Infusions of Allogeneic Natural Killer Cells As Cancer Therapy

Clinical Cancer Review Research

Infusions of Allogeneic Natural Killer Cells as Cancer Therapy

Wing Leung1,2

Abstract Natural 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, and novel augmentation techniques, to improve NK 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 human NK cells open up a new arena of 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 Interest W. 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' Disclosures The members of the planning committee have no real or apparent conﬂict of interest to disclose.

Learning Objectives Upon 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 Support This activity does not receive commercial support.

Introduction Infusions of purified natural killer (NK) cells are late- For many cancers, cure rates remain unacceptably low. comers in cellular immunotherapy, compared with den- Advances in genetics and immunology have led to biologic dritic cells (DC), lymphokine-activated killer (LAK) cells, therapies with non–cross-resistant mechanisms of action tumor-infiltrating lymphocytes, and conventional cytotoxic and no overlapping toxicities. Cancer immunotherapy is T cells. Many investigators consider that the cytolytic activ- possible with an array of effector mechanisms involving ity of LAK cells commonly used in the 1980s actually innate and adaptive immunity (1). Various cytokines, anti- represents the activity of IL2-activated NK cells, although bodies, and cellular therapies have been FDA approved or the products contained polyclonal T cells. Biologically, are in late-phase clinical trials. murine NK cells have been known to kill malignant cells without T-cell assistance or prior sensitization since their initial description in 1975 (2–5); however, the therapeutic Author's Affiliations: 1Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital; and 2Department potential of human NK cells was not revealed until their of Pediatrics, University of Tennessee, Memphis, Tennessee biology of target cell recognition was elucidated in the Corresponding Author: Wing Leung, St. Jude Children's Research Hos- 1990s and the antileukemia effect was observed in HLA- pital, 262 Danny Thomas Place, Memphis, TN 38105-3678. Phone: 901- haploidentical hematopoietic cell transplantation (HCT) in 595-2617; Fax: 901-521-9005; E-mail: [email protected] the early 2000s (6, 7). This review covers NK cell biology doi: 10.1158/1078-0432.CCR-13-1766 relevant to cancer immunotherapy and outlines avenues 2014 American Association for Cancer Research. being explored in clinical trials.

