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Keywords:

  • cancer immunotherapy;
  • clinical trials;
  • cytokines;
  • ex vivo expansion;
  • gene therapy;
  • natural killer cells

Abstract.

  1. Top of page
  2. Abstract.
  3. Natural killer cells: a historical background
  4. NK cells in cancer
  5. Potential of NK cells in cancer immunotherapy
  6. Concluding remarks
  7. Conflict of interest statement
  8. Acknowledgements
  9. References

As our understanding of the molecular mechanisms governing natural killer (NK) cell activity increases, their potential in cancer immunotherapy is growing increasingly prominent. This review analyses the currently available preclinical and clinical data regarding NK cell-based immunotherapeutic approaches in cancer starting from a historical background and an overview of molecular mechanisms taking part in NK cell responses. The status of NK cells in cancer patients, currently investigated clinical applications such as in vivo modulation of NK cell activity, ex vivo purification/expansion and adoptive transfer as well as future possibilities such as genetic modifications are discussed in detail.


Abbreviations:
ADCC

antibody-dependent cellular cytotoxicity

ALL

acute lymphoblastic leukaemia

AML

acute myeloid leukaemia

BM

bone marrow

BMT

bone marrow transplantation

CML

chronic myelogenous leukaemia

CR

complete remission

CRC

colorectal carcinoma

DC

dendritic cell

DLI

donor lymphocyte infusion

G-CSF

granulocyte colony stimulating factor

GM-CSF

granulocyte-macrophage colony stimulating factor

GMP

good manufacturing practice

GvHD

graft-versus-host disease

HCC

hepatocellular carcinoma

HLA

human leukocyte antigen

HSCT

haematopoietic stem cell transplantation

IFN

interferon

IL

interleukin

KIR

killer-cell immunoglobulin-like receptor

LAK cells

lymphokine-activated killer cells

LGL

large granular lymphocyte

MHC

major histocompatibility complex

MM

multiple myeloma

NB

neuroblastoma

NCR

natural cytotoxicity receptor

PBMC

peripheral blood mononuclear cell

PBSC

peripheral blood stem cell

PHA

phytohaemagglutinin

PR

partial remission

RCC

renal cell carcinoma

ROS

reactive oxygen species

SCID

severe combined immunodeficiency

SCT

stem cell transplantation

TCR

T-cell receptor

TNF

tumour necrosis factor

TRAIL

TNF-related apoptosis inducing ligand

Treg

regulatory T cell

WBC

white blood cell

Natural killer cells: a historical background

  1. Top of page
  2. Abstract.
  3. Natural killer cells: a historical background
  4. NK cells in cancer
  5. Potential of NK cells in cancer immunotherapy
  6. Concluding remarks
  7. Conflict of interest statement
  8. Acknowledgements
  9. References

Initially regarded as an ‘experimental artifact’ in T-cell cytotoxicity assays, natural killer (NK) cells were first discovered in mice more than 30 years ago by Kiessling et al., who also named them natural killer cells [1, 2] and in parallel by Herberman et al. [3, 4]. Human NK cells were initially described as nonadherent, nonphagocytic, FcγR+, large granular lymphocytes (LGL) [5]. Later it was, however, appreciated that NK cells not only shared the LGL phenotype and some NK cells also displayed normal small lymphocyte morphology, depending on their activation status [6]. This made it difficult to detect the NK cell population just by the size and morphology. The identification of the NKR-Pl [7] and NK1.1 [8] made it possible to define the murine NK cells roughly as NK1.1+ TCR sIg CD16+. Today, human NK cells are defined as CD3CD56+ lymphocytes. They comprise ∼10–15% of all circulating lymphocytes and are also found in peripheral tissues, including the liver, peritoneal cavity and placenta. Resting NK cells circulate in the blood, but following activation by cytokines, they are capable of extravasation and infiltration into most tissues that contain pathogen-infected or malignant cells [9–11].

The discovery of NK cells suggested a possible effector mechanism behind the phenomenon of ‘hybrid resistance’. Skin and organ transplantations had shown that allogeneic grafts were rejected whilst syngeneic grafts were tolerated, i.e. rejection only took place when the grafts had MHC molecules differing from the host. This rejection was mediated by T cells, which could induce either a graft-versus-host or a host-versus-graft reaction. Irradiated (AxB)F1 mice rejected BM transplants from either parent, despite the fact that the transplant did not express any foreign MHC molecules. This was not in accordance with the reigning dogmas of T-cell-mediated rejection. The BM rejection could still be observed in severe combined immunodeficient (SCID) mice, which have no T and B cells but have functional NK cells [12].

Initially, it was not clear how NK cells distinguished the target cells they should kill from those that they should spare. When Kärre summarized his and other people’s work for his doctoral thesis, he found a common denominator not about what was commonly expressed on target cells but about what was commonly missing. This lead him to formulate the missing-self hypothesis, where he suggested that NK cells kill target cells lacking expression of self MHC class-I molecules although the mechanism was unclear [13, 14] (see Fig. 1). This model was later confirmed by the discovery of inhibitory receptors on NK cells. Missing-self could also explain the hybrid resistance phenomenon; the (AxB)F1 host killed cells from either parent A or B because these cells lacked complete self MHC expression (A+B). To further test the missing-self hypothesis, a MHC class I-deficient version of the tumour cell line RMA was established and named RMA-S. C57BL/6 mice inoculated with RMA-S cells rejected the tumours, whilst mice inoculated with RMA developed the tumour. By treating the mice with NK depleting anti-asialo GM1 antibody, the difference in tumour outgrowth disappeared [15]. This confirmed that NK cells-mediated the selective rejection of MHC lacking tumour growth.

image

Figure 1.  The recognition of tumour cells by NK cells. The figure presents four hypothetical scenarios for the encounter of an NK cell and a tumour cell. (a) Although the tumour cell does not express any inhibitory ligands, it cannot be killed by the NK cell because it also lacks the expression of any activating ligands. This target is practically invisible to the NK cell and no recognition takes place. (b) The tumour cell expresses ligands for inhibitory receptors, whereas it lacks ligands for activating receptors. The NK cell recognizes the inhibitory ligands and, therefore, no killing takes place. (c) The tumour cell has significantly downregulated or absent expression of inhibitory ligands along with sufficient expression of activating ligands. Missing-self recognition takes place and the target is killed. (d) The tumour cell expresses significant levels of both inhibitory and activating ligands. The NK cells recognize both types of ligands and the outcome of this interaction is determined by the balance of inhibitory and activating signals.

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Natural killer cells are separated into two subsets based on their CD56 antigen expression. Yet, this separation is not just phenotypic but rather has many functional outcomes. The majority (∼90%) of human NK cells have low-density expression of CD56 (CD56dim), whereas ∼10% of NK cells are CD56bright. Early functional studies of these subsets revealed that the CD56dim cells are more cytotoxic [16]. However, there are a number of other cell-surface markers that confer unique phenotypic and functional properties to CD56bright and CD56dim NK cell subsets. CD56bright subset is shown to exclusively express IL-2 receptor α chain (IL-2Rα/CD25), whilst they lack or express only at very low levels the FCγRIII (CD16). On the other hand, the CD56dim subset has high expression of CD16 and lacks CD25 expression. These properties set very different roles to the different subsets with regards to antibody dependent cellular cytotoxicity (ADCC) and response to IL-2 stimulation. In addition to distinct expression of adhesion molecules and cytokine receptors, the CD56bright NK cell has the capacity to produce high levels of immunoregulatory cytokines, but has low-level expression of killer-cell immunoglobulin-like receptors (KIRs) and is poorly cytotoxic. By contrast, the CD56dim NK cell appears to produce low levels of cytokines but has high-level expression of KIRs and is a potent cytotoxic effector cell. Such evidence suggests that the CD56bright and CD56dim subsets are distinct lymphocytes with unique roles in the immune system. Thus, studies of the biology of human NK cells are eventually approaching NK cells as separate CD56bright and CD56dim subsets rather than a homogenous population.

