Natural killer cell lines in tumor immunotherapy

Abstract

Natural killer (NK) cells are considered to be critical players in anticancer immunity. However, cancers are able to develop mechanisms to escape NK cell attack or to induce defective NK cells. Current NK cell-based cancer immunotherapy is aimed at overcoming NK cell paralysis through several potential approaches, including activating autologous NK cells, expanding allogeneic NK cells, usage of stable allogeneic NK cell lines and genetically modifying fresh NK cells or NK cell lines. The stable allogeneic NK cell line approach is more practical for quality-control and large-scale production. Additionally, genetically modifying NK cell lines by increasing their expression of cytokines and engineering chimeric tumor antigen receptors could improve their specificity and cytotoxicity. In this review, NK cells in tumor immunotherapy are discussed, and a list of therapeutic NK cell lines currently undergoing preclinical and clinical trials of several kinds of tumors are reviewed.

Keywords

natural killer cell / natural killer cell line / tumor immunotherapy / genetic modification

Introduction

Natural killer (NK) cells were initially described in 1975 and were originally defined as innate effector lymphocyte [1,2]. Our current understanding of human NK cell development lags limited compared with that of human T or B lymphocytes, and to date NK cell precursors (NKP) have not been clearly characterized in humans [3]. NK cells are widely distributed throughout the body, and can be found in the lungs, liver, peripheral blood mononuclear cells (PBMCs), spleen, bone marrow (BM) and lymph node (LN); in contrast, NK cells are almost undetectable in the thymus [4]. Based on their surface expression of CD56, human NK cells have been divided into CD56^bright and CD56^dim subsets and the majority (approximately 90%) are CD56^dim expressing high levels of Fcγ RIII (CD16), whereas a minority (approximately 10%) are CD56^bright and CD16^dim/neg [5-7]. Human NK cells have been shown to differentiate into NK1 and NK2 subsets, similar to the Th1 and Th2 subsets of CD4⁺ T cells [8]. The different NK cell subsets have distinct functions in immune responses.

NK cells are a key component of the innate immune system and are characterized by their rapid response to and strong cytolytic activity against virus-infected or transformed cells without pre-sensitization and restriction by major histocompatibility (MHC) molecules [9-11]. NK cell cytotoxicity is carried out by two main mechanisms. One is granule-dependent cytotoxicity, which is mediated by perforin and granzyme B, and is triggered by activating receptors or FcR. The other is triggering of the apoptosis pathway in the target cell through the expression of TNF-related apoptosis-inducing ligand (TRAIL) or Fas ligand (FasL) on the NK cell surface or tumor necrosis factor-α (TNF-α) secretion by NK cell [12]. NK cells act as cytolytic effector cells against target cells, and can also act as regulatory cells during immune responses and influence the subsequent adaptive immune responses. Under some physiologic and pathological conditions, NK cells are potent producers of several cytokines, such as interferon-γ (IFN-γ), TNF-α, interleukin (IL)-10, and growth factors. Additionally, NK cells produce many chemokines that impact dendritic cells (DCs), macrophages and neutrophils during an immune response [13], thus endowing NK cells with regulatory functions. More importantly, recent studies have revealed that NK cells also have “memory” properties that were previously ascribed only to T and B cells [14].

In this review, the interactions between NK cells and cancer are discussed, and the current NK cell-based immunotherapies are reviewed, including a list of therapeutic NK cell lines in preclinical and clinical trials against several kinds of tumors.

NK cells and cancer

NK cell cytotoxicity is thought to be regulated by a balance of signals between inhibitory and activating receptors. NK cells are currently known to express at least three families of receptors: C-type lectin-like receptors (NKG2D, CD94/NKG2), natural cytotoxicity receptors (NCRs) and killer cell immunoglobulin-like receptors (KIRs). The activating receptors include the NCRs (identified as NKp46, NKp30 and NKp44), a C-type lectin receptor (NKG2D) and KIRs; the inhibitory receptors include KIRs and CD94/NKG2. The KIRs can induce either stimulatory or inhibitory signaling through recognition of HLA-A, HLA-B and HLA-C alleles, depending on the sequence of their intracellular domain [15]. NK cells identify their targets through a set of activating and inhibitory receptors. Three recognition models have been proposed: “non-self recognition,” by which NK cells recognize pathogen-encoded molecules that are not expressed by the host; “stress induced-self recognition,” by which NK cells recognize self proteins that are upregulated in transformed or infected cells; and “missing-self recognition,” by which NK cells recognize self proteins that are expressed by normal cells but downregulated by infected or transformed cells [16,17]. The recognition of tumor cells by NK cells is complex, and all three recognition models might play a role. NK cell killing can be triggered when target cells exhibit decreased or altered self-MHC class I expression along with the sufficient expression of activating ligands; when both inhibitory and activating ligands are expressed on tumor cells, the outcome of NK cell is determined by the balance of the inhibitory and activating signals [12].

Nevertheless, malignant cells are able to develop mechanisms to escape from NK cell attack or to induce defective NK cells. Malignant cells utilize many strategies to counteract NK cells, such as the expression of FasL to induce NK cell apoptosis, secretion of immunosuppressive factors that inhibit NK cell proliferation, resistance to Fas- or perforin-mediated apoptosis and upregulation of MHC class I to inhibit NK cell activation upon ligation of certain inhibitory KIRs. Several NK cell abnormalities in cancer patients have been described, including decreased NK cell counts, defective NK cell proliferation, increased number of CD56^bright NK cells that display poor cytotoxicity against tumor cells, decreased cytotoxic activity of NK cells, defective expression of activating receptors, defective expression of intracellular signaling molecules, and defective NK cytokine production [12,18-24].

NK cell-based tumor immunotherapy

The NK cell is an important part of the innate immune system and is a key player in the first-line defense against malignancies. Therefore, the usage of NK cells in human cancer immunotherapy has been proposed and treatments along these cells have been recently entered clinical trials [25]. Current NK cell-based cancer immunotherapy aimed at overcoming NK cell paralysis in patients. NK cell-based cellular immunotherapy could potentially be implemented through the administration of cytokines or immunomodulatory drugs to activate endogenous NK cells; the adoptive transfer of ex vivo expanded autologous NK cells or donor-derived NK cells; the transplantation of induced alloreactive NK cells from allogeneic stem cells; and the adoptive transfer of an established NK cell line [25-29].