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NK Cell Biology autologous NK therapy (20). Among known NK receptors, NK cells were first named and characterized by Kiessling the KIR receptor family is one of the primary determinants at the Karolinska Institute (Stockholm, Sweden) and by of NK response. Subsets of T cells also expressed KIR (21). Herberman at the National Cancer Institute (Bethesda, Clinical KIR typing includes genotyping for gene content and A/B haplotype categorization, phenotyping for gene MD; refs. 2–5). The term "natural" referred to their natural þ existence in rodents and "killer" to their spontaneous expression and number of KIR cells, and allelotyping for killing of lymphoma and leukemia cells in nonimmune functional strength. animals. They were called "N-cells" or "K cells" by some The first level of KIR diversity is gene content (22, 23). investigators (3, 8). These cells were unique, as they lacked Only approximately 5% of people have all 15 gene family markers characteristic of B and T lymphocytes and were members; others lack one or more genes. The diversity of nonphagocytic, low-adhesive, and cytotoxic to IgG-coated gene content is based primarily on diversity in B haplo- targets. types, which typically contain more activating KIRs. The NK cells are generated in bone marrow and develop two hallmark genes for A haplotypes are KIR2DL3 and normally in athymic mice and in humans with DiGeorge KIR3DL1. They are segregated with KIR2DL2 and KIR3DS1 syndrome. Induction of cytotoxicity is possible in NK as alleles in the centromeric (Cen) and telomeric (Tel) cells within minutes, in contrast with the slower conven- motifs, respectively (Fig. 4A). On the basis of this pattern, a simplified typing and scoring method for B haplotype tional induction of the T-cell immune response (9). þ content can be derived (Fig. 4B), viz., KIR2DL3 / Approximately 5% to 15% of human blood lymphocytes þ þ þ KIR2DL2 , Cen-A/A; KIR2DL3 /KIR2DL2 , Cen-A/B; are NK cells (CD56 , CD3/14/19 ), 90% of which are þ þ þ KIR2DL3 /KIR2DL2 , Cen-B/B; KIR3DL1 /KIR3DS1 , CD56dim, with most of them being CD16 ,andtheyare þ þ Tel-A/A; KIR3DL1 /KIR3DS1 , Tel-A/B; and KIR3DL1 / responsible for early innate immunity and antibody- þ dependent cell-mediated cytotoxicity (ADCC) against KIR3DS1 , Tel-B/B. Because KIRs are encoded in chromo- infectionorcancerthroughIFNg, perforin, granzyme, some 19 and ligand HLAs are in chromosome 6, these gene FasL, or TRAIL (10). Approximately 10% of human blood families are segregated independently; thus, HLA geno- NK cells are CD56bright, and they participate in late (>16 types cannot be used to predict KIR content, and autolo- hours) inflammatory response by secreting IFNg,TNFa, gous KIR-HLA mismatch is possible. G-CSF, GM-CSF, and IL3. Interactions between DCs and The second level of diversity is gene expression (24). By NK cells are essential for priming adaptive immunity. NK real-time PCR and flow cytometry, more than 10-fold cells may also directly interact with T and B cells through variability in expression has been observed among donors. CD40 ligand and OX40 (11, 12). Nowadays, the easiest method to quantify NK cells expres- sing only one inhibitory KIR gene (i.e., negative for all other MHC inhibitory receptors) is multicolor flow cyto- Regulatory receptors þ Human NK cells are regulated by a sophisticated network metry (24–26). The number of single KIR cells is in direct of surface receptors (Fig. 1), allowing them to distinguish proportion to activity against target cells without the ligand (receptor–ligand mismatched). Although the num- normal from abnormal cells within seconds. Target cell þ lysis occurs when the activating signal dominates the ber of single KIR cells correlates with the presence of self- inhibitory signal (Fig. 2). Early studies revealed similarities ligand (25), the variability among donors renders predic- between murine NK cells and cells responsible for so-called tions based on the ligand alone unacceptable for clinical "hybrid resistance" to parental hematopoietic grafts (13, use. 14). The proposal that the resistance was related to recep- The third level of diversity is allele polymorphism, which tors recognizing the absence of MHC class I arose from has been observed in all inhibitory KIR genes (22, 23). For observations that murine lymphoma cells that were low in example, in the KIR2DL1 family, 25 alleles have been MHC expression were NK-susceptible (15). This led to the observed, and each has different strengths in inhibiting NK "missing-self" hypothesis in the 1980s (16). Break- cells and different durability of surface expression after throughs in human NK receptor biology were seen in the interaction with ligands (27). These differences correlate early 1990s, when the KIR gene family was found to with the intensity of inhibitory signaling through SHP2 and recognize HLA class I (17–19). Identification of other NK b arrestin-2, resulting in differences in immune synapse receptors, such as NCRs, NKG2D, DNAM, 2B4, and CD94/ formation. Mechanistically, the arginine residue at position NKG2A, collectively points to a sophisticated network that 245 in the transmembrane domain is important in inhibi- controls human NK activity. tion. Alleles with arginine in position 245 are stronger than those with cysteine in that position (27). On the basis of this molecular determinant, single-nucleotide polymorphism Step 1: KIR Typing for Donor Selection and assays have been developed for rapid, high-throughput Prognostication clinical typing (28). The same assay can be simultaneously Three general steps in NK-based therapy are summarized used for KIR ligand typing. Notably, this assay requires less in Fig. 3. KIR typing is a critical first step because KIR is than 0.1 mg of DNA and can be performed within 1 day. highly polymorphic. KIR typing is not only useful for Using this assay, investigations showed that patients who allogeneic donor selection but also for prognostication in received a donor graft containing the stronger KIR allele had

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IFNα IL1 IL2 IL12 IL15 IL18 IL21