As the name implies, NK cells can kill certain cells without prior sensitization, but they are also potent producers of various cytokines, such as IFN-γ, TNF-α, GM-CSF and IL-3 [17]. Therefore, NK cells are also believed to function as regulatory cells in the immune system, influencing other cells and responses and acting as a link between the adaptive and innate immune responses. For example, NK cells seem to participate in the development of the autoimmune disease, myasthenia gravis, by regulating both the autoreactive T and B cells through IFN-γ production [18]. Moreover, it has been observed that depletion of NK cells in C57Bl/6 mice leads to increased engraftment of neuroblastoma (NB) xenografts mainly because of dysregulation of Th1-oriented B-cell responses [19]. These data prove the significant impact of NK cells on adaptive immune responses. Other studies have also shown a close interaction between NK cells and dendritic cells (DC) [20]. In addition to their role as the initiators of antigen specific responses, DCs have been shown to support the activity of NK cells [21], whilst reciprocally, cytokine-preactivated NK cells have been shown to activate DCs and induce their maturation and cytokine production [22–24]. In vivo activation of NK cells by a DC vaccine consisting of autologous DCs loaded with a tumour-associated antigen has also been shown [25]. NK cells are also involved in the defence against virus infections and autoimmunity both of which have been elegantly reviewed elsewhere [26, 27].

Today, we know that NK cell cytotoxicity is the result of a complex balance between the inhibitory and activating receptors [28]. Table 1 provides a list of human NK cell activating and inhibitory receptors identified to our knowledge. Upon recognition of the ligands on the target cell surface by activating NK cell receptors, various intracellular signalling pathways drive NK cells towards cytotoxic action and this results in target cell cytolysis [29].

Table 1.   Activating and inhibitory receptors on human NK cells
CDAlternative nameType of signalLigandDistribution on NK cells
CD2LFA-2ActivationCD58 (LFA-3)All
CD7LEU-9ActivationSECTM1, GalectinAll
CD11aLFA-1ActivationICAM-1,-2,-3,-4,-5All
CD11bMac-1ActivationICAM-1, FibrinogenAll
CD16FcγRIIIActivationIgGMainly CD56dim Negative/dim on CD56bright
CD27TNFRSF7?CD70Mainly on CD56bright Negative/dim on CD56dim
CD44Hyalunorate receptorActivationHyalouronanAll
CD59ProtectinActivationC8, C9All
CD69CLEC2CActivationUnkownActivated
CD85jILT-2InhibitionHLA-A, -B, -GSubset
CD94/CD159aCD94/NKG2AInhibitionHLA-EMost
CD94/CD159cCD94/NKG2CActivationHLA-EMost
CD96TACTILEActivationCD155Activated low expression on resting
CD160BY55ActivationHLA-CAll
CD161NKR-P1Activation/InhibitionLLT1Subset
CD223Lag3ActivationHLA Class IIActivated
CD226DNAM-1ActivationCD112, CD155All
CD2442B4Activation/InhibitionCD48All
CD314NKG2DActivationMICA, MICB, ULB-1,-2,-3,-4All
CD319CRACCActivationCRACCMature NK cells
CD328Siglec-7InhibitionSialic acidSubset
CD329Siglec-9InhibitionSialic acidSubset
CD335NKp46ActivationViral haemagglutinin (?)All
CD336NKp44ActivationViral haemagglutinin (?)Activated
CD337NKp30ActivationViral haemagglutinin (?)All
VariousKIR2DS, KIR3DSActivationHLA Class ISubsets
VariousKIR2DL, KIR3DLInhibitionHLA Class ISubsets
NTB-AActivationNTB-AAll
KLRG1InhibitionE-,N-,P-cadherinAll

However, these processes are tightly controlled by a group of inhibitory receptors. These receptors act as negative regulators of NK cytotoxicity and inhibit the action of NK cells against ‘self’ targets. A main group of this type of receptors is KIRs, which are mainly specific for self MHC Class-I molecules. If the target cell is recognized by inhibitory KIRs, which means, it has sufficient amount of self MHC Class-I molecules on the cell surface, an inhibitory signal from KIRs stops the action of cytotoxic pathways triggered by activating receptors [30, 31]. The KIRs are type I (extracellular amino terminus) membrane proteins that contain either two or three extracellular Ig-like domains [32] and they are designated as KIR2D or KIR3D respectively. The cytoplasmic domains of the KIRs can be either short (S) or long (L), corresponding to their function as either activating or inhibitory receptors respectively. Members of the KIR family recognize HLA-A, HLA-B and HLA-C alleles and KIR2DL4 recognizes HLA-G [33]. The KIR receptors are clonally distributed on NK cells, which provides that even the loss of a single HLA allele (a common event in tumourigenesis and viral infections) can be detected by a pool of NK cells [33, 34].

The activating side of the balance also includes a series of different receptors (see Table 1). The main activating receptor group is called natural cytotoxicity receptors (NCRs) [29] and it is believed that the main control over the NK cell activating pathways is regulated by these receptors. Currently, there are three different NCRs identified: NKp30 [35], NKp44 [36] and NKp46 [37]. NKp30 and NKp46 are expressed both in activated and in nonactivated NK cells, whereas NKp44 expression is restricted to activated NK cells. Most activating receptors do not directly signal through their cytoplasmic tail, but instead associate noncovalently with other molecules containing immunoreceptor tyrosine-based activation motifs (ITAM) that serve as the signal transducing proteins. NKp30 and NKp46 are associated with CD3ζ, whereas NKp44 is associated with DAP12. NK cell activation has been studied extensively in recent years and is discussed elsewhere [38, 39].

Natural killer cells have been described as ‘large granular lymphocytes’ and their granularity is their means for target cell killing (see Fig. 2). These granules contain perforin and granzyme B [40] and it is postulated that granzymes and perforin both bind to the target surface as part of a single macromolecular complex [41]. When an NK cell encounters a target cell, perforin and granzyme B are released; granzyme enters the target cell and mediates apoptosis, whilst perforin disrupts endosomal trafficking [42, 43]. NK cells can also express FasL and TNF-related apoptosis-inducing ligand (TRAIL), which are both members of the TNF family and are shown to induce target cell apoptosis when they bind their receptors on target cells [44, 45]. TNF-α has also been suggested to mediate activation-induced cell death by NK cells [46].

image

Figure 2.  Mechanisms of NK cell cytotoxicity. The cytotoxicity of NK cells are carried out by two main mechanisms. The first mechanism is granule-dependent cytotoxicity where upon triggering by (a) activating receptors or (b) the Fc receptor (CD16), the cytotoxic granules in the cytosol of the NK cell are polarized towards the immunological synapse and the contents (mainly perforin and granzyme B) are unleashed upon the target cell by exocytosis. The second mechanism is the triggering of apoptosis pathways in the target cell via stimulation of death receptors on by (c) TRAIL or (d) Fas ligand expressed on the NK cell surface as well as (e) secretion of TNF-α.

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NK cells in cancer

  1. Top of page
  2. Abstract.
  3. Natural killer cells: a historical background
  4. NK cells in cancer
  5. Potential of NK cells in cancer immunotherapy
  6. Concluding remarks
  7. Conflict of interest statement
  8. Acknowledgements
  9. References

The development of any malignancy is under close surveillance by NK cells as well as other members of the immune system. Nevertheless, malignant cells obtain means to escape from the immune system and proliferate. General mechanisms include saturation of the immune system by the rapid growth of the tumour, inaccessibility of the tumour owing to defective vascularization, its large dimension or its localization in immune-privileged sites and resistance to the Fas- or perforin-mediated apoptosis. The expression of FasL by tumour cells as a counterattack strategy against immune effector such as T cells and NK cells is also common [47–49]. Additionally, the defective expression activation receptors and various intracellular signalling molecules by T cells and NK cells in cancer patients was observed and reported to correlate with disease progression [50]. It has also been shown that malignant cells secrete immunosuppressive factors that inhibit T and NK cell proliferation [51]. As a result of all these events, defective immunity secondary to tumour development has been a well-established phenomenon [52]. Table 2 presents a selection of previously defined NK cell abnormalities in cancer patients.