Many treatment strategies have been exploited to activate endogenous NK cell, promote NK cell proliferation or induce more potent NK cell-mediated anti-tumor responses. One major strategy is the systemic administration of cytokines involved in NK cell differentiation and activation, such as IL-2, IL-12, IL-15, IL-18, IL-21 and type-1 interferons (IFNs) [30-34]. Cytokines have successfully been used to treat several human cancers through the direct or indirect activation of NK cells. Upon cytokine stimulation, NK cells become LAK cells with upregulated expression of effector molecules, such as adhesion molecules, NKp44, perforin, granzymes, FasL and TRAIL, and enhanced proliferative ability and cytokines production [35-42]. In 1980, IL-2-activated NK cells were used in clinical trials to treat patients with solid primary or metastasized tumors [28]. Unfortunately, IL-2 treatment is associated with life-threatening toxicity, inducing vascular leak syndrome (VLS) [43]. Although low-dose IL-2 treatment has proven to be safe, there is no evidence of efficacy. Additionally, IL-2 also stimulates the expansion of another IL-2Rα-expressing regulatory subset of cells in vivo, Treg cells, which can suppress the anti-tumor activity mediated by immune effector cells [44,45].

Protocols for purifying and ex vivo expanding NK cells for adoptive immunotherapy have been studies with the goal of obtaining large numbers of NK cells [12]. Expanded NK cells stimulated with feeder cells, such as K562 cells or EBV-LCL, and cytokines, such as IL-2 and IL-15, show an activated phenotype with an upregulated expression of NKG2C, NKG2D, FasL, TRAIL and granzymes, and exhibit significantly stronger cytotoxicity against tumor cells than freshly isolated NK cells [46]. Due to the success of the adoptive transfer of autologous NK cells in experimental tumor models, this procedure has been evaluated for cancer immunotherapy in the clinical setting. Infusion with enriched autologous NK cells expanded and activated ex vivo, greatly improves the clinical responses in patients with metastatic RCC [47], malignant glioma [48], and breast cancer along with IL-2 infusions or not. The infusions were well tolerated without any obvious adverse effects [49]. However, under the stimulation of cytokines, the phenotype and function of endogenous NK cells from cancer patients are difficult to reverse effectively for tumor immunotherapy. These autologous NK cells are not maximally cytotoxic as determined by in vitro assays, and no consistent efficacy of autologous NK-cell therapy was detected in cancer patients when compared with cohorts of matched controls [50,51].

The NK cells from donors show higher cytotoxic activity against various KIR-ligand mismatched tumor cell lines than against KIR-ligand matched targets [52]. This mechanism is involved in the substantial GVL effects of HSCT with HLA-haplo-identical transplants. After establishing their efficacy in anti-leukemia response, haploidentical NK cells were evaluated for treating solid cancers. NK-enriched cell population were isolated from related haploidentical donor PBMCs, stimulated with IL-2, and adoptively transferred into patients with poor-prognosis AML, metastatic melanoma or metastatic renal cell carcinoma. The results showed that the donor NK cells could be expanded in vivo, dependent on the preparative treatment of more potent immunosuppressive regimen Hi-Cy/Flu and associated with high endogenous concentrations of IL-15 [53]. In a phase I clinical trial, haploidentical NK cells activated and expanded with IL-15 and hydrocortisone in combination with chemotherapy, were found to be safe and potentially clinically effective for NSCLC [54].

By comparison, there are some limitations to the approach of reinfusion with ex vivo expanded autologous or allogeneic NK cells [55]. NK cells isolated from patients with malignant diseases often showed impaired function, and the anti-tumor activity of autologous NK cells are inhibited by self MHC-I molecules [56]. In addition, endogenous NK and LAK cells might be insufficiently cytotoxic against advanced tumor cells [57]. Because NK cells only constitute a small proportion of PBMCs and do not continually proliferate, generating highly pure NK cells in a large enough number to meet clinical requirements sufficiently is both time consuming and cost intensive. Isolating and ex vivo expanding donor-derived NK cells on a large scale also risks the contamination with other lymphocytes, and T cell contamination poses a risk for graft versus host disease (GVHD). Until now, the expansion potential of activated NK cells has not yet been standardized, which results in the transfer of NK cells with different phenotypes in different clinical trials and affects the therapeutic potential of the technique and increases the risk for adverse reactions [56]. To overcome these difficulties, some researchers have dedicated themselves to establishing permanent NK cell line that demonstrates a high cytotoxicity against tumor cells, can be easily expanded to large numbers under good manufacturing practice (GMP) conditions and would be readily available on demand in a standardized quality for immunotherapy. Most importantly, the anti-tumor activities of NK cell lines can be further enhanced, making them more cytotoxic, and they can be easily expanded and maintained in vitro [56]. In Table 1, the ex vivo expansion of some NK cell lines for clinical application are shown. These lines are more practical for quality control and large-scale production.

NK cell lines and tumor immunotherapy

Seven malignant NK cell lines, including the NK-92, YT, NKL, HANK-1, KHYG-1, NK-YS and NKG cell lines have been established [58,59]. Among them, the YT cells were derived from a not further defined acute lymphoblastic lymphoma, and the others were derived from patients with various NK cell malignancies [58,59]. The YT and NK-YS cells were generated in Japan, the NKG cells were generated in China, and the other cell lines were established in western countries [59-65]. Three of the cell lines, YT, NK-YS and HANK-1 are Epstein-Barr virus (EBV)-positive (type II latency), making them useful for studying the biologic characteristics of EBV-associated lymphoma/leukemia, but it does not make them useful for studying anti-tumor activity [59,64-66]. The NK-92, KHYG-1, NKL and NKG cell lines do not carry the EBV genome, and their anti-tumor activities have been well documented [67]. These NK cell lines for adoptive cellular immunotherapy are introduced below.

NK-92 cell line

This cell line was established in 1994 from the peripheral blood of a 50-year-old male patient with rapidly progressive non-Hodgkin’s lymphoma, and has a CD56⁺CD2⁺CD57⁺CD3^- phenotype [62,68]. The growth of NK-92 cells is dependent on the presence of recombinant IL-2. The NK-92 cells express a large number of activating receptors including NKp30, NKp46, 2B4, NKGD/E and CD28, and high levels of molecules associated with cytotoxicity such as perforin, granzyme, FasL, TRAIL, TWEAK and TNF-α, while expressing few inhibitory receptors and lacking almost all of the inhibitory KIRs [69]. Therefore, NK-92 cells exhibit the characteristics of activated NK cells and are cytolytic to a wide array of malignant cells [70,71].

