infusions of allogeneic natural killer cells as cancer therapy · the proposal that the resistance...

Review

Infusions of Allogeneic Natural Killer Cells as CancerTherapy

Wing Leung1,2

AbstractNatural killer (NK) cells are normal white blood cells capable of killing malignant cells without prior

sensitization. Allogeneic NK cell infusions are attractive for cancer therapy because of non–cross-resistant

mechanisms of action and minimal overlapping toxicities with standard cancer treatments. Although NK

therapy is promising, many obstacles will need to be overcome, including insufficient cell numbers, failure

of homing to tumor sites, effector dysfunction, exhaustion, and tumor cell evasion. Capitalizing on the

wealth of knowledge generated by recent NK cell biology studies and the advancements in biotechnology,

substantial progress has been made recently in improving therapeutic efficiency and reducing side effects.

A multipronged strategy is essential, including immunogenetic-based donor selection, refined NK cell

bioprocessing, andnovel augmentation techniques, to improveNK function and to reduce tumor resistance.

Although data from clinical trials are currently limited primarily to hematologic malignancies, broader

applications to a wide spectrum of adult and pediatric cancers are under way. The unique properties of

humanNKcells openup anewarenaof novel cell-based immunotherapy against cancers that are resistant to

contemporary therapies. Clin Cancer Res; 20(13); 3390–400. �2014 AACR.

Disclosure of Potential Conflicts of InterestW. Leung is a co-inventor of a patent-pending diagnostic method assigned to St. Jude Children’s Research Hospital and licensed to

Insight Genetics.

CME Staff Planners' DisclosuresThe members of the planning committee have no real or apparent conflict of interest to disclose.

Learning ObjectivesUpon completion of this activity, the participant should have a better understanding of the biology of human natural killer (NK) cells

relevant to cancer immunotherapy, the general steps of allogeneic NK-based treatments, and the avenues of current and future strategies

being explored in NK cell therapy.

Acknowledgment of Financial or Other SupportThis activity does not receive commercial support.

IntroductionFor many cancers, cure rates remain unacceptably low.

Advances in genetics and immunology have led to biologictherapies with non–cross-resistant mechanisms of actionand no overlapping toxicities. Cancer immunotherapy ispossible with an array of effector mechanisms involvinginnate and adaptive immunity (1). Various cytokines, anti-bodies, and cellular therapies have been FDA approved orare in late-phase clinical trials.

Infusions of purified natural killer (NK) cells are late-comers in cellular immunotherapy, compared with den-dritic cells (DC), lymphokine-activated killer (LAK) cells,tumor-infiltrating lymphocytes, and conventional cytotoxicT cells. Many investigators consider that the cytolytic activ-ity of LAK cells commonly used in the 1980s actuallyrepresents the activity of IL2-activated NK cells, althoughthe products contained polyclonal T cells. Biologically,murine NK cells have been known to kill malignant cellswithout T-cell assistance or prior sensitization since theirinitial description in 1975 (2–5); however, the therapeuticpotential of human NK cells was not revealed until theirbiology of target cell recognition was elucidated in the1990s and the antileukemia effect was observed in HLA-haploidentical hematopoietic cell transplantation (HCT) inthe early 2000s (6, 7). This review covers NK cell biologyrelevant to cancer immunotherapy and outlines avenuesbeing explored in clinical trials.

Author's Affiliations: 1Department of Bone Marrow Transplantation andCellular Therapy, St. Jude Children's Research Hospital; and 2Departmentof Pediatrics, University of Tennessee, Memphis, Tennessee

Corresponding Author: Wing Leung, St. Jude Children's Research Hos-pital, 262 Danny Thomas Place, Memphis, TN 38105-3678. Phone: 901-595-2617; Fax: 901-521-9005; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1766