Table 2.   NK cell abnormalities in cancer patients
AbnormalityDisease
Decreased cytotoxic activity of NK cellsNonsmall cell lung cancer [215]
Hepatocellular carcinoma [216, 217]
Stage IV rectal cancer [218]
Head and neck cancer [219]
Breast cancer [219]
Cervical carcinoma [220]
Squamous cell carcinoma of the penis [221]
Bronchogenic carcinoma [222]
Ovarian cancer [223]
AML [224]
ALL [224, 225]
B-CLL [226]
CML [227]
MM [228]
Defective expression of activating receptorsHepatocellular carcinoma [216]
Metastatic melanoma [229]
AML [230]
MM [95, 231]
Defective NK cell proliferationMetastatic renal cell carcinoma [232]
Nasopharyngeal cancer [233]
CML [234]
Increased number of CD56bright NK cellsHead and neck cancer [219]
Breast cancer [219]
Defective expression of intracellular signalling moleculesCervical cancer [235]
Colorectal cancer [236]
Ovarian cancer [237]
Prostate cancer [238]
AML [239]
CML [239]
Decreased NK cell countsNasopharyngeal cancer [233]
CML [234]
Increased NK cell countsMM [228]
Defective cytokine productionAML [224]
ALL [224, 225]
CML [240]

Potential of NK cells in cancer immunotherapy

  1. Top of page
  2. Abstract.
  3. Natural killer cells: a historical background
  4. NK cells in cancer
  5. Potential of NK cells in cancer immunotherapy
  6. Concluding remarks
  7. Conflict of interest statement
  8. Acknowledgements
  9. References

Modulation of NK cell activity

IL-2 alone. The cDNA encoding for the human IL-2 gene was cloned in 1983 [53] after a long search starting in 1965 for the soluble factors in lymphocyte conditioned media that could sustain the proliferation of T cells in culture [54, 55]. It is now well known that IL-2 effects many types of cells in the immune system including cytotoxic T cells, helper T cells, regulatory T cells, B cells and NK cells. Currently, there are three distinct chains of the IL-2 receptor identified; the α (CD25), β (CD122) and γ (CD132) chains. The γ chain is shared amongst various interleukin receptors (IL-4, IL-7, IL-9, IL-15, IL-21), thus named the common γ chain and it is essential for lymphoid development [56]. The β chain is shared between IL-2 and IL-15 receptors [57, 58]. The β and~γ chains come together to form the intermediate affinity IL-2/15 receptor. The distinction between the high affinity receptors for IL-2 and IL-15 comes with the α chains. The IL-2Rα chain alone is regarded as the low affinity receptor and is believed to lack the capacity to deliver intracellular signals because of its short intracellular tail [59]. However, when the α chain forms a complex with the β and γ chains, the high affinity IL-2 receptor is formed. The co-expression of all three chains is confined to regulatory T cells, CD56bright NK cells as well as activated conventional CD4+ and CD8+ T cells [60]. Thus, these cells are expected to give a better response to the presence of low dose IL-2 as they have the capacity to form a high affinity IL-2 receptor complex.

It has been well defined that IL-2 activation of NK cells can result in cytotoxic activity against targets that were previously NK-resistant [61]. Observations on the interaction of autologous and allogeneic NK cells with fresh tumour cells have also proved that IL-2 activation in vitro enhances the tumour killing potential of NK cells [62, 63]. Early reports of IL-2-based treatment on animal models have established a solid basis for efficiency of this approach for cancer immunotherapy in many different settings [64–72]. Although cytotoxic T cells have been the primary point of interest, especially during the early phases of IL-2 use, the antitumour response triggered by IL-2 were frequently attributable to NK cells [73–77]. Whiteside et al. [75] have demonstrated in xenograft models of head and neck squamous cell carcinoma, that IL-2 activated NK cells are as potent as tumour specific CD8+ T cells in vivo. Egilmez et al. [76] have shown that sustained delivery of IL-2 using biodegradable microspheres can promote the NK cell-mediated rejection of lung tumour xenografts in SCID mice. Likewise, our group has demonstrated in a syngeneic murine model of multiple myeloma (MM) that NK cells are the primary mediators of IL-2 induced tumour rejection [77].

In the clinical setting, the pioneering work of Rosenberg et al. [78, 79], which has demonstrated the potent immunostimulatory effect of IL-2 in advanced cancer patients, resulted in a great interest for the use of cytokines and immune effector cells for the treatment of cancer. Further reports have shown that IL-2 treatment results in in vivo activation of NK cell cytotoxicity [80] and this effect is dependent on the dose and schedule of IL-2 administration [81]. Since then, such an approach of stimulating endogenous NK cells with cytokines in an attempt to promote in vivo killing of tumour cells has been used by many investigators.

It has been observed that IL-2 treatment of some cancer patients receiving a T-cell depleted allogeneic BMT was well tolerated, decreased relapse risk and increased survival compared to those not receiving IL-2 [82]. Such observations have drawn more and more interest for the use of NK cells in cancer immunotherapy. Other investigators have shown that IL-2 administration stimulates the ERK signalling pathway in especially CD56bright NK cells and CD14+ monocytes, but not CD3+ T cells, which suggests that it has a distinct way of acting on this lymphocyte subpopulation [83].

Interleukin-2 has received FDA approval for the treatment of metastatic renal cell carcinoma (RCC) in 1992 based on its ability to induce an objective response rate of 15–20% [84]. It has also been demonstrated that in RCC patients undergoing IL-2-based therapy and nephrectomy, a higher percentage of circulating NK cells is a predictor of response [85].

Natural killer cells have also been shown to play an important role in the effective treatment or prevention of AIDS-associated lymphoma using low-dose IL-2 infusions [86]. Treatment of patients with AIDS-associated malignancies results in an increase in absolute NK cell numbers, whilst no significant change in T-cell subsets or plasma HIV RNA level is seen [87].

The use of IL-2 alone has been attempted in many other tumour types, mostly as an adjuvant to chemotherapy or stem cell transplantation (SCT). Treatment of patients with breast cancer and lymphoma using IL-2 was shown to increase the number of circulating NK cells and their cytotoxicity against NK resistant breast cancer and lymphoma cell lines significantly [88]. Burns et al. have treated 23 lymphoma/breast cancer patients with daily s.c. IL-2 infusions supplemented with two i.v. bolus IL-2 infusions. As anticipated, the therapy resulted in a significant increase in total WBC count along with a more than 10-fold increase in circulating CD56bright NK cells. Yet, no improvement in survival or relapse was observed when compared with matched controls [89].

Kalwak et al. administered intermediate doses of i.v. IL-2 to 11 children with poor prognosis solid tumours following autologous SCT (ASCT). A significant increase in T- and NK-cell counts and an elevated level of NK cell cytotoxic activity was observed. Again, mainly CD56bright NK cells expanded in vivo and most of them expressed the inhibitory receptor CD94. Despite the increased NK cell activity, relapses occurred frequently [90]. In another study, IL-2 treatment of NB patients after intensive chemotherapy and autologous BMT resulted in a drastic increase of NK cell numbers and cytotoxicity during 1-year treatment. One of seven patients relapsed and that was the patient that showed only a slight increase in the NK cell subset in response to IL-2 treatment [91].