Figure 1. Surface receptors and NK cell their ligands. Cytokine receptors KIR2DL1/2/3 (HLA-C) KIR2DS1/S2 (HLA-C) are shown on top of a human NK cell. Other receptors are broadly KIR3DL1 (HLA-B) KIR2DS4 (HLA-A, -C) classiﬁed and color-coded on the KIR3DL2 (HLA-A) KIR2DL4 (HLA-G, HS) basis of their primary function (inhibitory receptors in red, CD94/NKG2A (HLA-E) CD94/NKG2C (HLA-E) Inhibitory Activating activating receptors in green, inhibitory coreceptors in red-black LIR-1 (HLA-A–G) KIR3DL2 (CpG) stripes, and activating coreceptors KLRG-1 (cadherins) CD16 (IgG) in green-black stripes). Their ligands are shown within CEACAM1 (CEACAM5) NKG2D (MIC, ULBP) parentheses. Many other known receptors are not shown, including TIGIT (PVR, PVRL2) NCRs (B7-H6, NKp44L) chemotactic receptors (CCR-2, -5, Coreceptor -7: CXCR-1, -3, -4, -6; CX3CR1; and Chem23R), adhesion Siglec –3, –7, –9 (sialic acid) DNAM-1 (PVR, Nectin-2) receptors (CD2 and b1 and b2 integrins), and activating LAIR-1 (collagen) 2B4 (CD48) coreceptors (CD96, CS1, and TLR). CD300A (PS) NKp80 NTBA (NTBA) (AICL)

fewer relapses, better survival, and less transplant-related acute lymphoblastic leukemia (ALL)], total body irradia- mortality (29). These effects were observed regardless of tion, T-cell depletion, and donor type. Statistically, there is primary disease [acute myelogenous leukemia (AML) vs. an interaction effect with HLA-C receptor–ligand mismatch:

Characteristics Figure 2. Dynamic equilibrium. After NK cell Target cell Outcome of target cell cell-to-cell contact, NK cell integrates signals from its surface A receptors in seconds, resulting in – either target-cell attack or no Normal MHC class I response and continual + No response No activating ligands immunosurveillance of other cells. A, healthy cells express normal amount of MHC class I ligands with no activating "stress" ligands. B, B downregulation or absence of MHC ligand for cognate inhibitory No MHC class I No response receptors is insufﬁcient to trigger No activating ligands NK cells. This happens in the physiologic setting with red blood cells and autologous KIR receptor– C ligand mismatch cells, and in pathologic conditions with adult No MHC class I lymphoblastic leukemia. C, Attack target activating ligands sufﬁcient activating ligands must be expressed on target cells to induce NK cell activity. D, if self-MHC D ligands are expressed in normal amount, the reactivity of the NK cell Normal MHC class I is ultimately dependent on the Dynamic equilibrium activating ligands balance of activating and inhibitory signals. Successful NK cell therapy relies on clinical strategies that optimize activation and avoid © 2014 American Association for Cancer Research inhibition by cancer cells. CCR Reviews

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Donor selection, including KIR typing Donor

105 0 0

104 Best

103 O Neg H 2 102 2DL1 2DL22DL33DL1 0 61.2 38.8 B

2 3 4 5 0 10 10 10 10 NK cell purification and processing ex vivo

C in vivo Patient NK cell augmentation QA and QC

Aug NK Tumor Tumor Aug NK NK Tumor Tumor Tumor NK NK Aug Tumor NK Aug Tumor Tumor NK

Figure 3. Three basic steps of allogeneic NK cell therapy. A, the first step includes donor selection based on health history and examination, infectious biomarkers, 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 cell purification and processing to enrich NK cells and to improve NK cell functions. The process needs vigorous steps for quality assurance (QA) and quality control (QC). C, after cell infusion, antitumor activity could be enhanced in vivo using various augmentation agents (Aug).