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(13) July 1, 20143390

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NK Cell BiologyNK cells were first named and characterized by Kiessling

at the Karolinska Institute (Stockholm, Sweden) and byHerberman at the National Cancer Institute (Bethesda,MD; refs. 2–5). The term "natural" referred to their naturalexistence in rodents and "killer" to their spontaneouskilling of lymphoma and leukemia cells in nonimmuneanimals. They were called "N-cells" or "K cells" by someinvestigators (3, 8). These cells were unique, as they lackedmarkers characteristic of B and T lymphocytes and werenonphagocytic, low-adhesive, and cytotoxic to IgG-coatedtargets.NK cells are generated in bone marrow and develop

normally in athymic mice and in humans with DiGeorgesyndrome. Induction of cytotoxicity is possible in NKcells within minutes, in contrast with the slower conven-tional induction of the T-cell immune response (9).Approximately 5% to 15% of human blood lymphocytesare NK cells (CD56þ, CD3/14/19�), 90% of which areCD56dim, with most of them being CD16þ, and they areresponsible for early innate immunity and antibody-dependent cell-mediated cytotoxicity (ADCC) againstinfection or cancer through IFNg , perforin, granzyme,FasL, or TRAIL (10). Approximately 10% of human bloodNK cells are CD56bright, and they participate in late (>16hours) inflammatory response by secreting IFNg , TNFa,G-CSF, GM-CSF, and IL3. Interactions between DCs andNK cells are essential for priming adaptive immunity. NKcells may also directly interact with T and B cells throughCD40 ligand and OX40 (11, 12).

Regulatory receptorsHumanNK cells are regulated by a sophisticatednetwork

of surface receptors (Fig. 1), allowing them to distinguishnormal from abnormal cells within seconds. Target celllysis occurs when the activating signal dominates theinhibitory signal (Fig. 2). Early studies revealed similaritiesbetweenmurineNK cells and cells responsible for so-called"hybrid resistance" to parental hematopoietic grafts (13,14). The proposal that the resistance was related to recep-tors recognizing the absence of MHC class I arose fromobservations that murine lymphoma cells that were low inMHC expression were NK-susceptible (15). This led to the"missing-self" hypothesis in the 1980s (16). Break-throughs in human NK receptor biology were seen in theearly 1990s, when the KIR gene family was found torecognize HLA class I (17–19). Identification of other NKreceptors, such as NCRs, NKG2D, DNAM, 2B4, and CD94/NKG2A, collectively points to a sophisticated network thatcontrols human NK activity.

Step 1: KIR Typing for Donor Selection andPrognosticationThree general steps in NK-based therapy are summarized

in Fig. 3. KIR typing is a critical first step because KIR ishighly polymorphic. KIR typing is not only useful forallogeneic donor selection but also for prognostication in

autologous NK therapy (20). Among known NK receptors,the KIR receptor family is one of the primary determinantsof NK response. Subsets of T cells also expressed KIR (21).Clinical KIR typing includes genotyping for gene contentand A/B haplotype categorization, phenotyping for geneexpression and number of KIRþ cells, and allelotyping forfunctional strength.

The first level of KIR diversity is gene content (22, 23).Only approximately 5% of people have all 15 gene familymembers; others lack one or more genes. The diversity ofgene content is based primarily on diversity in B haplo-types, which typically contain more activating KIRs. Thetwo hallmark genes for A haplotypes are KIR2DL3 andKIR3DL1. They are segregated with KIR2DL2 and KIR3DS1as alleles in the centromeric (Cen) and telomeric (Tel)motifs, respectively (Fig. 4A). On the basis of this pattern, asimplified typing and scoring method for B haplotypecontent can be derived (Fig. 4B), viz., KIR2DL3þ/KIR2DL2�, Cen-A/A; KIR2DL3þ/KIR2DL2þ, Cen-A/B;KIR2DL3�/KIR2DL2þ, Cen-B/B; KIR3DL1þ/KIR3DS1�,Tel-A/A; KIR3DL1þ/KIR3DS1þ, Tel-A/B; and KIR3DL1�/KIR3DS1þ, Tel-B/B. Because KIRs are encoded in chromo-some 19 and ligandHLAs are in chromosome 6, these genefamilies are segregated independently; thus, HLA geno-types cannot be used to predict KIR content, and autolo-gous KIR-HLA mismatch is possible.