The use of IL-2 for inducing NK cell-mediated killing of tumour cells has also been a popular approach in haematological malignancies. IL-2 has been shown to provide stimulation of PBMCs for killing of multiple myeloma (MM) cells [92]. Later studies have proved that NK cells have an effective cytotoxic activity against MM cell lines and tumour cells from MM patients [93]. Our group has recently demonstrated that NK cells from MM patients can be expanded ex vivo using GMP-compliant components, and they show high cytotoxic activity against autologous MM cells whilst retaining their tolerance against normal cells of the patient [94]. Other researchers have also shown that HLA Class I molecules, NCRs and NKG2D take part in the recognition of myeloma cells by autologous and allogeneic NK cells [95, 96]. Likewise, NK cells from AML patients at remission have also been expanded ex vivo and showed cytotoxic activity against allogeneic and autologous AML blasts, which could be further enhanced by IL-2 [97]. In a clinical AML study, where IL-2 was used alone, patients older than 60 years in first complete remission after induction and consolidation chemotherapy were randomly assigned to no further therapy (n = 82) or a 90-day regimen (n = 81) of 14-day cycles of low-dose rIL-2, aimed at expanding NK cells, followed by 3 day higher doses aimed to induce cytotoxicity of expanded NK cells. No prolongation of disease-free or overall survival was seen and the authors concluded that low-dose IL-2 maintenance immunotherapy alone is not a successful strategy to treat older AML patients [98]. Other researchers have observed that IL-2 activated autologous NK cells from CML patients can suppress primitive CML progenitors in long-term culture [99].

Overall, data from the reports reviewed above clearly demonstrates that although promising outcomes have been observed, low-dose IL-2 treatment is not an optimal strategy for most indications. In most cases of low-dose IL-2 administration (picomolar serum concentrations), a specific expansion of the CD56bright NK cell subset, that is known to have a regulatory rather than cytotoxic activity, is observed [59]. Within the NK cell population, IL-2Rα that confers high affinity for IL-2 is uniquely expressed by CD56bright cells [100], which could explain their selective expansion in response to low-dose IL-2. Although such treatment has proven to be safe, there is yet no convincing proof of efficacy. Likewise, the in vivo expansion of another IL-2Rα expressing regulatory cell subset; Treg cells could also overwhelm and/or suppress the antitumour activity that is to be carried out by immune effector cells. The potential of Treg cells to dampen NK cell-mediated antitumour responses has primarily been suggested in a murine leukaemia model [101]. The effect of Treg cells in cancer immunotherapy has now been well recognized [102, 103] and attempts to circumvent such suppression are underway [104].

IL-2 in combination with other factors. Studies have shown that IL-2, IL-12 and IL-15 stimulate NK cell cytotoxicity in vitro and show synergy when used in combination [105, 106]. Such cytokines have been widely used for in vitro studies to define requirements of NK cell activation that could potentially be used in cancer immunotherapy. As the β and γ chains of IL-2 and IL-15 receptors are shared, the signalling pathways triggered by these cytokines overlap to a great extent. The main signalling events triggered by engagement of the IL-2/15Rβγ heterodimer include phosphorylation of STAT3 and STAT5 by Jak1 and Jak3 respectively [107] and their subsequent translocation to the nucleus as transcription factors. Moreover, IL-2/15 engagement also results in activation of NF-κB [108] and the anti-apoptotic protein BCL-2 [107] confirming the close relationship between IL-2 and IL-15 derived signals, it has been shown that ex vivo expanded NK cells from paediatric ALL patients showed cytotoxic activity against autologous ALL blasts when activated by IL-2 or IL-15 or both of them [109].

The IL-2Rα chain has not been acknowledged to deliver any intracellular signals yet. However, the IL-15Rα chain has been shown to signal independently from the β/γ heterodimer, which in part explains the different effects of the two cytokines. Such signalling events include recruitment of TRAF2 to the short cytoplasmic tail of IL-15Rα [110], which results in NF-κB activation, and protection from TNF-α induced apoptosis as well as activation of the tyrosine kinase Syk [111] that provides a survival benefit for the cells. It is imperative to note that these signalling events by the IL-15R α chain have not been confirmed in NK cells yet. The selective modulation of protein kinase C isoforms has also been reported to take part in NK cell activation and is suggested to be a common mechanism used by IL-12, IL-2 and IL-15 [112].

Interleukin-12 differs from these two cytokines because of the fact that IL-12 receptor lacks common γ chain. The IL-12 receptor is composed of two subunits: IL-12Rβ1 and IL-12Rβ2, which bind Tyk2 and Jak2 respectively. Upon engagement, these kinases transduce the signal via a number of STAT molecules, STAT4 being the best identified and essential for IL-12 signalling in NK cells [113, 114]. IL-2 activated PBMCs showed increased cytotoxic activity against autologous primary lung cancer cells, which was further augmented with the addition of IL-12 [115]. This may be explained by the observation that IL-2 upregulates the expression of IL-12Rβ1 and IL-12Rβ2 as well as STAT4 [116]. The increased expression of IL-12R on NK cells in patients treated with low dose IL-2 also confirms this explanation [116]. NK cells have been shown to kill NB cell lines when activated with IL-2 and/or IL-12 [117] and molecules such as LFA-3 and ICAM-1 are suggested as important modulators of the susceptibility of NB cells to NK cell-mediated killing [118].

A major hurdle in tumour immunotherapy has been the various mechanisms by which tumours induce dysfunction or tolerance of local immune cells [52]. The immunosuppressive effect of reactive oxygen species (ROS) derived from tumour cells, tumour-derived macrophages or monocytes activated by cytokine therapy has been well defined and addressed in various tumours [52, 119]. Monocyte/macrophage derived ROS have been shown to induce apoptosis and anergy to IL-2 activation in T cells and NK cells [120]. Affirmatively, supplementation of the anti-oxidant vitamin E upregulates NKG2D expression and enhances NK cell function in CRC patients [121]. Other researchers have tried combination of IL-2 with histamine, an inhibitor of ROS synthesis in monocytes/macrophages [122], to counteract the negative effect of ROS originating mainly from the expanding monocyte/macrophage population. In a randomized phase II trial where 63 patients with metastatic RCC were treated with IL-2 and histamine [123], addition of histamine resulted in a decrease of monocyte expansion and number of intratumoural macrophages as well as an increase in the number of intratumoural NK cells and CD8+ T cells. The same study also demonstrated a likely correlation between the number circulating NK cells and cytotoxicity. Likewise, histamine has also been shown to synergize with IL-2 [124] and reverse the inhibition of NK cell cytotoxicity against heterologous AML blasts by monocyte-derived ROS [125]. Hellstrand et al. have treated 22 AML patients (mean age 59) with IL-2 and histamine. The treatment was well tolerated and showed an impressive survival benefit. The authors reported that 15/22 patients have achieved a CR and 7/7 evaluable patients have achieved a duration of CR that exceeds the foregoing remission [126], which stands out as a promising finding to counterweigh the ineffectiveness of IL-2 treatment in certain settings.

Other investigators have combined of IL-2 and IFN-α in the clinical setting. It has been demonstrated that perioperative treatment with IFN-α significantly increases NK cell activity although number of cells are decreased [127]. Atzpodien et al. have infused IL-2 along with interferon-α to 47 patients with cancer. They have observed a significant relation between the increase of circulating NK cell numbers (CD56+) and the response [128]. Molto et al. have observed in eight metastatic RCC patients undergoing immunotherapy with low dose s.c. IFN-α-2b and IL-2 following radical nephrectomy, that patients who achieved complete or partial responses had higher NK cell cytotoxic activity than those who remained in progression [129]. In another study by Moroni et al., 25 patients with metastatic RCC were treated with low-dose IL-2 and IFN-α. 6/25 had objective response (CR or PR) and 12/24 had stable disease. Significant increase in total lymphocyte counts as well as CD4+ cell, CD8+ cells, CD25+ cells, NK cells and eosinophils were observed [130]. In a similar study, low-dose IL-2 for 5 days per week and IFN-α twice weekly for four consecutive weeks were applied to 27 patients with advanced renal cell carcinoma. A significant increase in total lymphocytes, eosinophils, CD25+ cells and NK cells was observed. Within the NK cells, the CD56bright population had a higher expansion rate. Yet, no clinical benefit was observed [131].