As shown in Table 2, studies of NK-92 cells for tumor immunotherapy were carried out in mouse models and clinical trials. Immunotherapy treatment of malignant melanoma with NK-92 cells was first demonstrated in an SCID mouse model [69]. NK-92 cells were highly cytotoxic to human melanoma cells, including MEWO melanoma cells and WM1341 cell line both in vitro and in vivo. NK-92 cells reduced the WM1341 primary tumor size by 40%–90% and the MEWO tumors by 30%–75% in the xenografted SCID mice [69]. Following preclinical mouse studies and ex vivo applications such as purging of leukemia, lymphoma and CML, the NK-92 cell line has also been used in direct infusions of patients [56,72-74]. NK-92 cells have achieved FDA approval for testing in patients with advanced malignant melanoma and renal cell carcinoma in Europe and the United States [56]. This therapy is safe and has generated anti-tumor effects in advanced renal cell carcinoma and malignant melanoma [25,75,76]. Irradiation of NK-92 cells with 5 Gy prevents further cell division, and the substantial cytotoxicity can be maintained with up to 10 Gy irradiation for 48 h in vitro [25,77]. No toxicity against nonmalignant allogeneic cells has been reported. Data from these trials suggest that infusion with NK-92 cells may be safe and potentially beneficial and excellent candidate for adoptive cellular immunotherapy. NK-92 is, thus far, the only NK cell line that has entered clinical trials. The NK-92 cell line will serve as a platform for the future study of NK cell-based tumor immunotherapy.

KHYG-1 cell line

The KHYG-1 cell line was established from the blood of a patient with aggressive NK leukemia, possessing a p53 point mutation. It was also established by culturing peripheral leukemia cells with recombinant IL-2. The KHYG-1 cells show LGL morphology with an immunophenotype of CD1^-, CD21^-, surface CD3^-, cytoplasmic CD3e⁺, CD7⁺, CD8α⁺, CD16^-, CD25^-, CD33⁺, CD34^-, CD56⁺, CD57^-, CD122⁺, CD132⁺, and TdT^-. The KHYG-1 cells displayed NK cell activity and IL-2-dependent proliferation in vitro, suggesting that they were of NK cell origin [67]. This was the first case of human NK leukemia from which a cell line with an aberrant p53 gene has been established [67]. The KHYG-1 cell line induces apoptosis in tumor cells by the granzyme M/perforin pathway in addition to the activation of NKp44. Furthermore, the KHYG-1 line was observed to exhibit greater cytotoxicity than the NK-92 cells [78]. The novel activation receptor, NKp44 and its adaptor, DAP12, NKG2D, and constitutively phosphorylated ERK2 may be associated with the enhanced cytotoxicity of KHYG-1 [78]. The irradiation (at least 10 Gy) of KHYG-1 cells inhibits their proliferation but does not diminish their enhanced cytolytic activity against several tumor targets, suggesting that KHYG-1 cells may be a feasible immunotherapeutic agent in the treatment of cancers [79]. The study that examined the usage of KHYG-1 cells against tumors is summarized in Table 2.

NKL cell line

NKL cell line was established from the peripheral blood of a patient with CD3^-CD16⁺CD56⁺ large granular lymphocyte (LGL) leukemia in 1996. The NKL cell line also grows in the presence of IL-2 at a concentration of 100 pM; however, IL-1, IL-4, IL-6, IL-7, IL-12, TNF-α, IFN-α and IFN-γ do not support the growth of NKL cells. The morphology of NKL cells resembles that of normal activated NK cells, with phenotypes similar to CD16^- CD56^dim NK cells. NKL cells express CD2, CD6, CD11a, CD26, CD27, CD29, CD38, CD43, CD58, CD81, CD94, CD95, class II MHC, and the C1.7.1 antigen, but do not express detectable levels of CD3, CD4, CD5, CD8, CD14, CD19, CD20, CD28, or αβ or γδ TCR on the cell surface. When cultured for prolonged periods of time in vitro, the density of the CD16, CD56, and CD57 antigens on NKL cells declined markedly [63]. The NKL cells showed very similar natural killing, antibody-dependent cellular cytotoxicity (ADCC) and proliferative responses as the CD16^-CD56^dim NK cells [63]. Compared with primary NK-92 cells, the NKL cells have a different anti-tumor spectrum and exhibit greater cytotoxicity against certain human cancer cells such as the human gastric carcinoma cell line, SGC7901 [80]. Among all of the NK cell lines, NKL appears to have retained the most original features of NK cells. The anti-tumor activity of NKL cells is shown in Table 2, suggesting that they may potentially be useful as the effector cells in adoptive immunotherapy against tumors [81,82].

NKG cell line

Recently, a novel human NK cell line NKG was described by our research team [59]. It is the first NK cell line established in China, and was isolated from a Chinese male patient with rapidly progressive non-Hodgkin’s lymphoma. The growth of NKG cells is IL-2-dependent and similar to NK-92 cells. However, the doubling time of the NKG cells (2-3 days) was longer than that of NK-92 cells (24–36h) [56]. The NKG cells showed LGL morphology and were phenotypically identified as CD56^bright NK cells that were CD16^-, CD27^-, CD3^-, αβTCR^-, γδTCR^-, CD4^-, CD8^-, CD19^-, CD161^-, CD45⁺, CXCR4⁺, CCR7⁺, CXCR1^-, and CX3CR1^-. With high levels of adhesive molecules (CD2, CD58, CD11a, CD54, CD11b, and CD11c), and an array of activating receptors (NKp30, NKp44, NKp46, NKG2D, and NKG2C) and cytolysis-related receptors and molecules (TRAIL, FasL, granzyme B, perforin, and IFN-γ), NKG cells were highly cytotoxic against a series of tumor cell lines including Ho8910, K562, SGC7901, A549, Hep2, HepG2, HCT116, SBKR3, LoVo, and Daudi cells [59]. Compared with NK-92 cells, the only NK cell line that has entered clinical trials, the NKG cells exhibited stronger cytotoxicity against tumor cells, including both MHC-I negative cell lines (LoVo and Daudi) and MHC-I positive cell lines (Ho8910 and K562). The higher expression levels of NKG2D and NKp30 on the NKG cells may explain the differences in the cytotoxicity against tumor cells. The irradiation of NKG cells with 8 Gy inhibits their proliferation, and maintains their cytotoxicity against tumor cells within 48 h, indicating that 8 Gy would be viable dose for NKG cell irradiation. After adoptive transfer, the NKG cells were undetectable in the peripheral blood, liver, spleen, lymph node, kidney or bowel of nude mice, demonstrating their safety. Irradiated NKG cells were successfully used for immunotherapy against human ovarian cancer in a xenograft mouse model, and they significantly inhibited the ovarian tumor growth, decreased the mortality rate and prolonged the survival time, even for advanced-stage ovarian tumors [59]. Irradiated NKG cells could migrate into the ovarian tumor tissue to kill the tumor cells in vivo. Thus, NKG cells are also a promising candidate for clinical cellular immunotherapy to control human cancer. Further preclinical and clinical testing of NKG cells for tumor immunotherapy is ongoing.