Patients with the best survival were those with a stronger the donor but absent in the recipient, the donor is mis- KIR allele and receptor–ligand mismatch from donors. The matched. Because B haplotypes contain more genes than A second-best group was those with a stronger KIR allele but haplotypes, mismatch typically signiﬁes a B haplotype in no receptor–ligand mismatch. The worst survival was in the donor. The third model is the receptor–ligand mismatch those with the weaker allele, regardless of mismatch. model proposed by the Memphis group (31). This model Donor KIR typing is not necessary in some KIR mismatch requires donor KIR typing and recipient HLA typing. models (Fig. 4C). The ﬁrst model was proposed by the A donor is mismatched if an inhibitory receptor is present Perugia group (7); the ligand mismatch model requires in the donor but the ligand is absent in the recipient. typing HLA in both donor and recipient. A donor is mis- Notably, the receptor–ligand mismatch model is the only matched if a ligand is present in the donor but absent in the model applicable to autologous transplantation and anti- recipient. This ligand mismatch model does not require body therapy; studies have shown that the model is useful donor or recipient KIR typing. The second model was for predicting relapse in both therapeutic settings (32–34). proposed by the Nantes group (30); in this model, KIR is Not all potential donors require all three levels of KIR typing typed in both donor and recipient. If a receptor is present in (Fig. 4D).

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A Cen A Tel A 3DL3 2DL3 2DL1 3DP1 2DL4 3DL1 2DS4 3DL2

3DL3 2DS2 2DL2 2DL5B 2DS3/5 2DL1 3DP1 2DL4 3DS1 2DL5A 2DS3/5 2DS1 3DL2

Cen B Tel B B KIR2DL3 KIR3DL1 Centromeric B score Telomeric B score Positive Negative Positive Negative

Positive 12 Positive 12 KIR2DL2 KIR3DS1 Negative 0 Does not Negative 0 Very rare exist Total B score = Centromeric B score + Telomeric B score = (______) *Total B scores range from 0–4 C

Applicable to Applicable to Mismatch Definition of Relationship Test ordered Example autologous antibody model mismatch cell therapy therapy Donor Recipient Donor KIRL Missing KIRL in Ligand vs. Donor HLA a recipient for a C2 C2 No No mismatch recipient recipient KIRL that is HLA KIRL present in donor C1 C2

Donor Recipient Missing KIR in Donor KIR Donor KIR Receptor a recipient for a vs. recipient No No mismatch KIR that is recipient KIR KIR present in donor 2DS/Bx Donor Recipient Missing KIRL in Receptor– Donor KIR Donor KIR a recipient for a C2 ligand vs. recipient KIR that is Yes Yes mismatch recipient HLA KIRL present in donor 2DL2

D C245+ R245– Donor KIR2DL1 allelotyping Not selected

C245– R245+ BW4+ C1+ C2+ Recipient HLA typing Donor KIR genotyping Select donor with highest B score

Missing ≥ 1 ligand

Donor KIR phenotyping Receptor–ligand match Donor KIR genotyping Select donor with highest B score by flow cytometry