In a study combining IL-2, IFN-α and histamine, mononuclear cells in peripheral blood and tumour biopsies from 13 patients with metastatic malignant melanoma were followed and a trend towards a gradual increase in the absolute number of circulating NK cells in patients maintaining stable disease during therapy was noted. The extent of leukocyte infiltration in tumour tissues prior to treatment correlated with the response. Additional NK cell infiltration during treatment was seen only in responding patients [132].

Combination of IL-2 and IFN-α with GM-CSF has been shown to increase significantly in total lymphocytes, activated T cells (CD4+ and CD8+), NK cells, monocyte DR expression, neutrophils and eosinophils. Four of 18 patients had partial or complete remission, whilst 10/18 patients had stable disease [133].

In a study by Re et al., NK cells from healthy donors were expanded and activated ex vivo through stimulation with IL-2 and PHA in the presence of feeder cells. The authors have observed that tumour samples from nine different patients with solid tumours were susceptible to lysis by NK cells when a KIR-ligand mismatch was present [134]. In a similar study, Castriconi et al. have observed that primary NB cells are susceptible to lysis by allogeneic NK cells that have been expanded in the presence of IL-2, PHA and feeder cells. They have also identified that susceptibility to lysis is dependent on the expression of PVR on tumour cells (the ligand of NK cell activating receptor DNAM-1) [135].

Taken together, the overall experience from supporting IL-2 treatment with secondary factors seems to be promising. It is obvious that if the results from these studies are evaluated carefully in the design of future clinical trials, such combination approaches may provide a solid basis for the use of IL-2 for stimulation of endogenous NK cells against tumours in many different indications.

Other factors that alter endogenous NK cells. Several other factors influencing the antitumour activity of NK cells have been questioned in animal models. The role of NK cells in xenograft models of breast cancer have been demonstrated in the interesting report by Burd et al. where the authors report that hyperthermia per se can induce changes in tumour vasculature, leading to an increased lymphocyte recruitment to the tumour site and subsequently diminished tumour growth in an NK cell dependent manner [136]. Okamoto et al. have analysed the effects of OK-PSA, a lipoteichoic acid-related molecule that enhances anti-tumour immunity, in mice. Along with general improvement of immune parameters, OK-PSA administration resulted in significant NK cell dependent suppression of tumour growth [137]. In another study, it has been shown that prolactin stimulation increases NK cell activity against YAC-1 targets, improves NK cell development during reconstitution after syngeneic bone marrow transplantation in mice and enhances anti-tumour activity by adoptively transferred syngeneic NK cells against colon cancer development in BALB/c mice [138].

To achieve an efficient in vivo NK cell activity, concomitant drug administration should be considered carefully. Markasz et al. investigated the effect of frequently used chemotherapeutic agents on NK cells and demonstrated that chlorambucil, MG-132, docetaxel, cladribine, paclitaxel, bortezomib, gemcitabine and vinblastine effectively inhibit NK cell activity. Oxaliplatin, dactinomycin, cytarabine, daunorubicin, vincristine, and topotecan were only marginally suppressive, whilst most NK cell lines were not affected by bevacizumab, bleomycin, doxorubicin, epirubicin, vinorelbine, carboplatin, methotrexate, ifosphamide, etoposide, hydroxyurea, asparaginase, 5-fluorouracil, 6-mercaptopurine, streptozocin and cyclophosphamide [139]. It has also been demonstrated that methylprednisolone inhibits expression of NCRs, proliferation and natural cytotoxicity in response to IL-2 and IL-15 [140]. Purified NK cells from healthy donors are found to be susceptible to lysis by anti-thymocyte globulin (ATG) even more than T cells, whilst G-CSF has a minimal effect on natural cytotoxicity [141].

The effects of various drugs in modulating tumour susceptibility to NK cell-mediated lysis have also been investigated by various researchers. Combinations of 5-aza-2′-deoxycytidine, trichostatin A, vitamin D3, bryostatin-1 and all-trans-retinoic acid, used together with myeloid growth factors and interferon-γ, were shown to increase expression of UL16-binding proteins (ULBP) up to 10-fold in the AML cell line HL60 and in primary AML blasts. Higher ULBP expression increased NKG2D-dependent sensitivity of HL60 cells to NK-mediated killing [142]. IFN-γ treatment of primary CD133+ glioblastoma cells, which normally express very low levels of MHC Class I and NK cell activating ligands, was shown to increase expression of both and were sensitized to killing by primary NK cells from healthy donors [143]. In another study, the authors have demonstrated that treatment with arsenic trioxide up-regulates NKG2D ligands, such as ULBP1 and sensitizes to NK cell-mediated killing in K562, NB4 and MCF7 cell lines [144]. Treatment with the histone deacetylase inhibitor valproic acid was found to up-regulate NKG2D ligands and sensitize the tumour cells to NK cell-mediated killing in 66 AML patients at diagnosis [145], whilst treatment with another histone deacetylase inhibitor trichostatin A induced expression if MICA and MICB in leukaemia cell lines and primary leukaemia cells from eight patients and increased susceptibility to NK cell-mediated killing [146].

Unlike T cells, NK cells don’t express a unique, antigen specific receptor. A strategy to target NK cells to tumour cells specifically is by making use of NK cells’ ADCC capabilities in vivo. ADCC by NK cells is mediated through binding of immunoglobulin G complexes or antibody-coated targets to the low-affinity Fc receptor for IgG, CD16. Antigen density, structure and specificity of Fc binding are the critical components for efficient induction of ADCC [147]. Human NK cells have been shown to exhibit ADCC using murine monoclonal antibodies of several isotypes (IgG1, IgG2a, IgG2b, IgG3) [148, 149]. A comprehensive review regarding monoclonal antibody-based targeted therapy is discussed elsewhere [150].

As virtually all ADCC activity in PBMCs is mediated by NK cells [151–153], it is important to determine how many target cells an NK cell can kill before it must revive to continue. Bhat and Watzl reported that IL-2-activated NK cells can engage and kill four target cells in 16 h; after this time, the cells appear to be exhausted, with reductions in available perforin and granzyme B, which is reversible by IL-2 treatment [154].

Adoptive transfer of NK cells

Sources of NK cells for adoptive transfer. In the clinical setting, the number, purity and state of proliferation/activation of NK cells to be used, are the key factors to consider. Regarding purification, single step GMP protocols to deplete CD4+/CD8+αβT cells are possible and result in passive enrichment of innate lymphocytes such as NK cells and γδT cells, which seem to preserve their proliferative and cytotoxic capacity [155]. Simple purification of NK cells by a single-step or two-step procedure may be enough for some applications. Leung et al. [141] have demonstrated that donor NK cells, purified by a clinical-scale two-step immunomagnetic separation method, have normal expression of cell surface markers, intracellular cytokines, perforin and granzyme B. Table 3 summarizes a selection of reports that study the isolation of clinical grade NK cells for adoptive immunotherapy.

Table 3.   Clinical scale NK cell purification
ReportStrategy nPurity (% NK cells)Recovery (%)Viability (%)CD3 (%)CD14 (%)CD19 (%)
Iyengar [241]CliniMACS CD3 depletion CliniMACS CD56 enrichment129149NR0.17.70.2
Passweg [176]CliniMACS CD3 depletion CliniMACS CD56 enrichment69735NR<0.01NRNR
Lang [242]CliniMACS CD56 enrichment DynaBeads CD3 depletion49942>90NRNRNR
McKenna [243]CliniMACS CD3 depletion3638798613126
McKenna [243]CliniMACS CD3 depletion CliniMACS CD56 enrichment139019850.2150.67
Koehl [244]2× CliniMACS CD3 depletion CliniMACS CD56 enrichment119533950.015NR
Frohn [245]SuperMACS NK cell selection kit108550NR18.50.3

The actual obstacle in clinical studies with adoptive transfer of NK cells originates from the fact they are normally present only in low numbers in PBMCs and effector cell preparations such as lymphokine activated killer (LAK) cells. Obtaining a large number of NK cells is an influential albeit difficult task that underlies the most significant challenge to the development of successful NK cell adoptive transfer protocols. Thus, many researchers have worked on the expansion of NK cells. Table 4 summarizes a selection of studies reporting successful ex vivo expansion of NK cells for adoptive immunotherapy applications. Some of these NK cell-based products have already been used in the clinic and will be discussed in more detail.