Using NK cell lines for allogeneic NK cell transfer has a strong potential for beneficial efficacy, due to the lack of suppressive KIR ligand(s) (ie. HLA) in the recipient [83]. However, significant differences in the HLA molecules between ethnicities might induce HLA-specific antibody production in the recipient, which could inhibit any beneficial effects. That is to say NK cell lines established in western countries, such as NK-92 cell line, might not be suitable for Chinese patients. As the HLA genotype of NKG cells showed common HLA compatibility to the Han-population in China, this cell line would be more appropriate for use in cancer patients of Chinese descent.

Gene modified NK cell lines

Genetic modification of NK cells may provide new possibilities for cancer immunotherapy by improving NK cell function and specificity or endowing them with additional capabilities. Genetic modification can be applied in a variety of approaches, ranging from inducing NK cell proliferation and survival via cytokine gene modification to the specific targeting of NK cells to certain tissues or malignant cells [12]. The overexpression of activating receptors by genetic modification could increase tumor cell killing efficiency in NK cells when the tumor cells express the specific ligand, and the silencing of inhibitory receptors expression by RNAi could increase tumor cell killing efficiency in NK cells when the tumor cells express high levels of inhibitory ligands. The use of a chimeric receptor that binds a tumor-specific antigen and delivers intracellular signals for the activation of NK cell cytotoxicity can induce the retargeting of NK cells [12]. Currently, there are several gene-modified NK cell lines that are being assessed for tumor immunotherapy (Table 3).

Cytokine gene-modified NK cell lines

NK cells can be modified with a cytokine transgene to enhance the NK cell function by directly providing the necessary cytokine. Several cytokines, including IL-2, IL-15 and stem cell factor (SCF) have been used in gene-modified NK cell lines to augment the activation NK cells and their cytotoxicity toward tumor cells.

IL-2 acts as a growth factor for NK cell progenitors and mature NK cells, and enhances NK cell killing activity by inducing the production of molecules related to cytotoxicity. IL-2 is necessary for NK-92 cell proliferation, survival and cytotoxicity. During immunotherapy with NK-92 cells, exogenous IL-2 was co-administered to prolong the treatment. However, the data indicated that IL-2 also promoted the expansion of Tregs and resulted in the direct suppression of both T and NK cell responses [44,45]. To avoid this side effect, IL-2-independent NK-92 cells were established through human IL-2 gene transduction [77,84,85]. Nagashima et al. demonstrated that IL-2 gene-modified NK-92 cells proliferated in an IL-2 independent manner and exhibited significantly stronger cytotoxicity against tumor targets than the parental cells by secreting IFN-γ and TNF-α in vitro. Furthermore, NK-92 cells that were engineered to produce IL-2 were therapeutic against liver and melanoma metastasis in the experimental mouse model [84]. These studies suggested that IL-2 gene modified NK-92 cells are promising for clinical applications and their use may avoid the drawbacks of administering exogenous IL-2.

IL-15 is a pleiotropic cytokine that plays an important role in both the innate and adaptive immune systems. IL-15 is absolutely required for NK cell development, expansion and survival in vivo [86]. This cytokine is also a potent regulator of NK cell cytolytic activity [87,88], and more efficient than IL-2 in expanding NK cells [89,90]. Based on this, our group established human IL-15 gene-modified NK-92 cells (NK-92-IL15) [91]. The IL-15 gene modified NK cells continuously produced a high level of IL-15, which promoted NK cell proliferation more rapidly with low doses of IL-2 or IL-15, and inhibited cell apoptosis by upregulating the anti-apoptosis genes, Bcl-2, Bcl-xl and Mcl-1, and downregulating the apoptosis genes Bim and Noxa. Moreover, IL-15 gene-modified NK cells exerted significantly stronger cytotoxicity than the parent NK cells against a broad range of target tumor cells via increasing the expression of cytotoxic effector molecules, such as perforin, FasL and IFN-γ, and upregulating the expression of activating NK cell receptor, NKG2D, and downregulating the inhibitory NK cell receptors, NKG2A/CD94. IL-15 altered the phenotype of NK-92 cells and also enhanced NK-92 cytotoxicity. Similar results were obtained when the human IL-15 transgene was used to modify the NKL cell line [92]. Human IL-15 gene modification also promoted NKL cell proliferation at low doses of IL-2 and inhibited cell apoptosis by upregulating the anti-apoptosis genes, Bcl-2, Bcl-xl and Mcl-1, and downregulating the apoptosis genes Bim and Noxa. The anti-tumor activity of human IL-15 gene-modified NKL cells was enhanced, at least partly, through increasing the expression of IFN-γ, perforin and FasL [92].

NK-92 cells are typical immature NK cells with a standard CD56^brightCD16^- phenotype. Because stem cell factor (SCF) is an important early-acting cytokine in NK cell development that can increase NK cell differentiation and proliferation by upregulating the IL-2Rßc expression in NK progenitor cells, we speculated that inducing the expression of SCF might promote NK-92 cell differentiation and proliferation. We established NK92-SCF cells and found that these cells proliferated more rapidly than the parent NK-92 cells, especially in the presence of IL-15, possibly via the upregulation of IL-2Rßc expression. The SCF expression also increased the NK-92 cell cytotoxicity, which was, at least partly, through upregulating the production of cytotoxic effector molecules, including perforin and FasL [93].

Recently, our group investigated human IFN-α gene-modified NKL cells. These cells stably secrete a high level of IFN-α. Unlike IL-2 and IL-15, IFN-α is not necessary for the survival of NK cells. IFN-α expression did not influence NK cell proliferation, but did promote the expression of perforin, grazyme, TNF-α and IFN-γ, resulting in stronger cytolytic activity than the parental NKL cells against HCC in vitroand in vivo. Furthermore, the IFN-α gene-modified NKL cells upregulated Fas expression in HCC through the production of TNF-α and IFN-γ (Zhang et al. unpublished data).

Chimeric receptor gene-modified NK cell lines

Unlike T cells, NK cells do not express individual antigen specific receptors. NK cells cannot respond to many tumors such as carcinomas or melanoma, and particularly nonhematopoietic tumors, even though these tumor cells lose their surface MHC class I expression. Genetically modifying NK cells to recognize tumor targets directly could enhance the therapeutic potential of NK cells against a broader range of cancers. Based on the strategy of generating tumor specific T cells by receptor gene modification, NK cells could be directed to tumor cells by engineering the expression of chimeric antigen receptor (CAR) genes [94-99]. A CAR consists of a single-chain Fv (scFv) fragment from an antibody specific for a surface antigen, fused to a signal-transducing chain like the γ chain of the FcϵRI receptor or the CD3ζ chain, triggering a cytolytic response through intracellular signaling molecules.