Receptor–ligand mismatch

Select donor with largest amount of receptor–ligand mismatched cells

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Step 2: NK Cell Processing and Quality Another way to increase NK potency is using chimeric Assurance antigen receptor (CAR). Receptor insertion can be mediated using retroviral, lentiviral, mRNA, or Sleeping Beauty trans- NK cell purification poson/transposase systems (49–51). Investigations have The second step for therapy is NK cell processing and shown that NK cells transduced with anti-CD19 CAR or preinfusion quality assessment, including cell count, via- NKG2D are potent against B-lineage and osteosarcoma cells bility, sterility, phenotype, function, and purity (35). Stud- þ in vivo and in vitro (49, 52). CAR cells have been generated ies in the 1980s with autologous LAK cells consisted against GD2 in neuroblastoma and CD33/CD123 in mye- primarily of expanded polyclonal T cells with low NK loid leukemia (51, 53, 54). Clinical trials are ongoing, percentages (36). The easiest way to select highly purified including NCT00995137 and NCT01974479, for B-lineage NK cells is immunomagnetic cell separation (37). After hematologic malignancies. separation, the product typically contains more than 90% For centers without gene modification capabilities, NK NK cells with very few T cells, B cells, and monocytes. Thus, cells could be activated by culturing in the presence of the risks of GVHD, regulatory T cell (Treg) suppression, unmodified CD56 cells and stimulating cytokines. Recent- cytokine competition, Epstein-Barr virus–lymphoprolifera- ly, investigators showed that this method expanded and tive disease, passenger lymphocyte syndrome, cytokine activated NK cells from donors and patients with neuro- storm, and suppression by myeloid suppressor cells or blastoma (55). These cells have great efficacy toward neu- blood DCs are minimized (38–40). The NK phenotype is roblastoma in vitro and in vivo through NCR, DNAM-1, unaltered, and cells retain extensive proliferative capacity perforin, and granzyme B without risk of GVHD. in vivo with potent antitumor response (41). Importantly, One obstacle in NK therapy is insufficient homing to purified NK cells can be infused in small volumes, and þ tumor sites. Recently, investigators used CCR7 cells to studies have shown that purified cells are safe, do not cause transfer CCR7 onto NK cells via trogocytosis, resulting in GVHD, and are detectable in the recipient’s blood for better homing of NK cells to the tumor site in a mouse approximately 2 to 4 weeks after infusion with preferential model and in Transwell migration experiments (56). expansion of KIR-mismatched NK cells (42). Another way to facilitate adhesion of NK cells to tumor cells is using immunocytokines such as antibody against NK cell bioprocessing ex vivo GD2 conjugated to IL2 (57). This chimeric protein attaches Donor NK cells can be manipulated ex vivo using a to GD2 on neuroblastoma cells on one side and to IL2 combination of strategies to optimize efficacy and spec- receptor on NK cells on the other. ificity (Fig. 5). One challenge has been NK exhaustion, as In addition to mature NK cells, human embryonic stem adoptively transferred cells rapidly lose activating recep- cells (hESC), induced pluripotent stem cells, and NK leu- tor expression and IFNg production capability through kemia cell lines may be used as starting cells (58–60). hESC- downregulation of transcription factors T-bet and eome- þ derived NK cells carry the CD94 CD117low/ phenotype, sodermin (43). Furthermore, a highly acidic tumor envi- which has potent antitumor activity (58). Cytokine-based ronment may render NK cells nonfunctional (44). A culture systems, in particular, enhance the expansion of common strategy to prime and activate cells is using þ NKG2D/NCR NK cells from umbilical cord blood (59). soluble factors such as IL2, -12, -15, -18, and -21 or type Because of the technical difficulty in producing cellular I IFN (45). Studies have shown that IFNa and IL2 syn- products at treatment centers, the National Heart, Lung, and ergistically augment NK cells against solid tumors. Sim- Blood Institute (NHLBI; Bethesda, MD) sponsored the ilarly, IL12 with either IL2 or IL18 can be used to over- Production Assistance for Cellular Therapies program come resistance (31). In cytokine-based culture systems, (PACT; ref. 61). Using this approach, apheresis cells have massive expansion of mature NK cells is possible. In a been sent to remote processing centers and purified, and study of 7 patients with newly diagnosed, untreated activated NK cells were sent to the transplant centers for multiple myeloma, the number of NK cells expanded on infusion into patients (62). average by 1,600-fold after 20 days of culture with con- siderable increase in cytotoxicity (46). Step 3: NK Augmentation In Vivo Alternatively, NK cells could be stimulated using artificial presenting cells with membrane-bound molecules such as Combination with therapeutic antibody or chimeric IL15, IL21, and 41BB ligand (47, 48). Animal studies have proteins shown that these cells are potent against Ewing sarcoma, One mechanism of antibodies in cancer therapy is ADCC. rhabdomyosarcoma, and neuroblastoma and showed pro- NK cells carry high-affinity Fc receptors that mediate this longed survival without GVHD. function. 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 that are 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 total B scores. C, all three mismatch models are biologically sound but are fundamentally different in principles and in clinical usages, including the relationship 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.