The second level of diversity is gene expression (24). Byreal-time PCR and flow cytometry, more than 10-foldvariability in expression has been observed among donors.Nowadays, the easiest method to quantify NK cells expres-sing only one inhibitory KIR gene (i.e., negative for allother MHC inhibitory receptors) is multicolor flow cyto-metry (24–26). The number of single KIRþ cells is in directproportion to activity against target cells without theligand (receptor–ligand mismatched). Although the num-ber of single KIRþ cells correlates with the presence of self-ligand (25), the variability among donors renders predic-tions based on the ligand alone unacceptable for clinicaluse.

The third level of diversity is allele polymorphism, whichhas been observed in all inhibitory KIR genes (22, 23). Forexample, in the KIR2DL1 family, 25 alleles have beenobserved, and each has different strengths in inhibiting NKcells and different durability of surface expression afterinteraction with ligands (27). These differences correlatewith the intensity of inhibitory signaling through SHP2 andb arrestin-2, resulting in differences in immune synapseformation. Mechanistically, the arginine residue at position245 in the transmembrane domain is important in inhibi-tion. Alleles with arginine in position 245 are stronger thanthosewith cysteine in that position (27).On the basis of thismolecular determinant, single-nucleotide polymorphismassays have been developed for rapid, high-throughputclinical typing (28). The same assay can be simultaneouslyused for KIR ligand typing. Notably, this assay requires lessthan 0.1 mg of DNA and can be performed within 1 day.Using this assay, investigations showed that patients whoreceived a donor graft containing the stronger KIR allele had

NK Infusions as Cancer Therapy

www.aacrjournals.org Clin Cancer Res; 20(13) July 1, 2014 3391



fewer relapses, better survival, and less transplant-relatedmortality (29). These effects were observed regardless ofprimary disease [acute myelogenous leukemia (AML) vs.

acute lymphoblastic leukemia (ALL)], total body irradia-tion, T-cell depletion, and donor type. Statistically, there isan interaction effectwithHLA-C receptor–ligandmismatch:

© 2014 American Association for Cancer Research

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2B4 (CD48)

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KIR2DS1/S2 (HLA-C)

IFNα

NCRs (B7-H6, NKp44L)

DNAM-1 (PVR, Nectin-2)

Figure 1. Surface receptors andtheir ligands. Cytokine receptorsare shown on top of a human NKcell. Other receptors are broadlyclassified and color-coded on thebasis of their primary function(inhibitory receptors in red,activating receptors in green,inhibitory coreceptors in red-blackstripes, and activating coreceptorsin green-black stripes). Theirligands are shown withinparentheses. Many other knownreceptors are not shown, includingchemotactic receptors (CCR-2, -5,-7: CXCR-1, -3, -4, -6; CX3CR1;and Chem23R), adhesionreceptors (CD2 and b1 and b2integrins), and activatingcoreceptors (CD96,CS1, and TLR).


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Figure 2. Dynamic equilibrium. Aftercell-to-cell contact, NK cellintegrates signals from its surfacereceptors in seconds, resulting ineither target-cell attack or noresponse and continualimmunosurveillance of other cells.A, healthy cells express normalamount of MHC class I ligands withno activating "stress" ligands. B,downregulation or absence ofMHCligand for cognate inhibitoryreceptors is insufficient to triggerNK cells. This happens in thephysiologic setting with red bloodcells and autologous KIR receptor–ligand mismatch cells, and inpathologic conditions with adultlymphoblastic leukemia. C,sufficient activating ligandsmust beexpressed on target cells to induceNK cell activity. D, if self-MHCligands are expressed in normalamount, the reactivity of the NK cellis ultimately dependent on thebalance of activating and inhibitorysignals. Successful NK cell therapyrelies on clinical strategies thatoptimize activation and avoidinhibition by cancer cells.

