Review |
`
`Natural killer cell-based immunotherapy in cancer: current
`insights and future prospects
`
`doi: 10.1111/j.1365-2796.2009.02121.x
`
`T. Sutlu & E. Alici
`
`From the Division of Haematology, Department of Medicine, Karolinska University Hospital Huddinge, Karolinska Institutet,
`Stockholm, Sweden
`
`Abstract. Sutlu T, Alici E (Karolinska Institutet, Stock-
`holm, Sweden). Natural killer cell-based immunother-
`apy in cancer:
`current insights and future prospects
`(Review). J Intern Med 2009; 266: 154–181.
`
`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
`in vivo
`investigated clinical applications
`such as
`modulation of NK cell activity, ex vivo purifica-
`tion ⁄ expansion and adoptive transfer as well as
`future possibilities such as genetic modifications are
`discussed in detail.
`
`Keywords: cancer immunotherapy, clinical trials, cyto-
`kines, ex vivo expansion, gene therapy, natural killer
`cells.
`
`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 anti-
`gen; 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 cytotoxic-
`ity receptor; PBMC, peripheral blood mononuclear cell;
`PBSC, peripheral blood stem cell; PHA, phytohaemag-
`glutinin; PR, partial remission; RCC, renal cell carci-
`noma; ROS, reactive oxygen species; SCID, severe
`combined immunodeficiency; SCT, stem cell transplan-
`tation; 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
`
`in
`Initially regarded as an ‘experimental artifact’
`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, FccR+,
`large granular
`lymphocytes (LGL) [5]. Later it was, however, appre-
`ciated that NK cells not only shared the LGL pheno-
`type and some NK cells also displayed normal small
`lymphocyte morphology, depending on their activa-
`tion 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
`
`154 ª 2009 Blackwell Publishing Ltd
`
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`T. Sutlu & E. Alici |
`
`Review: Natural killer cell-based immunotherapy in cancer
`
`it possible to define the murine NK cells roughly as
`)
`)
`NK1.1+ TCR
`CD16+. Today, human NK cells
`sIg
`)
`CD56+ lymphocytes. They com-
`are defined as CD3
`prise 10–15% of all circulating lymphocytes and are
`also found in peripheral tissues, including the liver,
`peritoneal cavity and placenta. Resting NK cells cir-
`culate in the blood, but following activation by cyto-
`kines,
`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 dif-
`fering 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 accor-
`dance 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 Ka¨rre 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 com-
`monly missing. This lead him to formulate the miss-
`ing-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).
`the missing-self hypothesis, a MHC
`To further test
`
`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 lack-
`ing tumour growth.
`
`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
`the CD56dim cells are more
`subsets revealed that
`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 a chain
`(IL-2Ra ⁄ CD25), whilst
`they lack or express only
`at very low levels the FCcRIII
`(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 cellu-
`lar cytotoxicity (ADCC) and response to IL-2 stim-
`ulation.
`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
`the CD56dim NK cell appears to produce
`contrast,
`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 lympho-
`cytes 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.
`
`ª 2009 Blackwell Publishing Ltd Journal of Internal Medicine 266; 154–181 155
`
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`T. Sutlu & E. Alici |
`
`NK
`cell
`
`Tumour
`cell
`
`Review: Natural killer cell-based immunotherapy in cancer
`
`(a)
`
`(b)
`
`(c)
`
`(d)
`
`Inhibitory
`receptor
`
`Inhibitory
`ligand
`
`Activating
`receptor
`
`Activating
`ligand
`
`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-c, TNF-a,
`GM-CSF and IL-3 [17]. Therefore, NK cells are also
`
`Fig. 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 recog-
`nizes the inhibitory ligands and, therefore, no killing takes
`place. (c) The tumour cell has significantly downregulated
`or absent expression of inhibitory ligands along with suffi-
`cient expression of activating ligands. Missing-self recogni-
`tion 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.
`
`believed to function as regulatory cells in the immune
`system, influencing other cells and responses and act-
`ing as a link between the adaptive and innate immune
`responses. For example, NK cells seem to participate
`in the development of the autoimmune disease, myas-
`thenia gravis, by regulating both the autoreactive T and
`B cells through IFN-c production [18]. Moreover, it
`has been observed that depletion of NK cells in
`C57Bl ⁄ 6 mice leads to increased engraftment of neuro-
`blastoma (NB) xenografts mainly because of dysregu-
`lation of Th1-oriented B-cell responses [19]. These
`data prove the significant impact of NK cells on adap-
`tive 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 recipro-
`cally, cytokine-preactivated NK cells have been shown
`to activate DCs and induce their maturation and cyto-
`kine 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 acti-
`vating receptors [28]. Table 1 provides a list of
`human NK cell activating and inhibitory receptors
`identified to our knowledge. Upon recognition of the
`
`156 ª 2009 Blackwell Publishing Ltd Journal of Internal Medicine 266; 154–181
`
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`

`T. Sutlu & E. Alici |
`
`Review: Natural killer cell-based immunotherapy in cancer
`
`ligands on the target cell surface by activating NK
`cell
`receptors, various intracellular signalling path-
`ways drive NK cells towards cytotoxic action and this
`results in target cell cytolysis [29].