Table 4. Ex vivo NK cell expansion
StudynStart materialMediumSerumFeeder cellsAdditionTime (days)SystemNK Fold expansionPurity (% NK)Cytotoxicity
  1. HS, human AB serum; FBS, foetal bovine serum; AP, autologous plasma; FFP, fresh-frozen plasma; aFor the first 24 h only; bfor the first five days only; cnot reported in the paper, this data was taken from reference [248].

Escudier [159] and Hercend [246]22 RCC patientsCD3 depleted nonadherent PBMCsDMEM8% HSLAZ 388200 U mL−1 IL-2 2 mmol L−1l-glutamine 1 mmol L−1 sodium pyruvate 0.2% NaOH 100 U mL−1 penicillin 0.1 mg mL−1 streptomycin13–21V-bottom microplates55>90Cytotoxic against Daudi
Miller [247]4 donorsCD5 and CD8 depleted PBSCsRPMI-164010% HSNo1000 U mL−1 IL-2 2 mmol L−1l-glutamine 1000 U mL−1 penicillin 100 U mL−1 streptomycin21Polystyrene Cell Factories Teflon bags Polyelofin bags33c 21c 12c88Cytotoxic against K562 and Raji
Lister [162]11 lymphoma 1 BrCa patientsAdherent activated NK cellsRPMI-164010% HSAllogeneic MNC6000 U mL−1 IL-214–18Flasks3185Cytotoxic against Daudi
Pierson [248]7 donorsCD5 and CD8 depleted PBMCs2 : 1 DMEM : Ham’s F12-based NK medium10% HSNo1000 U mL−1 IL-2 20 μmol L−1 2-mercaptoethanol 50 μmol L−1 ethanolamine 20 mg mL−1 L-ascorbic acid 5 μg L−1 sodium selenite 100 U mL−1 penicillin 33Stirred-tank bioreactor (n = 1) Spinner flasks (n = 3) 24-well plates (n = 3)352 120 5196 96 95Cytotoxic against K562 and Raji
Carlens [249]7 donorsTotal PBMCsCellGro SCGM5% HSNo500 U mL−1 IL-2 10 ng mL−1 OKT3b21Flasks∼70055Cytotoxic against K562
Luhm [165]37 donorsNK cells purified with MACS NK cell kitX-VIVO 20NoAllogeneic MNC100 U mL−1 IL-2 10 U mL−1 IL-15 100 μg mL−1 PHAa 1 μmol mL−1 ionomycina14–21Teflon bags10092Cytotoxic against Daudi, U266, NCI cell lines but not K562
Torelli [97]13 AML patientsNonadherent PBMCsRPMI-164010% FBSRPMI 886650 U mL−1 IL-210–1224-well plates3580Cytotoxic against allogeneic and autologous AML blasts
Guven [250]6 B-CLL patientsTotal PBMCsCellGro SCGM5% HSNo500 U mL−1 IL-2 10 ng mL−1 OKT3b21Flasks24374Cytotoxic against K562
Klingemann [251]5 donorsCD56 enriched PBMCsX-VIVO 1010% HSNo200 mmol L−1l-glutamine 500 U mL−1 IL-2 10 ng mL−1 IL-1514NR5–20NRCytotoxic against K562 and Raji
Ishikawa [160]9 glioma patientsTotal PBMCsRHAMα5% APHFWT200 U mL−1 IL-26–724-well platesNR86Cytotoxic against HFWT
Koehl [244]15 donorsSee Table 3X-VIVO 105% FFPNo1000 I mL−1 IL-210–14Flasks, Teflon bags5NRCytotoxic against K562 and allogeneic primary leukaemia cells
Torelli [109]26 ALL patientsNonadherent PBMCsRPMI-164010% FBSRPMI 886650 U mL−1 IL-210–1224-well plates35–4590Cytotoxic against autologou ALL blasts
Alici [94]7 MM patientsTotal PBMCsCellGro SCGM5% HSNo500 U mL−1 IL-2 10 ng mL−1 OKT3b21Flasks162565Cytotoxic against K562 and autologous MM cells

Adoptive transfer of autologous NK cells. Adoptive transfer studies with NK cells have proven to be efficient in various animal models. Basse et al. have published a detailed protocol about the evaluation of cancer immunotherapy with IL-2 activated NK cells in mice [156]. Evans et al. have reported that daily administration of IL-15 to cyclophosphamide injected C57BL/6J mice bearing rhabdomyosarcoma prolongs remission and enhances the efficacy of NK cell adoptive immunotherapy [157]. Furthermore, our group has reported successful NK cell adoptive immunotherapy of multiple myeloma in a syngeneic immunocompetent mouse model [77]. Additionally, it has also been demonstrated that activated NK cells from AML patients are cytotoxic against autologous AML blasts in vivo in a NOD/SCID model [158]. Supported by the achievements of adoptively transferred NK cells in experimental tumour models, various groups have evaluated the adoptive transfer of autologous NK cells for cancer immunotherapy in the clinical setting. Escudier et al. have demonstrated that the infusion of autologous NK cells that have been expanded and activated ex vivo for 13–21 days, greatly improves clinical responses in patients with metastatic RCC who have previously achieved partial remission through IL-2 infusions [159]. Ishikawa et al. have treated nine patients with malignant glioma using IFN-β infusions along with autologous NK enriched effector cells and reported that the infusion of these cells were safe and partially effective [160]. In a similar fashion, deMagalhaes-Silverman et al. have treated five patients with breast cancer using autologous NK cells cultured ex vivo for 14 days along with IL-2 infusions right after ASCT. Infusions were well tolerated without any adverse effects [161].

Lister et al. have treated a breast cancer and 11 lymphoma patients with autologous NK cells that were cultured ex vivo for 14–18 days [162]. The treatment was reported to be feasible and well tolerated. However, although an early in vivo amplification of NK cell activity was observed, no clinical benefit was observed. Burns et al. have also followed an NK cell-based approach to treat 34 patients with lymphoma or breast cancer [89]. Prior to aphaeresis, patients were treated for 4–6 weeks with s.c. IL-2, which resulted in a significant increase in total WBC count along with a more than 10-fold increase in circulating CD56bright NK cells. After 16 h, ex vivo activation of aphaeresis material, a cell product of mean 45% NK cells, was infused back to patients. Although the authors report markedly increased cytotoxicity against Raji and MCF-7 targets after IL-2 treatment or ex vivo IL-2 activation, no improvement in survival or relapse was observed when compared with matched controls. In another trial by the same group (43 patients with metastatic breast cancer), addition of IL-2 to G-CSF for PBSC mobilization resulted in an increased number of NK cells and activated T cells in the PBSC product with an increased cytotoxic activity against the breast cancer cell line MCF-7. Yet again, no significant impact on engraftment time or efficacy was observed upon the infusion of these grafts [163]. Krause et al. have reported treatment of a lung cancer and 11 CRC patients using NK cells stimulated ex vivo with Hsp70 peptides along with low-dose IL-2 infusions [164]. The treatment was well tolerated and one patient showed stable disease during therapy.

As discussed earlier, tumourigenesis imposes various defects on the immune system. To use autologous NK cells effectively for tumour immunotherapy, a reversal of phenotypic and functional defects is of paramount importance. Unfortunately, most of the earlier clinical studies did not include a detailed phenotypic and functional characterization of NK cells at the time of harvest or before infusion to the patient. Future studies should provide better insights into the characterization of the defects in the NK cell compartment and provide answers to whether these defects have been overcome after ex vivo manipulation of the cells. Although testing NK cell cytotoxicity against cell lines and allogeneic targets gives an estimation about the functional capacity of the cells, the analysis of cytotoxicity against fresh autologous tumour cells, if available, may be more predictive.

