor biological characteristics of gastric cancer**

#### **2.1 Epidemiological characteristics of gastric cancer**

Gastric cancer is one of the most common tumors of the digestive tract and one of the more common cancers in terms of cancer-related morbidity and mortality worldwide. According to the latest statistics gastric cancer is the fourth most common cancer worldwide and the second most common cause of cancer death [20–22]. The treatment of gastric cancer is still based on a combination of surgical procedures. For patients with early-stage gastric cancer, the 5-year overall survival (OS) rate after treatment is 90%. However, approximately 50% of patients are already progressive at diagnosis, with approximately 40–60% of those who undergo radical resection experiencing recurrence. Approximately 30–35% of patients with gastric cancer present with distant metastases at diagnosis, yet R0 resection is unlikely to be achieved in those with peritoneal dissemination. Even when resection of metastases is feasible, patients with metastatic gastric cancer have an extremely high recurrence rate. For such patients, palliative systemic therapy is the gold standard, and median survival rarely exceeds 12 months [23–30].

#### **2.2 Risk factors for gastric cancer**

Risk factors for gastric cancer include a number of aspects including geographical environment, dietary lifestyle, *Helicobacter pylori* (Hp) infection, precancerous lesions and genetics [31–34]. There are significant geographical differences in the development of gastric cancer. Smoking, nitrites, fungal toxins and polycyclic aromatic hydrocarbon compounds are the main carcinogens in terms of dietary life. *H. pylori* infection is a biological causative factor in the development of gastric cancer. Gastric polyps, chronic atrophic gastritis and residual stomach after partial gastrectomy are lesions that may be associated with varying degrees of chronic inflammatory processes, intestinal epithelial metaplasia or atypical hyperplasia of the gastric mucosa, with the potential for transformation into cancer. Genetic and molecular biology studies have shown that the incidence of gastric cancer is four times higher in blood relatives of gastric cancer patients than in controls. The carcinogenesis of gastric cancer is a multifactorial, multi-step and multi-stage development process involving changes in oncogenes, oncogenes, apoptosis-related genes and metastasis-related genes, and the forms of genetic changes are also varied [35–37].

#### **2.3 Tumor microenvironment**

The tumor microenvironment (TME) is the internal and external environment in which tumourigenesis, growth and metastasis are associated with tumor cells. It contains cancer cells, stromal cells and macrophages, a large number of immune cells and cells secreting factors that are recruited from a distance. Through their autocrine and paracrine actions, they alter and maintain the conditions for their own survival and development, and promote tumor growth and development. Systemic and local tissues can also limit and influence tumor development and progression through metabolic, secretory, immune, structural and functional alterations [38–41]. In recent years, the tumor immune microenvironment has played a key role in the development of gastric cancer. The immune system recognizes cancer cells and inhibits tumor development and metastasis.. However, tumor cells evade immune attack and induce immunosuppressive TME through two main pathways. Firstly, cancer cells hide the expression of surface antigens to avoid recognition by cytotoxic T cells. Secondly, immune tolerance to TME is induced by the secretion of immunosuppressive molecules such as interleukin-10 and VEGF [42–44].

#### **2.4 Importance of the immune system in gastric cancer**

The development, progression and prognosis of gastric cancer are strongly related to the function of the immune system. The common immune cell components in gastric cancer tissues include T cells, B cells, DC cells, NK cells, macrophages M1 and M2, etc. [45–47]. T lymphocytes are derived from lymphatic stem cells from bone marrow, which are differentiated and matured in the thymus, and then distributed to immune organs and tissues throughout the body through lymphatic and blood circulation to perform cellular immune functions. B cells can differentiate into plasma cells when stimulated by antigens, which synthesize and secrete antibodies (immunoglobulins) and perform humoral immunity. Dendritic cells are the body's most powerful and specialized antigen presenting cells (APC), which are highly efficient in the uptake, processing and presentation of antigens. Natural killer cells are derived

from bone marrow lymphoid stem cells, which depend on the bone marrow and thymic microenvironment for their differentiation and development, and are mainly found in the bone marrow, peripheral blood, liver, spleen, lung and lymph nodes; NK cells, unlike T and B cells, lymphocytes of tumor cells and virus-infected cells, are a class of non-specific killer cells that do not require pre-sensitisation. Macrophages are differentiated from monocytes in the blood after they have penetrated blood vessels. Infection and chronic inflammation are key factors in the pathogenesis of gastric cancer, with *H. pylori* and other pathogens disrupting the M1 macrophage response, thereby inducing an M2-like activation state, which increases the risk of disease progression [48–52].