NK-92 cells have been successfully engineered to express CAR targeting tumor specific antigens expressed on the tumor cell surface such as CD19, CD20, disialoganglioside GD(2), and HER-2/neu, which can be specifically recognized by a CAR and then trigger the activation and cytotoxicity of CAR-modified NK cells [100-104]. The CD19 antigen is constitutively expressed on the chronic lymphocytic leukemia (CLL) cells, making it a suitable target for CAR. Boissel et al. transferred an mRNA coding for an anti-CD19 CAR into the NK-92 cell line by electroporation. NK-92 cells expressing anti-CD19 CAR killed previously resistant CD19⁺ B-precursor ALL cell lines, and primary B-CLL cells, and, therefore, may present a safe, cell-based, and targeted treatment for patients with CLL [104]. The CD20 antigen is expressed at high levels on most mature B cells and B cell lymphomas. Muller et al. have generated genetically modified NK-92 cells carrying a CD20-specific chimeric antigen receptor. The NK-92-scFv-CD20 cells displayed enhanced cytotoxicity toward CD20 ⁺ tumor targets, and increased cytotoxicity against primary CLL tumor cells isolated from patients [100]. Uherek et al. used a chimeric single-chain variable fragment (scFv) receptor specific for the human erbB2 tumor-associated antigen (TAA) to modify NK-92 cell line and demonstrated that the NK-92 cells expressing this chimera could specifically mediate the enhanced killing of erbB2⁺ T cell lymphoma cells, and breast, ovarian and squamous cell carcinomas [103]. These CAR-modified NK-92 cells overcome the resistance of B cell lymphomas, neuroblastomas, and solid tumor cells of various origins. The NK-92-CAR cells specifically mediated the enhanced killing of TAA⁺ tumor cells in vitro and in vivo even though no exogenous IL-2 was included in the treatment.

Conclusions

The promise of NK cells and their therapeutic potential in cancer are increasingly of interest. As a kind of allogeneic NK cells, NK cell lines have a strong potential for beneficial efficacy due to the lack of suppressive KIR ligand(s) (ie. HLA) in the recipient. NK cell lines are more amenable than autologous or allogeneic activated NK cells to expansion to a large number under GMP conditions, and are readily available for clinical adoptive therapy. Moreover, NK cell lines are a prime target for genetic modification to generate NK cells with enhanced specificity and cytolytic potential. Further studies will explore more effect molecules used by NK cells for gene-modification and the candidate genes for NK cell modification may include those that silence inhibitory receptor or overexpress activating receptor on NK cells. However, significant differences in the HLA molecules might induce HLA-specific antibody production in the recipient, which could inhibit the transferred NK cell functions. More investigation is required to optimize NK cell therapeutic approaches for cancer patients through preclinical and clinical trials. Overall, the use of NK cell lines for tumor immunotherapy in clinical application is both feasible and promising.

References

[1] Herberman RB, Nunn ME, Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic acid allogeneic tumors. I. Distribution of reactivity and specificity. Int J Cancer 1975; 16(2): 216-229

[2] Kiessling R, Klein E, Wigzell H. “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol 1975; 5(2): 112-117

[3] Di Santo JP. Natural killer cell developmental pathways: a question of balance. Annu Rev Immunol 2006; 24(1): 257-286

[4] Grégoire C, Chasson L, Luci C, Tomasello E, Geissmann F, Vivier E, Walzer T. The trafficking of natural killer cells. Immunol Rev 2007; 220(1): 169-182

[5] Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-cell subsets. Trends Immunol 2001; 22(11): 633-640

[6] Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri MA. Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood 2002; 100(6): 1935-1947

[7] Cooper MA, Fehniger TA, Fuchs A, Colonna M, Caligiuri MA. NK cell and DC interactions. Trends Immunol 2004; 25(1): 47-52

[8] Peritt D, Robertson S, Gri G, Showe L, Aste-Amezaga M, Trinchieri G. Differentiation of human NK cells into NK1 and NK2 subsets. J Immunol 1998; 161(11): 5821-5824

[9] Roder JC, Pross HF. The biology of the human natural killer cell. J Clin Immunol 1982; 2(4): 249-263

[10] Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nat Rev Immunol 2001; 1(1): 41-49

[11] Miller JS. The biology of natural killer cells in cancer, infection, and pregnancy. Exp Hematol 2001; 29(10): 1157-1168

[12] Sutlu T, Alici E. Natural killer cell-based immunotherapy in cancer: current insights and future prospects. J Intern Med 2009; 266(2): 154-181

[13] Bryceson YT, March ME, Ljunggren HG, Long EO. Activation, coactivation, and costimulation of resting human natural killer cells. Immunol Rev 2006; 214(1): 73-91

[14] Paust S, von Andrian UH. Natural killer cell memory. Nat Immunol 2011; 12(6): 500-508

[15] Vitale M, Sivori S, Pende D, Augugliaro R, Di Donato C, Amoroso A, Malnati M, Bottino C, Moretta L, Moretta A. Physical and functional independency of p70 and p58 natural killer (NK) cell receptors for HLA class I: their role in the definition of different groups of alloreactive NK cell clones. Proc Natl Acad Sci USA 1996; 93(4): 1453-1457

[16] Karlhofer FM, Ribaudo RK, Yokoyama WM. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 1992; 358(6381): 66-70

[17] Colonna M, Samaridis J. Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science 1995; 268(5209): 405-408

[18] Carrega P, Morandi B, Costa R, Frumento G, Forte G, Altavilla G, Ratto GB, Mingari MC, Moretta L, Ferlazzo G. Natural killer cells infiltrating human nonsmall-cell lung cancer are enriched in CD56 ^bright CD16^-cells and display an impaired capability to kill tumor cells. Cancer 2008; 112(4): 863-875

[19] Jinushi M, Takehara T, Tatsumi T, Hiramatsu N, Sakamori R, Yamaguchi S, Hayashi N. Impairment of natural killer cell and dendritic cell functions by the soluble form of MHC class I-related chain A in advanced human hepatocellular carcinomas. J Hepatol 2005; 43(6): 1013-1020

[20] Bauernhofer T, Kuss I, Henderson B, Baum AS, Whiteside TL. Preferential apoptosis of CD56^dim natural killer cell subset in patients with cancer. Eur J Immunol 2003; 33(1): 119-124

[21] Pierson BA, Miller JS. CD56^+bright and CD56^+dim natural killer cells in patients with chronic myelogenous leukemia progressively decrease in number, respond less to stimuli that recruit clonogenic natural killer cells, and exhibit decreased proliferation on a per cell basis. Blood 1996; 88(6): 2279-2287