Based on the clinical data mentioned above, it is evident that overnight or short-term activation of NK cells is not sufficient for phenotypic and functional recovery. The data on infusion of autologous NK cells presented above reveal that it may be more likely to see a clinical benefit with long-term ex vivo activated NK cells. Corroborating this hypothesis, we have observed that long-term expansion and activation of autologous NK cells from MM patients provide significantly superior cytotoxic activity against autologous tumour cells when compared to short-term activated autologous NK cells [94]. Moreover, the high level of IL-2Rα expression in long-term expanded NK cells [165, 166] (our unpublished observations), as compared with endogenous CD56dim or CD56bright NK cells and short-term activated NK cells, may provide a higher benefit from subsequent IL-2 administration to the patients.

Adoptive transfer of allogeneic NK cells. The use of allogeneic NK cells is tentatively alluring, given the current comprehensions of NK cell regulation. A provisional prerequisite for NK cell alloreactivity is that the recipient lacks one or more KIR ligands present in the donor. The donor may in such situations have NK cells that express inhibitory KIR for which there is no ligand on recipient cells. Therefore, a KIR ligand-mismatched donor is likely to give the best chances for clinical response [167–169]. In certain donor-recipient combinations, chances for missing-self may prevail, providing better possibilities for anti-tumour reactivity. Within the setting of NK cell-based immunotherapy, the KIR-ligand mismatch phenomenon has attracted great attention [170] after the ground-breaking retrospective analysis of haplotype mismatched haematopoietic stem cell transplants by Ruggeri et al. revealed delayed relapse, better engraftment and protection from GvHD in leukaemia patients [168]. Later studies have shown that NK cells from healthy donors and cancer patients show higher cytotoxic activity against various KIR-ligand mismatched tumour cell lines when compared with KIR-ligand matched targets [171]. On the other hand, different prospective studies have suggested that development of NK cells after haploidentical transplantation is hampered and these cells have phenotypic and functional deficiencies [172, 173].

In 2005, Miller et al. have reported the successful adoptive transfer and in vivo expansion of haploidentical NK cells in patients with cancer [169]. A total of 43 patients with advanced cancer (10 with metastatic melanoma, 13 with metastatic renal cell carcinoma, 1 with refractory Hodgkin disease and 19 with poor-prognosis AML) were given haploidentical NK cell infusions together with IL-2 in a nontransplantation setting to determine safety and in vivo NK cell expansion. In general, donor NK cell infusions were well tolerated without evidence for induction of GvHD. Moreover, with this protocol, five out of 19 AML patients with poor prognosis achieved complete remission. Only four of the 19 patients had donors with a predictable alloreactive NK cell repertoire, that is, they were KIR ligand-mismatched in the GVH direction. Interestingly, out of these four patients, three achieved complete remission.

Ren et al. have treated 11 patients with refractory metastatic chemotherapy resistant solid tumours using IL-2 activated haploidentical PBSCs. PBSCs were activated for 4 h ex vivo prior to infusion. Activated PBSC grafts had lower CD3+ cells and higher NK cells (9% compared to 30%) and had increased cytotoxicity against tumour cell lines. Six of 11 patients achieved a relief of symptoms. Total response rate (complete, partial, mild remission or stable disease) was 72.7%(8/11) [174]. Koehl et al. have infused haploidentical NK cells to paediatric patients (2 ALL, 1 AML) up to a dose of 34 × 106 cells kg−1 with no side effects or GvHD [175]. Passweg et al. infused NK cells as DLI following allo-SCT to five patients (4 AML, 1 CML) up to 14 × 106 cells kg−1. Infusion was well tolerated and no GvHD was observed. Two patients (one receiving an additional stem cell dose after NK-DLI) showed improved donor chimerism, one patient showed stable donor chimerism, two showed decreasing donor chimerism [176]. Our group has also evaluated the infusion of long-term ex vivo expanded allogeneic NK cells following allogeneic SCT to cancer patients (submitted). The patients (1 CRC, 1 HCC, 2 RCC, 1 B-CLL) included in this pilot trial had undergone allogeneic SCT and had progressive disease following allogeneic SCT. NK cells were expanded from PBMCs that were obtained from individuals who had earlier donated allogeneic stem cells grafted to the patients. Cell infusions were safe, whether administered alone or with IL-2 s.c. No severe side effects or signs of acute GvHD were observed. Furthermore, one patient with HCC showed markedly decreased serum alpha-feto protein levels following cell infusions.

In an interesting recent study by Shi et al., 10 patients with MM were treated with haploidentical NK cells before ASCT. Patients were given overnight activated donor derived NK cells a day after conditioning twice in 48 h intervals, were further supplemented with IL-2 infusions for 11 days and received a delayed autograft at day 14. The allogeneic NK cells persisted in the periphery of the patient reaching maximum at around 7 days and eventually fading away until they were undetectable by day 14. The increasing donor chimerism up to day 7 suggests a confirmation for Miller et al.’s observation of in vivo expansion. The treatment was well tolerated with no signs of GvHD or rejection of the autograft. Encouragingly, a complete remission rate of 50% was reported. Although the allogeneic NK cells were not detectable after day 14, the efficiency of this approach may suggest that even without long-term engraftment, a clinical benefit for the patients can be observed. This brings the question about the in vivo fate of adoptively transferred allogeneic NK cells. Brand et al. have conducted a study where the allogeneic NK cells are followed by using PCR and radioactive labelling. Donor NK cells were cultured ex vivo for 15 days resulting in an expansion of 80-to-100 fold and were then infused to six end-stage patients with metastatic renal cell carcinoma. Infused cells could be detected in circulation by PCR up to 3 days. NK cells radiolabelled by 111In were followed by scintigraphy and were detectable up to 6 days. A distribution to the whole body, with preference for liver, spleen and bone marrow, was observed after a short initial uptake in the lungs. A total of 2/4 evaluable metastases showed a clear accumulation of transfused NK cells. The half-life corrected activity in all body compartments remained almost constant over the 6-day observation period in concordance with the absence of any excretion of radioactivity suggesting the lack of immune destruction by the host cells [177].

Another approach in NK cell-based tumour immunotherapy is the use of the cell line NK-92 [178], which can be consistently grown under GMP conditions. This cell line does not express any KIR but still has a broad spectrum of activating receptors [179]. Following preclinical mouse studies [180, 181] and ex vivo applications such as purging of leukaemia, lymphoma and CML [182, 183], NK-92 cell line has also been used as direct infusions to patients [184, 185]. Data from these trials suggest that infusion of NK-92 may be safe and potentially beneficial.

Genetic modification of NK cells for cancer immunotherapy

Gene transfer into NK cells may open new possibilities for the immunotherapy of cancer in both autologous and allogeneic settings. Applications of genetic modification could include various approaches from induction of proliferation/survival via cytokine gene therapy to specific targeting of NK cells to certain tissues or malignant cells (see Fig. 3). Such investigations have primarily began from proof-of-principle studies that resulted in the optimization of NK cell genetic modification via various methods including electroporation [186, 187], nucleofection [188, 189], transduction by chimeric adenoviral [190], chimeric EBV/retroviral [191], retroviral [192, 193] and lentiviral [194] vectors.

image

Figure 3.  Genetic manipulation approaches for modulating NK cell–tumour cell interactions. (a) Overexpression of activating receptors by genetic modification can increase tumour cell killing efficiency provided that the tumour is expressing the specific ligand for that receptor. (b) Silencing of inhibitory receptor expression by RNA interference can lead to increased tumour cell killing efficiency especially when the tumour is known to express high levels of inhibitory ligands. (c) Retargeting NK cells by the use of a chimeric receptor that binds a tumour-specific antigen and delivers intracellular signals for the activation of NK cell cytotoxicity.