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NK cells Selection of KIR- Ex vivo NKp mNK cell derived from favorable development and purification ESC/IPS donor mNK expansion and activation

ESC NKp mNK IPS HSC

Augmentation strategies ↑ • Survival (IL21) Figure 5. Clinically feasible • ↑ Cytokine priming approaches to optimize NK cell (IFNα, IL2, IL12, IL18) therapy. Most protocols use mature • ↑ Homing/adhesion NK cells (mNK) as starting cells, (togocytosis, ICs) while a few use stem or progenitor • ↑ Specificity/potency cells such as ESC, induced pluripotent stem cells (IPS), and (mAb, BiKE, CAR) hematopoietic stem cells (HSC) to • ↑ CD16 (ADAM17 inhibitor) generate NK precursors (NKp). NK ↑ • Activating receptor cells can be augmented through (NKG2D, DNAM, NCR) various strategies to obtain large • ↑ Lysis (lenalidomide, numbers of activated NK cells TKI, decitabine, CTLA4) (aNK). After infusion, the NK cells Sensitization • ↓ Inhibition may kill cancer cells directly or strategies (anti-KIR, anti-NKG2a, mediate the development of anti-A2A, anti-PD1 mAb) adaptive immunity via interactions • HDAC inhibitor with immature DCs (iDC), B cells, • ADAM inhibitor T-helper cells (Th), cytotoxic T Tumor Tumor • Proteasome aNK iDC lymphocytes (CTL), and mature inhibitor DCs (mDC). Tumor cells can be • Demethylation DC editing sensitized by various agents to and maturation • RXRγ agonist render them more susceptible to Lysis NK cells. Bold arrows indicate cell • DNA damage IFNγ B cell intrinsic changes. Thin arrows • JAK inhibitor indicate cell extrinsic interactions. Antitumor Ag, antigen. + antibody mDC CD8+ CD4 CTL Th

Tumor-specific Tumor Ag uptake adaptive immunity and presentation

attractive to administer NK therapy concurrently with anti- Combination with supplemental medication bodies such as those against CD19, CD20, CD22, CD33, After NK infusion, many regimens include low-dose CD123, HER2, EGFR, and GD2 (63, 64). Typing for recip- IL2 injections. Newer agents are being investigated. Lena- ient and donor FcgRIII 158V/F polymorphism is important lidomide enhances ADCC in rituximab treatment of in optimizing ADCC (65). Stimulating NK cells with an non-Hodgkin lymphoma and B-cell chronic lymphocytic agonistic monoclonal antibody specific for CD137 or with leukemia through enhanced granzyme B and FasL expres- blocking antibodies against KIR/NKG2A may increase cell sion (71). The effects of lenalidomide are partly related þ killing (66–68). Thus, using a second antibody that acti- to CD4 T-cell production of IL2 (72). Imatinib triggers vates donor NK cells may improve the efficacy of antibodies DC-mediated NK activation (73), whereas dasatinib against tumor-associated antigens. enhances NK expansion via eomesodermin (74). Ligation Another method to activate CD16 is a bispecific or tri- of CD86 on NK cells with CTLA4-Ig fusion protein or specific killer cell engager (BiKE or TriKE). A fully humanized blockade of A2A adenosine receptor interaction with CD16-CD33 BiKE has been created that strongly activates CD73 increases tumor killing (75, 76). þ NK cells against CD33 AML blasts (69). Pretreatment with During NK therapy, concurrent suppressive medications ADAM17 inhibitor prevented CD16 shedding and overcame should be avoided, including agents such as azacytidine and inhibition of class I MHC-recognizing inhibitory receptors. sorafenib, which substantially impair PI3K and ERK phos- Incubating cancer cells with BiKE and TriKE increased NK phorylation and hamper NK reactivity (77, 78). Sunitinib cytolytic activity against tumor targets and production of does not inhibit NK cells, and decitabine may augment NK IFNg,TNFa, GM-CSF, IL8, MIP-1a, and RANTES (70). reactivity (78).