#### **3. Relationship between NK cells and gastric cancer**

#### **3.1 Biological properties of NK cells**

Natural killer cells are important immune cells in the body, not only in the fight against tumors, viral infections and immune regulation, but also in the development of hypersensitivity reactions and autoimmune diseases in some cases, being able to recognize target cells and kill mediators [53]. In the innate immune system, NK cells are specialized immune effector cells that are the first line of anti-tumor lymphocytes and play an important role in tumor immunosurveillance. In previous clinical studies, it has been shown that low NK cell activity correlates with higher tumor incidence and high metastasis rates. nK cells are cytotoxic to tumor cells without prior activation and can modulate various immune responses by secreting immunomodulatory cytokines and chemokines. The combination of activating and inhibiting signals regulates the antitumor effects of NK cells [54]. NK cells are mainly differentiated from CD34+ bone marrow progenitor cells and are mostly present in peripheral blood accounting for 5–10% of PBMC, while active NK are also present in lymph nodes and bone marrow but at lower levels than in peripheral blood [55]. NK cells function differently from T cells and NKT cells due to the lack of expression of TCR and CD3-related complexes. Based on the expression of surface proteins CD56 and CD16, NK cells can be divided into two categories: CD56 + CD16- (immunomodulatory, cytokine production) and CD56-CD16+ (cytotoxic). However, recent advances in high-throughput sequencing and single-cell proteogenomics have revealed that NK cells may exhibit greater phenotypic heterogeneity outside of these two subpopulations, leading to the differentiation of distinct cell populations with different cellular functions [56].

#### **3.2 Functions of NK cells**

#### *3.2.1 Cytotoxic functions of NK cells*

The killing activity of NK cells is called natural killing activity because it is not restricted by MHC and does not depend on antibodies. NK cells are rich in cytoplasm and contain large asplenophilic granules, the amount of which is positively correlated with the killing activity of NK cells, which appear early after the action of NK cells on target cells, with killing effects seen in vitro at 1 hour and in vivo at 4 hours [57]. NK cells are highly cytotoxic and trigger an effective response by releasing cytolytic particles and cytotoxic cytokines after forming an immune synapse with a target.

The main mediators of killing are perforin, NK cytotoxic factor and TNF. In addition, they can recognize antibody-coated cellular γRIIIA (CD16) receptors via Fc and trigger antibody-dependent cytotoxicity (ADCC) and cytokine production. Killer cell immunoglobulin-like receptors (KIR) and natural killer group 2A (NKG2A) are two major inhibitory receptors that recognize HLA molecules [58, 59].

#### *3.2.2 Immunomodulatory functions of NK cells*

NK cells can synthesize and secrete a variety of cytokines to directly kill target cells and are also able to influence the function of B cells, T cells, dendritic cells, macrophages and neutrophils through the production of chemokines. These properties pave the way for it to play a key role in immunotherapy [60].

#### *3.2.3 Memory function of NK cells*

Natural killer cells play an important anti-tumor and anti-viral role in vivo, and recent studies have revealed that NK cells have acquired immune cell characteristics and are capable of forming immune memories. NK cell development, differentiation, homeostatic maintenance and memory formation are therefore essential for the implementation of NK cell function [61]. Early studies reported a similar memory response of NK cells in mouse models of cytomegalovirus infection, a behavior not normally associated with innate immune cells. In these studies, when stimulated with a combination of IL-12 and IL-18, mouse NK cells acquired a functional phenotype characterized by increased IFN production γ. Interestingly, after a resting phase, these cells were able to reactivate with the involvement of cytokine stimulation or activation receptors and exhibited enhanced IFNγ-like responses resembling the memorylike properties of adaptive immune cells. Later, Todd Fehniger's group hypothesized that human NK cells should similarly have memory-like properties. Consistent with this hypothesis, their studies showed that human NK cells pre-activated with IL-12, IL-15 and IL-18 and then rested for 1–3 weeks were able to generate a strong response driven by enhanced IFNγ produced after subsequent exposure to cytokines or K562 leukemia cells. Since then, additional research groups have described similar memory-like functions in various immune settings, including the observation of such responses in humans [62, 63].