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Clin Cancer Res; 20(13) July 1, 2014 Clinical Cancer Research3392



Patients with the best survival were those with a strongerKIR allele and receptor–ligand mismatch from donors. Thesecond-best group was those with a stronger KIR allele butno receptor–ligand mismatch. The worst survival was inthose with the weaker allele, regardless of mismatch.Donor KIR typing is not necessary in some KIRmismatch

models (Fig. 4C). The first model was proposed by thePerugia group (7); the ligand mismatch model requirestyping HLA in both donor and recipient. A donor is mis-matched if a ligand is present in the donor but absent in therecipient. This ligand mismatch model does not requiredonor or recipient KIR typing. The second model wasproposed by the Nantes group (30); in this model, KIR istyped in both donor and recipient. If a receptor is present in

the donor but absent in the recipient, the donor is mis-matched. Because B haplotypes contain more genes than Ahaplotypes, mismatch typically signifies a B haplotype inthe donor. The thirdmodel is the receptor–ligandmismatchmodel proposed by the Memphis group (31). This modelrequires donor KIR typing and recipient HLA typing.A donor is mismatched if an inhibitory receptor is presentin the donor but the ligand is absent in the recipient.Notably, the receptor–ligand mismatch model is the onlymodel applicable to autologous transplantation and anti-body therapy; studies have shown that the model is usefulfor predicting relapse in both therapeutic settings (32–34).Not all potential donors require all three levels of KIR typing(Fig. 4D).


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Figure 3. Three basic steps of allogeneic NK cell therapy. A, the first step includes donor selection based on health history and examination, infectiousbiomarkers, and KIR typing, because KIRs are highly polymorphic. All three levels of KIR typing should be done, including genotyping, phenotyping,and allelotyping (depicted from left to right are representative outputs from flow cytometry, RT-PCR, and allelotyping analyses). B, the second step is NK cellpurification and processing to enrich NK cells and to improve NK cell functions. The process needs vigorous steps for quality assurance (QA) andquality control (QC). C, after cell infusion, antitumor activity could be enhanced in vivo using various augmentation agents (Aug).






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3DL3 2DL3 2DL1 2DL4 3DL1 2DS4 3DL2

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Step 2: NK Cell Processing and QualityAssuranceNK cell purificationThe second step for therapy is NK cell processing and

preinfusion quality assessment, including cell count, via-bility, sterility, phenotype, function, and purity (35). Stud-ies in the 1980s with autologous LAK cells consistedprimarily of expanded polyclonal T cells with low NKpercentages (36). The easiest way to select highly purifiedNK cells is immunomagnetic cell separation (37). Afterseparation, the product typically contains more than 90%NK cells with very few T cells, B cells, and monocytes. Thus,the risks of GVHD, regulatory T cell (Treg) suppression,cytokine competition, Epstein-Barr virus–lymphoprolifera-tive disease, passenger lymphocyte syndrome, cytokinestorm, and suppression by myeloid suppressor cells orblood DCs are minimized (38–40). The NK phenotype isunaltered, and cells retain extensive proliferative capacityin vivo with potent antitumor response (41). Importantly,purified NK cells can be infused in small volumes, andstudies have shown that purified cells are safe, do not causeGVHD, and are detectable in the recipient’s blood forapproximately 2 to 4 weeks after infusion with preferentialexpansion of KIR-mismatched NK cells (42).