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Combination with tumor cell modulation NK therapy without HCT After CD16, NKG2D is the second most potent activating For allogeneic NK therapy in a nontransplant setting, receptor expressed on NK cells. Unfortunately, NKG2D transient suppression of the host immune system is ligand expression in most cancer cells is low (79). Recently, required to allow time for donor-derived NK expansion various methods have been used to upregulate NKG2D and therapeutic effect. Even in the autologous setting, ligand expression or prevent its shedding from the cell induction of transient leukopenia may promote homeo- surface, including epigenetic modulation with histone dea- static expansion of NK cells and reduce suppressive effects cetylase (HDAC) inhibitors (which may induce glycogen from Tregs and myeloid suppressor cells (32, 33, 92). One synthase kinase-3 activity; ref. 80), proteosome inhibitors, common outpatient regimen includes fludarabine and and demethylating agents. These agents may also work low-dose cyclophosphamide, and this regimen is well together to induce expression of Fas and TRAIL-R2 (DR5; tolerated even in children, with 10 of 10 patients surviving refs. 81, 82). Recently, high-throughput screening of 5,600 free of leukemia (42). The rare occurrence of complica- bioactive compounds revealed spironolactone as a novel tions results in the cost of fresh NK therapy being less RXRg agonist that activates the ATM–CHK2 pathway to than 10% of that for conventional HCT. Infusions of upregulate transcription of all classes of NKG2D ligands purified NK cells were feasible after similar conditioning for cancer prevention and control (83). In another study in 13 elderly patients including septuagenarians (93). using a lentiviral shRNA library targeting more than 10,000 In a study of haploidentical related-donor NK infusion, a human genes, silencing of JAK1 and JAK2 increased high-intensity inpatient regimen of high-dose cyclophos- the susceptibility of a variety of tumor cell types to NK- phamide and fludarabine resulted in a marked increase in mediated lysis (84). endogenous IL15, expansion of donor NK cells, and induction of complete hematologic remission in 5 of 19 patients Clinical Considerations with poor-prognosis AML (94). In a subsequent phase II NK cell therapy is applicable to various forms of cancer study for recurrent ovarian and breast cancer, 20 patients (85, 86), but its current use has been focused primarily in underwent the regimen of fludarabine and cyclophospha- hematologic malignancies (20, 63, 87, 88). NK cells can be mide with or without low-dose total body irradiation (95). used for patients with refractory leukemia and can be given After cell infusion, IL2 was administered subcutaneously for 7 before conventional HCT for induction of remission, after 2 weeks. With a mean NK cell dose of 2.16 10 cells/kg, 9 HCT as consolidation, or in place of HCT. of 13 (69%) patients without irradiation and 6 of 7 with In an early feasibility study, IL2-stimulated NK cells were irradiation had donor DNA detectable after NK cell infu- infused post-HCT into 3 pediatric patients who had persis- sions. In another study of 6 patients with advanced B-cell tent leukemia blasts at the time of transplant (89). No non-Hodgkin lymphoma, 4 patients showed an objective significant toxicity was observed, and complete remission clinical response (96). Unfortunately, Tregs also expanded and complete donor chimerism were observed within 1 in vivo in these two studies; therefore, future strategies month after HCT. In a subsequent prospective phase II should include novel techniques to suppress the expansion study, preemptive immunotherapy with purified NK cells of Tregs. after haploidentical HCT was investigated in 16 patients (90). The patients received 29 NK infusions 3, 40, and Conclusions 100 days after transplantation. The median dose of NK In summary, NK cells are promising in cancer therapy, 7 cells per product was 1.21 10 /kg. With a median fol- considering their speed of action, potency in cancer cell low-up of 5.8 years, 4 of the 16 patients with high-risk killing, applicability to many types of cancer, lack of adverse leukemia remained alive. effects, ease of preparation and administration, availability, In 10 patients with advanced multiple myeloma receiving permissibility of donor selection, affordability, and com- autologous HCT, haploidentical T cell–depleted, KIR plimentary actions with other therapies. The unique prop- ligand–mismatched NK cells were infused (91). No GVHD erties of NK cells open a new arena of novel cell-based or failure of autologous stem cell reconstitution was immunotherapy against cancers that are resistant to con- observed. Encouragingly, 50% of the patients achieved near temporary therapies. complete remission. These results set the stage for future studies of KIR ligand–mismatched NK therapy in the autol- Received October 29, 2013; revised February 6, 2014; accepted February ogous setting. 25, 2014; published online July 1, 2014.

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