#### **3.3 Rationale of NK cell anti-tumor**

NK cells may have a more important role in immune surveillance and killing of mutated tumor cells than T cells. Patients with certain diseases, such as Chediak-Higashi or X-linked lymphoproliferative syndrome, are particularly susceptible to malignant lymphoproliferative diseases due to a deficiency in NK function [64]. The essence of tumor immunotherapy is to enhance the surveillance and clearance of tumor cells by the patient's immune system through various means [65]. T cells and natural killer (NK) cells are the two most important effector cells that recognize and destroy tumor cells. Precise recognition of both positive signals provided by tumor antigens and negative signals provided by immune checkpoints can be used to determine tumor-specific T-cell activation. Similarly, NK cell activation is dependent on the integration of activating and inhibiting signals. Thus, disrupting the balance by blocking negative and enhancing positive signals from T and NK cells may be beneficial for cancer patients [66]. To date, many therapies have been

reported for blocking T and NK cell inhibitory receptors, such as the checkpoint molecules CTLA-4 or PD-1/PD-L1. However, some evidence suggests that these strategies provide benefit to only a limited number of patients with tumors and that most patients continue to experience disease progression. To date, strategies to enhance the anti-tumor function of T cells and NK cells have yielded encouraging results. As the understanding of neoantigens presented by MHC molecules expands, research and clinical implementation of neoantigen-based therapies, including peripatetic T-cell therapies and cancer vaccines, are full of potential for clinical application [67]. The synergistic action of many NK cell surface receptors determines the NK cell state. Tumor cells can be made "invisible" to NK cells by up-regulating inhibitory signals or/and down-regulating activation signals on NK cells, and NK cell function can even be altered by altering the affinity of KIR-MHC interactions through different peptide libraries provided by MHC molecules. Interfering with activating and inhibiting signals has been used in a variety of therapeutic techniques to enhance NK cell function. However, changes in the affinity of the KIR-peptide/MHC complex interactions may occur due to different components of the peptides provided by MHC molecules in tumor cells and may require more research [68].

#### **3.4 Application of NK cells in the treatment of gastric cancer**

In gastric cancer patients, NK cells usually exhibit a dysfunctional phenotype characterized by an altered gene expression profile and reduced cytotoxic function, which reduces the feasibility of autologous NK cell therapeutic applications. In addition, the reduced number of autologous NK cells is a major cause of tumor progression. Current NK cell therapy relies on allogeneic sources, namely peripheral blood mononuclear cells, umbilical cord blood, immortalized cell lines, hematopoietic stem and progenitor cells (HSPC) and induced pluripotent stem cells (iPSC). All sources can provide clinically meaningful doses of cells suitable for CAR recipient engineering and have transitioned to human studies. However, they have unique advantages and challenges, and may have different potential transcriptional, phenotypic, and functional properties [69].

#### *3.4.1 NK-92 cells*

NK-92, the first NK cell-based immunotherapy to receive U.S. Food and Drug Administration (FDA) clinical trial approval, is a homogenous immortalized NK lymphoma cell line that can be expanded ex vivo to achieve large cell numbers. It was found that NK-92 cells can kill tumor cell lines and primary tumor cells cultured in vitro, and in addition they are not susceptible to immunosuppression, making them promising for tumor cell therapy. However, its oncogenic risk and lack of ADCCmediated cell killing ability have limited its application [70].

#### *3.4.2 NK cells after in vitro differentiation of CD34+ progenitor cells*

Dolstra et al. showed in their trial that transfer of HSPC-NK cells into elderly patients with acute myelogenous leukemia (AML) resulted in better outcomes. However, the safety and efficacy of its application in other tumors needs further validation. Future work will also need to address whether HSPC-NK cells can be effectively designed to achieve enhanced tumor specificity [71].

*Advances in Natural Killer Cells and Immunotherapy for Gastric Cancer DOI: http://dx.doi.org/10.5772/intechopen.109695*

#### *3.4.3 NK cells after differentiation of iPSCs*

Induced pluripotent stem cells (iPSCs) have played an important role in disease modeling, drug discovery and cell therapy, and have contributed to the development of the disciplines of cell biology and regenerative medicine. Currently, iPSCs technology has become an important tool in the study of pathological mechanisms, new drugs are being developed using iPSCs technology, and the number of clinical trials using iPSCs-derived cells is growing. iPSCs are an attractive source of NK cells because of their clonal growth and high expansion capacity, as well as their ability to differentiate in vitro, allowing the manufacture of a large number of homogeneous NK cell products. A potential drawback is that iPSC-derived NK cells typically express low levels of endogenous CD16, although this can be mitigated by genetic engineering. Another possible concern is that iPSCs may have a DNA methylation profile consistent with their somatic cellular tissue origin. This "epigenetic memory" may affect the development of specific cell lineages that differ from the donor cells and should be considered when using iPSC platforms. Nevertheless, a growing number of genetically engineered iPSC-NK cell candidates are emerging from preclinical studies, some of which have already transitioned to clinical trials [72].

#### *3.4.4 NK cells from peripheral blood or from umbilical cord blood*

Primary NK cells can be collected from peripheral blood (PB-NK cells) or from umbilical cord blood (CB-NK cells.) CB-NK cells can be readily available frozen through blood banks, whereas PB-NK cells require single harvest and donor-specific collection from healthy donors. in 2005, in work led by Dario Campana, PB-NK cells served as the first successful CAR construct platform for somatic introduction into NK cells, and today, PB-NK cells provide the basis for a variety of current products [73].