NK cell bioprocessing ex vivoDonor NK cells can be manipulated ex vivo using a

combination of strategies to optimize efficacy and spec-ificity (Fig. 5). One challenge has been NK exhaustion, asadoptively transferred cells rapidly lose activating recep-tor expression and IFNg production capability throughdownregulation of transcription factors T-bet and eome-sodermin (43). Furthermore, a highly acidic tumor envi-ronment may render NK cells nonfunctional (44). Acommon strategy to prime and activate cells is usingsoluble factors such as IL2, -12, -15, -18, and -21 or typeI IFN (45). Studies have shown that IFNa and IL2 syn-ergistically augment NK cells against solid tumors. Sim-ilarly, IL12 with either IL2 or IL18 can be used to over-come resistance (31). In cytokine-based culture systems,massive expansion of mature NK cells is possible. In astudy of 7 patients with newly diagnosed, untreatedmultiple myeloma, the number of NK cells expanded onaverage by 1,600-fold after 20 days of culture with con-siderable increase in cytotoxicity (46).Alternatively, NK cells could be stimulated using artificial

presenting cells with membrane-bound molecules such asIL15, IL21, and 41BB ligand (47, 48). Animal studies haveshown that these cells are potent against Ewing sarcoma,rhabdomyosarcoma, and neuroblastoma and showed pro-longed survival without GVHD.

Another way to increase NK potency is using chimericantigen receptor (CAR). Receptor insertion can bemediatedusing retroviral, lentiviral, mRNA, or Sleeping Beauty trans-poson/transposase systems (49–51). Investigations haveshown that NK cells transduced with anti-CD19 CAR orNKG2Dare potent against B-lineage and osteosarcoma cellsin vivo and in vitro (49, 52). CARþ cells have been generatedagainst GD2 in neuroblastoma and CD33/CD123 in mye-loid leukemia (51, 53, 54). Clinical trials are ongoing,including NCT00995137 and NCT01974479, for B-lineagehematologic malignancies.

For centers without gene modification capabilities, NKcells could be activated by culturing in the presence ofunmodifiedCD56� cells and stimulating cytokines. Recent-ly, investigators showed that this method expanded andactivated NK cells from donors and patients with neuro-blastoma (55). These cells have great efficacy toward neu-roblastoma in vitro and in vivo through NCR, DNAM-1,perforin, and granzyme B without risk of GVHD.

One obstacle in NK therapy is insufficient homing totumor sites. Recently, investigators used CCR7þ cells totransfer CCR7 onto NK cells via trogocytosis, resulting inbetter homing of NK cells to the tumor site in a mousemodel and in Transwell migration experiments (56).Another way to facilitate adhesion of NK cells to tumorcells is using immunocytokines such as antibody againstGD2 conjugated to IL2 (57). This chimeric protein attachesto GD2 on neuroblastoma cells on one side and to IL2receptor on NK cells on the other.

In addition to mature NK cells, human embryonic stemcells (hESC), induced pluripotent stem cells, and NK leu-kemia cell linesmay be used as starting cells (58–60). hESC-derived NK cells carry the CD94þCD117low/� phenotype,which has potent antitumor activity (58). Cytokine-basedculture systems, in particular, enhance the expansion ofNKG2D/NCRþ NK cells from umbilical cord blood (59).

Because of the technical difficulty in producing cellularproducts at treatment centers, theNationalHeart, Lung, andBlood Institute (NHLBI; Bethesda, MD) sponsored theProduction Assistance for Cellular Therapies program(PACT; ref. 61). Using this approach, apheresis cells havebeen sent to remote processing centers and purified, andactivated NK cells were sent to the transplant centers forinfusion into patients (62).

Step 3: NK Augmentation In VivoCombination with therapeutic antibody or chimericproteins

Onemechanismof antibodies in cancer therapy is ADCC.NK cells carry high-affinity Fc receptors that mediate thisfunction. Conceptually, to overcome resistance, it would be

Figure 4. KIR haplotype map, B scoring, mismatch model, and typing algorithm. A, simplified genomic maps of A and B haplotypes. KIR genes thatare present in some but not all cen-B motifs are color-coded in green and the two loci that are used for simplified B-scoring are in red. B, calculation of totalB scores. C, all three mismatch models are biologically sound but are fundamentally different in principles and in clinical usages, including therelationship in consideration, the blood tests required, the definition of mismatches, and the applicability to antibody therapy and autologous settings.D, donor typing and selection algorithms.