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Liu et al. have reported transfection of CD18 gene into a clone of the NK cell line YT-1 that lacks functional CD18 expression. They have demonstrated that upon genetic modification, the cell line restores its cytotoxic capacity against a B-cell lymphoma line [195]. It has also been shown that the delivery of IL-15 gene to NK cell lines increases proliferative rate and cytotoxic capacity [196, 197]. Likewise, delivery of the IL-12 gene to mouse NK cells have increased their survival capacity and in vivo anti-tumour activity [198].

Systemic IL-2 administration frequently causes undesirable side effects [199, 200], e.g. the activation of other immune cell populations. More specifically, activated T cells increase the chance of GvHD [201], whilst the stimulation of immunosuppressive Treg cells is suboptimal for cancer patients [202]. In settings where IL-2 is given primarily to enhance NK activity, administration in a form that stimulates NK cells, without unwanted side effects, would be ideal. There have been various reports on IL-2 gene delivery via retroviral transduction [203] or particle-mediated [204] transfection to the IL-2 dependent NK cell line NK-92. Stable transduction of the IL-2 gene increased cytotoxic activity against tumour cell lines in vitro. Such a modification enabled the secretion of IL-2 by the NK92 cells and saved the cells from the dependency on exogenous IL-2 supplementation. Moreover, the IL-2 transduced cells showed greater in vivo antitumour activity in mice [203]. Similarly, Miller et al. have reported that IL-2 transduced mouse NK cells sustained proliferation in the absence of exogenously supplied IL-2 [205]. However, the expression of IL-2 in a secreted manner by NK cells may affect neighbouring cells or have the potential to cause a systemic IL-2 effect in patients. This risk prompted us to continue investigation to seek alternative approaches for IL-2 delivery retained in NK cells in a controlled and localized manner. Our group has constructed an endoplasmic reticulum-retained IL-2 gene that is not secreted but still confines autocrine growth stimulation to NK-92 cells [206]. Such an approach may be useful for future applications where secretion of high levels of IL-2 by the adoptively transferred NK cells might cause side effects.

Another approach to genetic modification of NK cells for cancer immunotherapy is retargeting of the NK cells to tumour cells via the expression of a chimeric antigen specific receptors. This is generally performed by using a single-chain variable fragment receptor specific for a certain tumour-associated antigen fused to the intracellular portion of the signalling molecule CD3ζ. Such receptors have been used by many different groups and have proven to be efficiently working in NK cells. Chimeric receptors against CEA [207], CD33 [208] and Her2/neu [209–211], have been successfully delivered to NK cell lines and were shown to increase antigen specific cytotoxic activity of NK cells both in vitro and in vivo.

These improvements have rapidly been translated to settings of primary NK cells and experimental models. Pegram et al. have gene modified primary mouse cells to express a chimeric receptor against Her2/neu and observed that the adoptive transfer of these cells to mice bearing Her2+ tumours inhibits tumour progression in vivo [212]. Likewise, Kruschinski et al. have modified primary NK cells from human donors to express a chimeric receptor against Her2/neu and observed high level of cytotoxic activity against Her2+ cell lines in both in vitro and xenograft models with RAG2−/− mice [213]. Moreover, Imai et al. have successfully demonstrated that NK cells from B-lineage ALL patients genetically modified to express a chimeric receptor against CD19 efficiently kill autologous leukaemic cells in vitro [214]. Taken together, these data indicate that the adoptive transfer of chimeric antigen-specific bearing NK cells might be an efficient approach in cancer immunotherapy.

Concluding remarks

  1. Top of page
  2. Abstract.
  3. Natural killer cells: a historical background
  4. NK cells in cancer
  5. Potential of NK cells in cancer immunotherapy
  6. Concluding remarks
  7. Conflict of interest statement
  8. Acknowledgements
  9. References

There is an escalating awareness on the importance of NK cells and their therapeutic potential in cancer and virus infections. The clinical importance of NK cell alloreactivity-mediated anti-leukaemic responses has been established in patients with AML by Ruggeri et al. and was subsequently demonstrated for other types of malignancies. In these studies, donor-versus-recipient NK cell alloreactivity reduced the risk of relapse whilst improving engraftment, with no incidence of GvHD. Unfortunately, the results of KIR-ligand incompatibility in recipients of unrelated donor HSCT are not unequivocal. This discrepancy remains unexplained, but might be related to differences in pretransplant conditioning regimens, the extent of HLA match, the features of the tumour, the stem cell graft or the immunosuppressive treatment regimen.

Preclinical and clinical data reviewed in this study clearly indicates that NK cell infusions in allogeneic and autologous settings are safe with possible anti-tumour effect. Nevertheless, further investigations are needed to fine-tune and select an optimal NK cell therapy scheme. Whilst all approaches, including endogenous modulation of NK cells and their adoptive transfer seem to be feasible and efficacious, it is evident that one scheme will not be suitable for all malignancies and therefore, all possible approaches need to be evaluated (see Fig. 4). Literature collected under this review seems to support that there is a NK cell dose dependent response in adoptive transfer settings. Besides, lessons from the monoclonal antibody-based targeted therapies clearly indicate that NK cells are one of the main effector populations exerting ADCC and thus clearing tumour. Nevertheless, current therapy may be limited by exhaustion of cellular cytotoxicity mediated by NK cells and tissue macrophages and therefore, the full potential of antibody-based immunotherapies may not yet be fully realized. Ineffective killing of antigen-positive cells may furthermore lead to substantial reductions in surface expression (i.e. shaving). Strategies to address these limitations include the development of immune modifying drugs that mobilize or replenish effector functions or supplementing with more cells to replenish the effector cell pool. Furthermore, several groups consider that immunotherapy with ex vivo expanded autologous NK cells or gene modified NK cells could potentially help these patients in raising an immune response against the tumour and increase the chances of a remission. However, there is still much more to learn about NK cells in terms of their development, differentiation, receptor acquisition, role of different subsets, ex vivo expansion kinetics, biodistribution after expansion and gene modification. One of the critical drawbacks in NK cell immunotherapy has been the lack of a large-scale clinical grade NK cell expansion method. Ability to expand such cells ex vivo presents a prospect to study their exact role, especially in cancers.

image

Figure 4.  Natural killer cell immunotherapy in cancer. The figure presents an overview of current and future approaches to NK cell-based immunotherapeutic strategies in the treatment of cancer. A critical prerequisite for efficient NK cell-based immunotherapy seems to be the reduction of the tumour mass by surgical removal, chemotherapy or radiotherapy to give the effector cells a numerical advantage. The yellow shaded upper left panel represents the in vivo modulation of NK cell activity against tumour via (a) stimulation with cytokines and/or (b) infusion of tumour-specific monoclonal antibodies to trigger an ADCC response. The green shaded lower left panel and the gray shaded right panel present approaches for adoptive transfer of autologous or allogeneic NK cells respectively. Autologous or donor NK cells can be transferred after (c & h) ex vivo short-term activation, (d & g) ex vivo long-term activation and expansion or (e & f) genetic modification. Infusion of (I) purified unstimulated donor NK cells is also under investigation.

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Acknowledgements

  1. Top of page
  2. Abstract.
  3. Natural killer cells: a historical background
  4. NK cells in cancer
  5. Potential of NK cells in cancer immunotherapy
  6. Concluding remarks
  7. Conflict of interest statement
  8. Acknowledgements
  9. References

We would like to thank Prof. Gösta Gahrton for his critical reading of this manuscript. The research in our lab is supported by Swedish pediatric cancer foundation and Clinigene NoE. Tolga Sutlu is supported by the International PhD Fellowship of the Scientific and Technological Research Council of Turkey.

References

  1. Top of page
  2. Abstract.
  3. Natural killer cells: a historical background
  4. NK cells in cancer
  5. Potential of NK cells in cancer immunotherapy
  6. Concluding remarks
  7. Conflict of interest statement
  8. Acknowledgements
  9. References
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