`
`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 spe-
`cific 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 infec-
`tions) can be detected by a pool of NK cells [33, 34].
`
`The activating side of the balance also includes a ser-
`ies of different receptors (see Table 1). The main acti-
`vating receptor group is called natural cytotoxicity
`receptors (NCRs) [29] and it is believed that the main
`control over the NK cell activating pathways is regu-
`lated by these receptors. Currently, there are three dif-
`ferent 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 immu-
`noreceptor
`tyrosine-based activation motifs (ITAM)
`that serve as the signal transducing proteins. NKp30
`and NKp46 are associated with CD3f, 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 gran-
`ular lymphocytes’ and their granularity is their means
`for target cell killing (see Fig. 2). These granules con-
`tain 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, perfo-
`rin and granzyme B are released; granzyme enters the
`target cell and mediates apoptosis, whilst perforin dis-
`rupts 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-a has also been suggested to mediate acti-
`vation-induced cell death by NK cells [46].
`
`NK cells in cancer
`
`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 immuno-
`suppressive 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.
`
`ª 2009 Blackwell Publishing Ltd Journal of Internal Medicine 266; 154–181 157
`
` 13652796, 2009, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2796.2009.02121.x, Wiley Online Library on [08/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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`

`T. Sutlu & E. Alici |
`
`Review: Natural killer cell-based immunotherapy in cancer
`
`Table 1 Activating and inhibitory receptors on human NK cells
`
`Alternative name
`
`Type of signal
`
`Ligand
`
`Distribution on NK cells
`
`CD
`
`CD2
`
`CD7
`
`CD11a
`
`CD11b
`
`CD16
`
`CD27
`
`CD44
`
`CD59
`
`CD69
`
`CD85j
`CD94 ⁄ CD159a
`CD94 ⁄ CD159c
`CD96
`
`CD160
`
`CD161
`
`BY55
`
`NKR-P1
`
`LFA-2
`
`LEU-9
`
`LFA-1
`
`Mac-1
`FccRIII
`
`Activation
`
`Activation
`
`Activation
`
`Activation
`
`Activation
`
`TNFRSF7
`
`?
`
`Hyalunorate receptor
`
`Protectin
`
`CLEC2C
`
`ILT-2
`CD94 ⁄ NKG2A
`CD94 ⁄ NKG2C
`TACTILE
`
`Activation
`
`Activation
`
`Activation
`
`Inhibition
`
`Inhibition
`
`Activation
`
`Activation
`
`Activation
`Activation ⁄
`Inhibition
`
`CD58 (LFA-3)
`
`SECTM1, Galectin
`
`ICAM-1,-2,-3,-4,-5
`
`ICAM-1, Fibrinogen
`
`IgG
`
`CD70
`
`Hyalouronan
`
`C8, C9
`
`Unkown
`
`HLA-A, -B, -G
`
`HLA-E
`
`HLA-E
`
`CD155
`
`HLA-C
`
`LLT1
`
`All
`
`All
`
`All
`
`All
`Mainly CD56dim
`Negative ⁄ dim on CD56bright
`Mainly on CD56bright
`Negative ⁄ dim on CD56dim
`All
`
`All
`
`Activated
`
`Subset
`
`Most
`
`Most
`
`Activated low expression
`
`on resting
`
`All
`
`Subset
`
`Activated
`
`CD223
`
`CD226
`
`CD244
`
`CD314
`
`CD319
`
`CD328
`
`CD329
`
`CD335
`
`CD336
`
`CD337
`
`Various
`
`Various
`
`–
`
`–
`
`Lag3
`
`DNAM-1
`
`2B4
`
`NKG2D
`
`CRACC
`
`Siglec-7
`
`Siglec-9
`
`NKp46
`
`NKp44
`
`NKp30
`
`KIR2DS, KIR3DS
`
`KIR2DL, KIR3DL
`
`NTB-A
`
`KLRG1
`
`Activation
`
`Activation
`Activation ⁄
`Inhibition
`
`Activation
`
`Activation
`
`Inhibition
`
`Inhibition
`
`Activation
`
`Activation
`
`Activation
`
`Activation
`
`Inhibition
`
`Activation
`
`Inhibition
`
`HLA Class II
`
`CD112, CD155
`
`CD48
`
`MICA, MICB,
`
`ULB-1,-2,-3,-4
`
`CRACC
`
`Sialic acid
`
`Sialic acid
`
`Viral haemagglutinin (?)
`
`All
`
`All
`
`All
`
`Mature NK cells
`
`Subset
`
`Subset
`
`All
`
`Viral haemagglutinin (?)
`
`Activated
`
`Viral haemagglutinin (?)