attractive to administer NK therapy concurrently with anti-bodies such as those against CD19, CD20, CD22, CD33,CD123, HER2, EGFR, and GD2 (63, 64). Typing for recip-ient and donor FcgRIII 158V/F polymorphism is importantin optimizing ADCC (65). Stimulating NK cells with anagonistic monoclonal antibody specific for CD137 or withblocking antibodies against KIR/NKG2A may increase cellkilling (66–68). Thus, using a second antibody that acti-vates donor NK cells may improve the efficacy of antibodiesagainst tumor-associated antigens.

Another method to activate CD16 is a bispecific or tri-specific killer cell engager (BiKEor TriKE). A fully humanizedCD16-CD33 BiKE has been created that strongly activatesNK cells against CD33þ AML blasts (69). Pretreatment withADAM17 inhibitor prevented CD16 shedding and overcameinhibition of class I MHC-recognizing inhibitory receptors.Incubating cancer cells with BiKE and TriKE increased NKcytolytic activity against tumor targets and production ofIFNg , TNFa, GM-CSF, IL8, MIP-1a, and RANTES (70).

Combination with supplemental medicationAfter NK infusion, many regimens include low-dose

IL2 injections. Newer agents are being investigated. Lena-lidomide enhances ADCC in rituximab treatment ofnon-Hodgkin lymphoma and B-cell chronic lymphocyticleukemia through enhanced granzyme B and FasL expres-sion (71). The effects of lenalidomide are partly relatedto CD4þ T-cell production of IL2 (72). Imatinib triggersDC-mediated NK activation (73), whereas dasatinibenhances NK expansion via eomesodermin (74). Ligationof CD86 on NK cells with CTLA4-Ig fusion protein orblockade of A2A adenosine receptor interaction withCD73 increases tumor killing (75, 76).

During NK therapy, concurrent suppressive medicationsshould be avoided, including agents such as azacytidine andsorafenib, which substantially impair PI3K and ERK phos-phorylation and hamper NK reactivity (77, 78). Sunitinibdoes not inhibit NK cells, and decitabine may augment NKreactivity (78).


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NK cells

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Figure 5. Clinically feasibleapproaches to optimize NK celltherapy.Most protocols usematureNK cells (mNK) as starting cells,while a few use stem or progenitorcells such as ESC, inducedpluripotent stem cells (IPS), andhematopoietic stem cells (HSC) togenerate NK precursors (NKp). NKcells can be augmented throughvarious strategies to obtain largenumbers of activated NK cells(aNK). After infusion, the NK cellsmay kill cancer cells directly ormediate the development ofadaptive immunity via interactionswith immature DCs (iDC), B cells,T-helper cells (Th), cytotoxic Tlymphocytes (CTL), and matureDCs (mDC). Tumor cells can besensitized by various agents torender them more susceptible toNK cells. Bold arrows indicate cellintrinsic changes. Thin arrowsindicate cell extrinsic interactions.Ag, antigen.

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Combination with tumor cell modulationAfter CD16, NKG2D is the secondmost potent activating

receptor expressed on NK cells. Unfortunately, NKG2Dligand expression in most cancer cells is low (79). Recently,various methods have been used to upregulate NKG2Dligand expression or prevent its shedding from the cellsurface, including epigenetic modulation with histone dea-cetylase (HDAC) inhibitors (which may induce glycogensynthase kinase-3 activity; ref. 80), proteosome inhibitors,and demethylating agents. These agents may also worktogether to induce expression of Fas and TRAIL-R2 (DR5;refs. 81, 82). Recently, high-throughput screening of 5,600bioactive compounds revealed spironolactone as a novelRXRg agonist that activates the ATM–CHK2 pathway toupregulate transcription of all classes of NKG2D ligandsfor cancer prevention and control (83). In another studyusing a lentiviral shRNA library targeting more than 10,000human genes, silencing of JAK1 and JAK2 increasedthe susceptibility of a variety of tumor cell types to NK-mediated lysis (84).