#### **4. Conclusion**

Immunotherapy is a novel and effective therapeutic strategy that has emerged as a new hope for many patients with gastric cancer. Although NK cell-based immunotherapy is considered to be a safe off-the-shelf antitumor therapy, important issues remain resolved and still a large proportion of gastric cancer patients do not benefit from immunotherapeutic agents. With the development of immunotherapy, it will be important to study the abnormal alterations in the biological phenotype and transcriptomic profile of NK cells, the NK cell subsets and their interrelationship with the prognosis of gastric cancer patients, and to elucidate the key parameters that determine the potency and persistence of NK cells. In addition, to maximize the lifespan and expand the efficacy of NK cells, multiple therapeutic strategies need to be combined [74]. Finally, to ensure NK cell quality, methods for NK cell proliferation and preservation need to be optimized. We believe that great breakthroughs will be achieved in the future in the study of NK cells for immunotherapy of gastric cancer.

#### **Acknowledgements**

The authors would like to thank the members in National Postdoctoral Research Station of Gansu Provincial Hospital, Institute of Biology & Hefei Institute of

Physical Science, Chinese Academy of Sciences, and Institute of Health-Biotech, Health-Biotech (Tianjin) Stem Cell Research Institute Co., Ltd. for their technical support. We also thank the staff in Beijing Yunwei Biotechnology Development Co., LTD for their language editing service. This study was supported by the following fund projects: Key Laboratory of Gastrointestinal Tumor Diagnosis and Treatment of National Health and Health Commission (2019PT320005); the National Natural Science Foundation of China (82260031); Key Laboratory of Molecular Diagnosis and Precision Therapy of Surgical Tumors in Gansu Province (18JR2RA033); Gansu Provincial Key Talent Project of Gansu Provincial Party Committee Organization Department (2020RCXM076); Basic Research Innovation Group of Gansu Province (22JR5RA709); Natural Science Foundation of Gansu Province (21JR11RA186, 20JR10RA415); Gansu Provincial Hospital Intra-Hospital Research Fund Project (21GSSYB-8, 20GSSY5-2); The 2021 Central-Guided Local Science and Technology Development Fund (ZYYDDFFZZJ-1); China Postdoctoral Science Foundation (2019 M661033), Science and technology projects of Guizhou Province (QKH-J-ZK[2021]-107), the National Science and Technology Major Projects of China for "Major New Drugs Innovation and Development" (2014ZX09508002-003), Major Program of the National Natural Science Foundation of China (81330015), the project Youth Fund funded by Shandong Provincial Natural Science Foundation (ZR2020QC097), Natural Science Foundation of Tianjin (19JCQNJC12500), Jiangxi Provincial Novel Research & Development Institutions of Shangrao City (2020AB002), Jiangxi Provincial Natural Science Foundation (20224BAB206077, 20212BAB216073), Jiangxi Provincial Key New Product Incubation Program from Technical Innovation Guidance Program of Shangrao City (2020G002).

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Notes/thanks/other declarations**

Not applicable.

#### **Appendices and nomenclature**


*Advances in Natural Killer Cells and Immunotherapy for Gastric Cancer DOI: http://dx.doi.org/10.5772/intechopen.109695*


### **Author details**

Shixun Ma1† , Li Li1† , Jintang Yin2 , Xiaohu Wang1 , Chongya Yang3 , Leisheng Zhang1,4,5,6\*, Tiankang Guo1 \* and Hui Cai1 \*

1 Key Laboratory of Molecular Diagnostics and Precision Medicine for Surgical Oncology in Gansu Province and NHC Key Laboratory of Diagnosis and Therapy of Gastrointestinal Tumor, Gansu Provincial Hospital, Lanzhou, China

2 Department of Clinical Laboratory, Jiuquan Central Blood Station, Jiuquan, China

3 Department of Geriatrics, Lintao County Hospital of Traditional Chinese Medicine, Dingxi, China

4 Jiangxi Research Center of Stem Cell Engineering, Jiangxi Health-Biotech Stem Cell Technology Co., Ltd., Shangrao, China

5 CAS Key Laboratory of Radiation Technology and Biophysics in Institute of Biology & Hefei Institute of Physical Science, Chinese Academy of Sciences, Hefei, China

6 Institute of Health-Biotech, Health-Biotech (Tianjin) Stem Cell Research Institute Co., Ltd, Tianjin, China

\*Address all correspondence to: leisheng\_zhang@163.com; tiankangguo2020@163.com and caialonteam@163.com

† Co-first author.

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3