`
`HLA Class I
`
`HLA Class I
`
`NTB-A
`
`E-,N-,P-cadherin
`
`All
`
`Subsets
`
`Subsets
`
`All
`
`All
`
`Potential of NK cells in cancer immunotherapy
`
`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 start-
`ing in 1965 for the soluble factors in lymphocyte con-
`
`ditioned 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 a
`
`158 ª 2009 Blackwell Publishing Ltd Journal of Internal Medicine 266; 154–181
`
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`

`T. Sutlu & E. Alici |
`
`Review: Natural killer cell-based immunotherapy in cancer
`
`Granule-dependent
`killing
`
`Killing by
`death ligands
`
`(b)
`
`(e)
`
`(d)
`
`(c)
`
`(a)
`
`Activating
`receptor
`
`Activating
`ligand
`
`CD16
`
`Antibody
`
`Cell surface
`antigen
`
`Cytotoxic
`granules
`
`Fas
`
`FasL
`
`TRAIL
`receptor
`
`TRAIL
`
`TNF-α
`
`TNF-αα
`receptor
`
`Fig. 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 apopto-
`sis 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-a.
`
`(CD25), b (CD122) and c (CD132) chains. The c
`chain is shared amongst various interleukin receptors
`(IL-4, IL-7, IL-9, IL-15, IL-21), thus named the com-
`mon c chain and it is essential for lymphoid develop-
`ment [56]. The b chain is shared between IL-2 and
`IL-15 receptors [57, 58]. The b and~c chains come
`to form the intermediate affinity IL-2 ⁄ 15
`together
`receptor. The distinction between the high affinity
`receptors for IL-2 and IL-15 comes with the a chains.
`The IL-2Ra 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 a chain forms a complex
`with the b and c chains, the high affinity IL-2 receptor
`is formed. The co-expression of all three chains is con-
`fined 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 acti-
`vation in vitro enhances the tumour killing potential of
`NK cells [62, 63]. Early reports of IL-2-based treat-
`ment on animal models have established a solid basis
`for efficiency of this approach for cancer immunother-
`apy in many different settings [64–72]. Although cyto-
`toxic T cells have been the primary point of interest,
`especially during the early phases of IL-2 use, the an-
`titumour 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 mul-
`tiple myeloma (MM) that NK cells are the primary
`mediators of IL-2 induced tumour rejection [77].
`
`In the clinical setting, the pioneering work of Rosen-
`berg et al.
`[78, 79], which has demonstrated the
`
`ª 2009 Blackwell Publishing Ltd Journal of Internal Medicine 266; 154–181 159
`
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`T. Sutlu & E. Alici |
`
`Review: Natural killer cell-based immunotherapy in cancer
`
`Table 2 NK cell abnormalities in cancer patients
`
`Abnormality
`
`Disease
`
`Decreased cytotoxic
`
`Nonsmall cell lung cancer [215]
`
`activity of NK cells
`
`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
`
`Hepatocellular carcinoma [216]
`
`of activating receptors
`
`Metastatic melanoma [229]
`
`AML [230]
`
`MM [95, 231]
`
`Defective NK cell
`
`Metastatic renal cell carcinoma [232]
`
`proliferation
`
`Nasopharyngeal cancer [233]
`
`Increased number of
`CD56bright NK cells
`Defective expression
`
`CML [234]
`
`Head and neck cancer [219]
`
`Breast cancer [219]
`
`Cervical cancer [235]
`
`of intracellular
`
`Colorectal cancer [236]
`
`signalling molecules
`
`Ovarian cancer [237]
`
`Prostate cancer [238]
`
`AML [239]
`
`CML [239]
`
`Decreased NK cell
`
`Nasopharyngeal cancer [233]
`
`counts
`
`Increased NK cell
`
`counts
`
`CML [234]
`
`MM [228]
`
`Defective cytokine
`
`AML [224]
`
`production
`
`ALL [224, 225]
`
`CML [240]
`
`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 can-
`cer 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 immu-
`notherapy. Other investigators have shown that IL-2
`administration stimulates the ERK signalling pathway
`in especially CD56bright NK cells and CD14+ mono-
`cytes, 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 treat-
`ment 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 dem-
`onstrated 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 preven-
`tion 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 chemo-
`therapy 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
`
`160 ª 2009 Blackwell Publishing Ltd Journal of Internal Medicine 266; 154–181
`
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`
`UPenn Ex. 2064
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`T. Sutlu & E. Alici |
`
`Review: Natural killer cell-based immunotherapy in cancer
`
`cancer patients with daily s.c. IL-2 infusions supple-
`mented with two i.v. bolus IL-2 infusions. As antici-
`pated, 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 treat-
`ment 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]. Like-
`wise, 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 remis-
`
`sion 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 progeni-
`tors 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 opti-
`mal strategy for most indications. In most cases of
`low-dose IL-2 administration (picomolar serum con-
`centrations), 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-2Ra that confers high
`affinity for IL-2 is uniquely expressed by CD56bright
`cells [100], which could explain their selective expan-
`sion in response to low-dose IL-2. Although such
`treatment has proven to be safe, there is yet no con-
`vincing proof of efficacy. Likewise, the in vivo expan-
`IL-2Ra expressing regulatory cell
`sion of another
`subset; Treg cells could also overwhelm and ⁄ or sup-
`press 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 immu-
`notherapy 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 b and c chains of IL-2
`
`ª 2009 Blackwell Publishing Ltd Journal of Internal Medicine 266; 154–181 161
`
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