Clinical ConsiderationsNK cell therapy is applicable to various forms of cancer

(85, 86), but its current use has been focused primarily inhematologic malignancies (20, 63, 87, 88). NK cells can beused for patients with refractory leukemia and can be givenbefore conventional HCT for induction of remission, afterHCT as consolidation, or in place of HCT.In an early feasibility study, IL2-stimulated NK cells were

infused post-HCT into 3 pediatric patients who had persis-tent leukemia blasts at the time of transplant (89). Nosignificant toxicity was observed, and complete remissionand complete donor chimerism were observed within 1month after HCT. In a subsequent prospective phase IIstudy, preemptive immunotherapy with purified NK cellsafter haploidentical HCT was investigated in 16 patients(90). The patients received 29 NK infusions 3, 40, and100 days after transplantation. The median dose of NKcells per product was 1.21 � 107/kg. With a median fol-low-up of 5.8 years, 4 of the 16 patients with high-riskleukemia remained alive.In 10patientswith advancedmultiplemyeloma receiving

autologous HCT, haploidentical T cell–depleted, KIRligand–mismatched NK cells were infused (91). No GVHDor failure of autologous stem cell reconstitution wasobserved. Encouragingly, 50%of the patients achieved nearcomplete remission. These results set the stage for futurestudies of KIR ligand–mismatched NK therapy in the autol-ogous setting.

NK therapy without HCTFor allogeneic NK therapy in a nontransplant setting,

transient suppression of the host immune system isrequired to allow time for donor-derived NK expansionand therapeutic effect. Even in the autologous setting,induction of transient leukopenia may promote homeo-static expansion of NK cells and reduce suppressive effectsfrom Tregs and myeloid suppressor cells (32, 33, 92). Onecommon outpatient regimen includes fludarabine andlow-dose cyclophosphamide, and this regimen is welltolerated even in children, with 10 of 10 patients survivingfree of leukemia (42). The rare occurrence of complica-tions results in the cost of fresh NK therapy being lessthan 10% of that for conventional HCT. Infusions ofpurified NK cells were feasible after similar conditioningin 13 elderly patients including septuagenarians (93).

In a study of haploidentical related-donor NK infusion, ahigh-intensity inpatient regimen of high-dose cyclophos-phamide and fludarabine resulted in a marked increase inendogenous IL15, expansion of donor NK cells, and induc-tion of complete hematologic remission in 5 of 19 patientswith poor-prognosis AML (94). In a subsequent phase IIstudy for recurrent ovarian and breast cancer, 20 patientsunderwent the regimen of fludarabine and cyclophospha-mide with or without low-dose total body irradiation (95).After cell infusion, IL2was administered subcutaneously for2 weeks. With a mean NK cell dose of 2.16� 107 cells/kg, 9of 13 (69%) patients without irradiation and 6 of 7 withirradiation had donor DNA detectable after NK cell infu-sions. In another study of 6 patients with advanced B-cellnon-Hodgkin lymphoma, 4 patients showed an objectiveclinical response (96). Unfortunately, Tregs also expandedin vivo in these two studies; therefore, future strategiesshould include novel techniques to suppress the expansionof Tregs.

ConclusionsIn summary, NK cells are promising in cancer therapy,

considering their speed of action, potency in cancer cellkilling, applicability tomany types of cancer, lack of adverseeffects, ease of preparation and administration, availability,permissibility of donor selection, affordability, and com-plimentary actions with other therapies. The unique prop-erties of NK cells open a new arena of novel cell-basedimmunotherapy against cancers that are resistant to con-temporary therapies.

Received October 29, 2013; revised February 6, 2014; accepted February25, 2014; published online July 1, 2014.

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