[22] Fauriat C, Mallet F, Olive D, Costello RT. Impaired activating receptor expression pattern in natural killer cells from patients with multiple myeloma. Leukemia 2006; 20(4): 732-733

[23] Kono K, Ressing ME, Brandt RM, Melief CJ, Potkul RK, Andersson B, Petersson M, Kast WM, Kiessling R. Decreased expression of signal-transducing zeta chain in peripheral T cells and natural killer cells in patients with cervical cancer. Clin Cancer Res 1996; 2(11): 1825-1828

[24] Tajima F, Kawatani T, Endo A, Kawasaki H. Natural killer cell activity and cytokine production as prognostic factors in adult acute leukemia. Leukemia 1996; 10(3): 478-482

[25] Ljunggren HG, Malmberg KJ. Prospects for the use of NK cells in immunotherapy of human cancer. Nat Rev Immunol 2007; 7(5): 329-339

[26] Semino C, Martini L, Queirolo P, Cangemi G, Costa R, Alloisio A, Ferlazzo G, Sertoli MR, Reali UM, Ratto GB, Melioli G. Adoptive immunotherapy of advanced solid tumors: an eight year clinical experience. Anticancer Res 1999; 19(6C): 5645-5649

[27] Margolin KA. Interleukin-2 in the treatment of renal cancer. Semin Oncol 2000; 27(2): 194-203

[28] Rosenberg SA, Lotze MT, Yang JC, Topalian SL, Chang AE, Schwartzentruber DJ, Aebersold P, Leitman S, Linehan WM, Seipp CA, White DE, Steinberg SM. Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer. J Natl Cancer Inst 1993; 85(8): 622-632

[29] Chan JK, Hamilton CA, Cheung MK, Karimi M, Baker J, Gall JM, Schulz S, Thorne SH, Teng NN, Contag CH, Lum LG, Negrin RS. Enhanced killing of primary ovarian cancer by retargeting autologous cytokine-induced killer cells with bispecific antibodies: a preclinical study. Clin Cancer Res 2006; 12(6): 1859-1867

[30] Farag SS, Caligiuri MA. Cytokine modulation of the innate immune system in the treatment of leukemia and lymphoma. Adv Pharmacol 2004; 51: 295-318

[31] Smyth MJ, Cretney E, Kershaw MH, Hayakawa Y. Cytokines in cancer immunity and immunotherapy. Immunol Rev 2004; 202(1): 275-293

[32] Becknell B, Caligiuri MA. Interleukin-2, interleukin-15, and their roles in human natural killer cells. Adv Immunol 2005; 86: 209-239

[33] Rosenberg SA. Interleukin-2 and the development of immunotherapy for the treatment of patients with cancer. Cancer J Sci Am 2000; 6(Suppl 1): S2-S7

[34] Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev 2002; 13(2): 155-168

[35] Bottino C, Moretta L, Pende D, Vitale M, Moretta A. Learning how to discriminate between friends and enemies, a lesson from Natural Killer cells. Mol Immunol 2004; 41(6-7): 569-575

[36] Moretta L, Bottino C, Pende D, Vitale M, Mingari MC, Moretta A. Different checkpoints in human NK-cell activation. Trends Immunol 2004; 25(12): 670-676

[37] Trinchieri G. Biology of natural killer cells. Adv Immunol 1989; 47: 187-376

[38] Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003; 3(2): 133-146

[39] Medvedev AE, Johnsen AC, Haux J, Steinkjer B, Egeberg K, Lynch DH, Sundan A, Espevik T. Regulation of Fas and Fas-ligand expression in NK cells by cytokines and the involvement of Fas-ligand in NK/LAK cell-mediated cytotoxicity. Cytokine 1997; 9(6): 394-404

[40] Johnsen AC, Haux J, Steinkjer B, Nonstad U, Egeberg K, Sundan A, Ashkenazi A, Espevik T. Regulation of APO-2 ligand/trail expression in NK cells-involvement in NK cell-mediated cytotoxicity. Cytokine 1999; 11(9): 664-672

[41] Mirandola P, Ponti C, Gobbi G, Sponzilli I, Vaccarezza M, Cocco L, Zauli G, Secchiero P, Manzoli FA, Vitale M. Activated human NK and CD8⁺ T cells express both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant to TRAIL-mediated cytotoxicity. Blood 2004; 104(8): 2418-2424

[42] Zamai L, Ahmad M, Bennett IM, Azzoni L, Alnemri ES, Perussia B. Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells. J Exp Med 1998; 188(12): 2375-2380

[43] Fehniger TA, Cooper MA, Caligiuri MA. Interleukin-2 and interleukin-15: immunotherapy for cancer. Cytokine Growth Factor Rev 2002; 13(2): 169-183

[44] van der Vliet HJ, Koon HB, Yue SC, Uzunparmak B, Seery V, Gavin MA, Rudensky AY, Atkins MB, Balk SP, Exley MA. Effects of the administration of high-dose interleukin-2 on immunoregulatory cell subsets in patients with advanced melanoma and renal cell cancer. Clin Cancer Res 2007; 13(7): 2100-2108

[45] Ghiringhelli F, Ménard C, Martin F, Zitvogel L. The role of regulatory T cells in the control of natural killer cells: relevance during tumor progression. Immunol Rev 2006; 214(1): 229-238

[46] Berg M, Lundqvist A, McCoy P Jr, Samsel L, Fan Y, Tawab A, Childs R. Clinical-grade exvivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells. Cytotherapy 2009; 11(3): 341-355

[47] Escudier B, Farace F, Angevin E, Charpentier F, Nitenberg G, Triebel F, Hercend T. Immunotherapy with interleukin-2 (IL2) and lymphokine-activated natural killer cells: improvement of clinical responses in metastatic renal cell carcinoma patients previously treated with IL2. Eur J Cancer 1994; 30A(8): 1078-1083

[48] Ishikawa E, Tsuboi K, Saijo K, Harada H, Takano S, Nose T, Ohno T. Autologous natural killer cell therapy for human recurrent malignant glioma. Anticancer Res 2004; 24(3b): 1861-1871

[49] deMagalhaes-Silverman M, Donnenberg A, Lembersky B, Elder E, Lister J, Rybka W, Whiteside T, Ball E. Posttransplant adoptive immunotherapy with activated natural killer cells in patients with metastatic breast cancer. J Immunother 2000; 23(1): 154-160

[50] Miller JS, Tessmer-Tuck J, Pierson BA, Weisdorf D, McGlave P, Blazar BR, Katsanis E, Verfaillie C, Lebkowski J, Radford J Jr, Burns LJ. Low dose subcutaneous interleukin-2 after autologous transplantation generates sustained in vivo natural killer cell activity. Biol Blood Marrow Transplant 1997; 3(1): 34-44

[51] Burns LJ, Weisdorf DJ, DeFor TE, Vesole DH, Repka TL, Blazar BR, Burger SR, Panoskaltsis-Mortari A, Keever-Taylor CA, Zhang MJ, Miller JS. IL-2-based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial. Bone Marrow Transplant 2003; 32(2): 177-186

[52] Igarashi T, Wynberg J, Srinivasan R, Becknell B, McCoy JP Jr, Takahashi Y, Suffredini DA, Linehan WM, Caligiuri MA, Childs RW. Enhanced cytotoxicity of allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells. Blood 2004; 104(1): 170-177

[53] Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA, Yun GH, Fautsch SK, McKenna D, Le C, Defor TE, Burns LJ, Orchard PJ, Blazar BR, Wagner JE, Slungaard A, Weisdorf DJ, Okazaki IJ, McGlave PB. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005; 105(8): 3051-3057

[54] Iliopoulou EG, Kountourakis P, Karamouzis MV, Doufexis D, Ardavanis A, Baxevanis CN, Rigatos G, Papamichail M, Perez SA. A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer. Cancer Immunol Immunother 2010; 59(12): 1781-1789

[55] Kobayashi N. Artificial cells for the development of cell therapy. Cell Transplant 2008; 17(1): 3-9

[56] Tonn T, Becker S, Esser R, Schwabe D, Seifried E. Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92. J Hematother Stem Cell Res 2001; 10(4): 535-544

[57] Smyth MJ, Hayakawa Y, Takeda K, Yagita H. New aspects of natural-killer-cell surveillance and therapy of cancer. Nat Rev Cancer 2002; 2(11): 850-861

[58] Drexler HG, Matsuo Y. Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma. Leukemia 2000; 14(5): 777-782

[59] Cheng M, Ma J, Chen Y, Zhang J, Zhao W, Wei H, Ling B, Sun R, Tian Z. Establishment, characterization and successful adaptive therapy against human tumors of NKG cell, a new human NK cell line. Cell Transplant2011 Jun 7. [Epub ahead of print].

[60] Fernandez LA, Pope B, Lee C, Zayed E. Aggressive natural killer cell leukemia in an adult with establishment of an NK cell line. Blood 1986; 67(4): 925-930

[61] Yodoi J, Teshigawara K, Nikaido T, Fukui K, Noma T, Honjo T, Takigawa M, Sasaki M, Minato N, Tsudo M. TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells). J Immunol 1985; 134(3): 1623-1630

[62] Gong JH, Maki G, Klingemann HG. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994; 8(4): 652-658

[63] Robertson MJ, Cochran KJ, Cameron C, Le JM, Tantravahi R, Ritz J. Characterization of a cell line, NKL, derived from an aggressive human natural killer cell leukemia. Exp Hematol 1996; 24(3): 406-415

[64] Yoneda N, Tatsumi E, Kawano S, Teshigawara K, Oka T, Fukuda M, Yamaguchi N. Detection of Epstein-Barr virus genome in natural-killer-like cell line, YT. Leukemia 1992; 6(2): 136-141

[65] Tsuchiyama J, Yoshino T, Mori M, Kondoh E, Oka T, Akagi T, Hiraki A, Nakayama H, Shibuya A, Ma Y, Kawabata T, Okada S, Harada M. Characterization of a novel human natural killer-cell line (NK-YS) established from natural killer cell lymphoma/leukemia associated with Epstein-Barr virus infection. Blood 1998; 92(4): 1374-1383

[66] Kagami Y, Nakamura S, Suzuki R, Iida S, Yatabe Y, Okada Y, Kobayashi T, Tsurumi T, Seto M, Ogura M, Taguchi O, Morishima Y. Establishment of an IL-2-dependent cell line derived from ‘nasal-type’ NK/T-cell lymphoma of CD2⁺, sCD3^-, CD3e⁺, CD56⁺ phenotype and associated with the Epstein-Barr virus. Br J Haematol 1998; 103(3): 669-677

[67] Yagita M, Huang CL, Umehara H, Matsuo Y, Tabata R, Miyake M, Konaka Y, Takatsuki K. A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation. Leukemia 2000; 14(5): 922-930

[68] Yan Y, Steinherz P, Klingemann HG, Dennig D, Childs BH, McGuirk J, O’Reilly RJ. Antileukemia activity of a natural killer cell line against human leukemias. Clin Cancer Res 1998; 4(11): 2859-2868

[69] Tam YK, Miyagawa B, Ho VC, Klingemann HG. Immunotherapy of malignant melanoma in a SCID mouse model using the highly cytotoxic natural killer cell line NK-92. J Hematother 1999; 8(3): 281-290

[70] Maki G, Klingemann HG, Martinson JA, Tam YK. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J Hematother Stem Cell Res 2001; 10(3): 369-383

[71] Komatsu F, Kajiwara M. Relation of natural killer cell line NK-92-mediated cytolysis (NK-92-lysis) with the surface markers of major histocompatibility complex class I antigens, adhesion molecules, and Fas of target cells. Oncol Res 1998; 10(10): 483-489

[72] Klingemann HG, Wong E, Maki G. A cytotoxic NK-cell line (NK-92) for ex vivo purging of leukemia from blood. Biol Blood Marrow Transplant 1996; 2(2): 68-75

[73] Maki G, Tam YK, Berkahn L, Klingemann HG. Ex vivo purging with NK-92 prior to autografting for chronic myelogenous leukemia. Bone Marrow Transplant 2003; 31(12): 1119-1125

[74] Arai S, Meagher R, Swearingen M, Myint H, Rich E, Martinson J, Klingemann H. Infusion of the allogeneic cell line NK-92 in patients with advanced renal cell cancer or melanoma: a phase I trial. Cytotherapy 2008; 10(6): 625-632

[75] Klingemann HG. Natural killer cell-based immunotherapeutic strategies. Cytotherapy 2005; 7(1): 16-22

[76] Malmberg KJ, Bryceson YT, Carlsten M, Andersson S, Björklund A, Björkström NK, Baumann BC, Fauriat C, Alici E, Dilber MS, Ljunggren HG. NK cell-mediated targeting of human cancer and possibilities for new means of immunotherapy. Cancer Immunol Immunother 2008; 57(10): 1541-1552

[77] Tam YK, Maki G, Miyagawa B, Hennemann B, Tonn T, Klingemann HG. Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy. Hum Gene Ther 1999; 10(8): 1359-1373

[78] Suck G, Branch DR, Smyth MJ, Miller RG, Vergidis J, Fahim S, Keating A. KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity. Exp Hematol 2005; 33(10): 1160-1171

[79] Suck G, Branch DR, Keating A. Irradiated KHYG-1 retains cytotoxicity: potential for adoptive immunotherapy with a natural killer cell line. Int J Radiat Biol 2006; 82(5): 355-361

[80] Zhang C, Zhang J, Niu J, Zhang J, Tian Z. Interleukin-15 improves cytotoxicity of natural killer cells via up-regulating NKG2D and cytotoxic effector molecule expression as well as STAT1 and ERK1/2 phosphorylation. Cytokine 2008; 42(1): 128-136

[81] García-Lora A, Martinez M, Pedrinaci S, Garrido F. Different regulation of PKC isoenzymes and MAPK by PSK and IL-2 in the proliferative and cytotoxic activities of the NKL human natural killer cell line. Cancer Immunol Immunother 2003; 52(1): 59-64

[82] Pedrinaci S, Algarra I, Garrido F. Protein-bound polysaccharide (PSK) induces cytotoxic activity in the NKL human natural killer cell line. Int J Clin Lab Res 1999; 29(4): 135-140

[83] Velardi A, Ruggeri L, Mancusi A, Aversa F, Christiansen FT. Natural killer cell allorecognition of missing self in allogeneic hematopoietic transplantation: a tool for immunotherapy of leukemia. Curr Opin Immunol 2009; 21(5): 525-530

[84] Nagashima S, Mailliard R, Kashii Y, Reichert TE, Herberman RB, Robbins P, Whiteside TL. Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo. Blood 1998; 91(10): 3850-3861

[85] Konstantinidis KV, Alici E, Aints A, Christensson B, Ljunggren HG, Dilber MS. Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 cells. Exp Hematol 2005; 33(2): 159-164

[86] Cooper MA, Bush JE, Fehniger TA, VanDeusen JB, Waite RE, Liu Y, Aguila HL, Caligiuri MA. In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells. Blood 2002; 100(10): 3633-3638

[87] Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood 2001; 97(1): 14-32

[88] He YG, Mayhew E, Mellon J, Niederkorn JY. Expression and possible function of IL-2 and IL-15 receptors on human uveal melanoma cells. Invest Ophthalmol Vis Sci 2004; 45(12): 4240-4246

[89] Waldmann TA, Dubois S, Tagaya Y. Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for immunotherapy. Immunity 2001; 14(2): 105-110

[90] Rodella L, Zamai L, Rezzani R, Artico M, Peri G, Falconi M, Facchini A, Pelusi G, Vitale M. Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells. Br J Haematol 2001; 115(2): 442-450

[91] Zhang J, Sun R, Wei H, Zhang J, Tian Z. Characterization of interleukin-15 gene-modified human natural killer cells: implications for adoptive cellular immunotherapy. Haematologica 2004; 89(3): 338-347

[92] Jiang W, Zhang J, Tian Z. Functional characterization of interleukin-15 gene transduction into the human natural killer cell line NKL. Cytotherapy 2008; 10(3): 265-274

[93] Zhang J, Sun R, Wei H, Zhang J, Tian Z. Characterization of stem cell factor gene-modified human natural killer cell line, NK-92 cells: implication in NK cell-based adoptive cellular immunotherapy. Oncol Rep 2004; 11(5): 1097-1106

[94] Kuwana Y, Asakura Y, Utsunomiya N, Nakanishi M, Arata Y, Itoh S, Nagase F, Kurosawa Y. Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions. Biochem Biophys Res Commun 1987; 149(3): 960-968

[95] Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci USA 1989; 86(24): 10024-10028

[96] Eshhar Z. Tumor-specific T-bodies: towards clinical application. Cancer Immunol Immunother 1997; 45(3-4): 131-136

[97] Uherek C, Groner B, Wels W. Chimeric antigen receptors for the retargeting of cytotoxic effector cells. J Hematother Stem Cell Res 2001; 10(4): 523-534

[98] Roberts MR, Qin L, Zhang D, Smith DH, Tran AC, Dull TJ, Groopman JE, Capon DJ, Byrn RA, Finer MH. Targeting of human immunodeficiency virus-infected cells by CD8⁺ T lymphocytes armed with universal T-cell receptors. Blood 1994; 84(9): 2878-2889

[99] Tran AC, Zhang D, Byrn R, Roberts MR. Chimeric zeta-receptors direct human natural killer (NK) effector function to permit killing of NK-resistant tumor cells and HIV-infected T lymphocytes. J Immunol 1995; 155(2): 1000-1009

[100] Müller T, Uherek C, Maki G, Chow KU, Schimpf A, Klingemann HG, Tonn T, Wels WS. Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells. Cancer Immunol Immunother 2008; 57(3): 411-423

[101] Esser R, Muller T, Stefes D, Kloess S, Seidel D, Gillies SD, Aperlo-Iffland C, Huston JS, Uherek C, Schonfeld K, Tonn T, Huebener N, Lode HN, Koehl U, Wels WS. NK cells engineered to express a GD(2) -specific antigen receptor display built-in ADCC-like activity against tumor cells of neuroectodermal origin. J Cell Mol Med2011<month>May</month><day>20</day>. [Epub ahead of print]

[102] Demirtzoglou FJ, Papadopoulos S, Zografos G. Cytolytic and cytotoxic activity of a human natural killer cell line genetically modified to specifically recognize HER-2/neu overexpressing tumor cells. Immunopharmacol Immunotoxicol 2006; 28(4): 571-590

[103] Uherek C, Tonn T, Uherek B, Becker S, Schnierle B, Klingemann HG, Wels W. Retargeting of natural killer-cell cytolytic activity to ErbB2-expressing cancer cells results in efficient and selective tumor cell destruction. Blood 2002; 100(4): 1265-1273

[104] Boissel L, Betancur M, Wels WS, Tuncer H, Klingemann H. Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells. Leuk Res 2009; 33(9): 1255-1259

[105] Tam YK, Martinson JA, Doligosa K, Klingemann HG. Ex vivo expansion of the highly cytotoxic human natural killer-92 cell-line under current good manufacturing practice conditions for clinical adoptive cellular immunotherapy. Cytotherapy 2003; 5(3): 259-272

[106] Klingemann HG, Miyagawa B. Purging of malignant cells from blood after short ex vivo incubation with NK-92 cells. Blood 1996; 87(11): 4913-4914

[107] Liu XC, Liang H, Tian Z, Ruan YS, Zhang L, Chen Y. Proteomic analysis of human NK-92 cells after NK cell-mediated cytotoxicity against K562 cells. Biochemistry (Mosc) 2007; 72(7): 716-727

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg