Several investigators have successfully obtained CIK cells from UCB units. The washouts of UCB units may yield approximately 500 *×* 106 CIK cells after a standardized 21-day expansion culture (159). Compared with PB-derived CIK cells, UCB CIK cells demonstrate lower immunogenicity and higher proliferative capacity and anti-tumor activity in pre-clinical models of cancer (160). Interestingly, UCB-derived CIK cells released higher amounts of IL-2 and IFN-γ and expressed higher levels of CCR6 and CCR7, pointing to a better ability to traffic to tumor sites and secondary lymphoid organs. CIK cells differentiated from PB and UCB also differ in receptor expression and in cytotoxic activity against ALL cells, suggesting that the source of CIK cells may impact on therapeutic efficacy (161).

# **Clinical Trials with CIK Cells**

An International Registry on CIK Cells (IRCC) has been established with the aim of reporting results from clinical trials centered on adoptively transferred CIK cells. In the first IRCC publication (167), eleven clinical trials with autologous or allogeneic CIK cells were identified, with 426 patients enrolled. Most trials included male patients with hepatocellular carcinoma, gastric cancer, and relapsed lymphoma. A clinical response was reported in 384 patients, who received up to 40 infusions of CIK cells. The total RR was 24% and a decrease of tumor volume was documented in three patients. DFS rates were significantly higher in patients treated with CIK cells than in a control group without CIK treatment. An update published in 2014 (168) enlists the results obtained in 2,729 patients from 45 phase I/II studies. A total of 1,520 patients with 22 different tumor entities were treated with CIK cells, either alone or in combination with chemotherapy. Allogeneic CIK cells were employed in the majority of the trials (41 out of 45). The number of CIK cells infused varied among the different trials, averaging 7.7 *×* 109 cells. Data on patient survival were available for 19 out of 45 trials; 15 of these 19 trials were paired, as they also included control patients receiving none or standard therapy alone.  High numbers of CIK cells at time of infusion were associated with a better prognosis. Quality of life was also improved in four of the five trials for which data were disclosed. Ancillary biological data were provided in 23 studies. The absolute numbers of T cells, as well as serum IFN-γ, were increased after immunotherapy compared with baseline. Some studies also reported a decrease of blood Treg cells after CIK infusions. Immunotherapy was generally well tolerated, with fever occurring in 41% of cases and headache and fatigue reported in 30% of cases (168). Mild GVHD was observed in seven of 52 patients treated with allogeneic CIK cells and was responsive to steroid therapy. Based on these findings, the IRCC currently recommends the use of at least 10 *×* 109 CIK cells with at least 30% CD3+CD56+ cells per infusion, every 2–4 weeks, for at least six times. The IRCC website4 (last accessed March 2015) lists 1,787 patients treated with CIK cells, mostly for hepatocellular carcinoma, gastric cancer, ovarian cancer, renal cell cancer, MM, and NSCLC.

China has the largest population of patients with malignant tumors and is the country where most clinical trials with CIK cells were conducted (169). Using the VIP database of Chinese scientific and technological journals5 , 24 articles were selected. Information on the total number of CIK cells used was available in 16 studies and ranged from 6 *×* 106 to 1.5 *×* 1010 in one single treatment course. As far as hematological malignancies are concerned, 1 study specifically dealt with non-Hodgkin's disease (12 patients) and 4 studies with AML (51 patients). Only 14 out of 24 studies contained details about clinical outcome. Of the reported 563 patients, 40 had a CR, 126 had a partial response, 125 had a minimal response, 135 had stable disease, and 58 had progressive disease. The remaining 76 patients did not reach an objective response. The overall RR was 51.7% (291/563) (169). Information on patient outcome was provided in 10 studies. Four studies reported a 1-year OS rate of 72.5%, six studies reported 2-year OS rate of 66.3%, one study reported 3-year overall RR of 75.5%, and two studies reported 5-year OS rate of 38.2%.

A phase I study of allogeneic CIK cells has been conducted in 11 patients with hematological malignancies relapsing after allogeneic HSCT (157). Six patients had received other salvage treatments before CIK administration, including DLIs, without significant clinical response. The median number of CIK cells infused was 12.4 *×* 106 /kg, with no infusion-related toxicities recorded. Acute GVHD occurred in four patients 30 days after the last CIK infusion, and progressed into extensive chronic GVHD in two cases. Disease progression and death were observed in six patients. One patient had stable disease, one experienced hematologic improvement, and three obtained complete responses. The same authors differentiated CIK cells from UCB samples and administered them to five patients relapsed after UCB transplantation (162). In three patients, chemotherapy had been given before CIK administration to reduce disease burden. Infusions of a median of 1.5 *×* 106 /kg CIK cells were provided early after leukemia relapse. Some clinical response was observed in one patient who also developed acute intestinal GVHD. The remaining four patients experienced disease progression and died. GVHD was managed with steroids and mesenchymal stromal cells but the patient ultimately succumbed to leukemia relapse (162).

In another study (170), CIK cells were generated for 24 patients with advanced-stage hematological malignancies, mostly from allogeneic donors, either under steady-state conditions or after stem cell mobilization. Overall, 55 infusions were given to 16 patients at doses ranging from 10 to 200 *×* 106 CD3+ cells/kg. Notably, the proportion of the CD3+CD56+ subset was higher in CIK cultures derived from patients than in those differentiated from healthy donors. The median expansion of CD3+ T cells and CD3+CD56+ NK-like T-cells was 9.33-fold and 27.77-fold, respectively. Responses attributable to CIK infusions were documented in five patients, including two with ALL, two with Hodgkin disease, and one with AML. In five patients, the response to CIK cells could not be assessed as salvage chemotherapy or dasatinib was concomitantly administered. Acute GVHD occurred in three patients and was manageable. For three of the six patients failing to respond, leukemia cells were available for *in vitro* killing assays, which showed only modest cytotoxicity of donor CIK cells against tumor targets. Overall, this study in advanced-stage hematological malignancies suggests that CIK administration may translate into some clinical efficacy with a modest toxicity and low incidence and severity of GVHD.

Finally, autologous immune effector cells generated by TG, IFN-γ, and IL-2 (ITG2) can be safely administered to patients with advanced and/or refractory solid tumors (**Figure 2**) (171). After 2–3 weeks in culture, a median of 4.65 *×* 106 immune effectors/kg of recipient's body weight was infused intravenously without observing any toxicity. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 *×* 106 CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. The median survival was 4.5 months (range 1–13) from the first infusion of CIK cells (171).

# **Closing Remarks**

Although the field of NK cell and CIK cell-based immunotherapy is rapidly advancing, some pre-clinical and clinical issues need to be clarified before this immunotherapy approach is widely offered to patients with hematological malignancies and solid tumors (172).

Cell therapy products enriched for NK cells using CD3 depletion and CD56 selection contain variable percentages of monocytes. It is now established that both monocytes and monocyte-derived DCs can support NK-cell proliferation and function (173). This implies that different NK-cell manufacturing protocols may affect the cellular composition of the final products and impact on NK-cell function. Also, the optimal number of NK cells to be infused remains to be determined. Patients reportedly tolerate target doses of 2 *×* 107 NK cells/kg without any serious side effect (78). This number of NK cells can be routinely obtained from a 1-day large-volume leukapheresis. However, it is possible that higher doses and/or multiple infusions of NK cells may be required for optimal clinical efficacy. Whether NK cells may exert their beneficial effects pre-emptively in patients with disease remission, or rather in the context of HSCT warrants particular attention (73). In this respect, KIR ligand mismatch is expected to mediate a more powerful effect in T-cell-depleted HSCT, since alloreactive T cells reportedly blunt NK reactivity

4 http://www.cik-info.org/index.php?kat=tableclinicaltrials

5 http://oldweb.cqvip.com

(37, 174). Finally, recent discoveries on NK-mediated allorecognition will guide the choice of the optimal NK-cell donor in order to maximally exploit the anti-tumor effect (33). The isolation of single KIR+ NK cells under GMP conditions is feasible and yields clinically applicable numbers of alloreactive NK cells (65).

# **References**

Current clinical evidence also points to CIK cells as a potentially useful immunotherapy approach for cancer patients. Similar to other forms of immunotherapy, the infusion of CIK cells may be more efficacious at disease stages where the tumor burden is relatively low or in an adjuvant setting, rather than for advanced disease (175).

expanded CD8+ NK-T cells in the treatment of lymphoma. *Biol Blood Marrow Transplant* (2001) **7**:532–42. doi:10.1016/S1083-8791(01)70014-6

but does not mediate tumor regression. *Clin Cancer Res* (2011) **17**:6287–97. doi:10.1158/1078-0432.CCR-11-1347

optimized manufacturing protocol. *Front Oncol* (2013) **3**:118. doi:10.3389/ fonc.2013.00118

patients relapsing after allogeneic stem cell transplantation: a phase I study. *Haematologica* (2007) **92**:952–9. doi:10.3324/haematol.11132

175. Rutella S, Locatelli F. Is there a role for cytokine-induced killer cells in cancer immunotherapy? *Immunotherapy* (2012) **4**:867–9. doi:10.2217/imt.12.89

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Pittari, Filippini, Gentilcore, Grivel and Rutella. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The Application of Natural Killer Cell Immunotherapy for the Treatment of Cancer

#### *Katayoun Rezvani1 \* and Rayne H. Rouce2,3*

*1Department of Stem Cell Transplantation, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA, 2Department of Pediatrics, Texas Children's Cancer and Hematology Centers, Baylor College of Medicine, Houston, TX, USA, 3Center for Cell and Gene Therapy, Baylor College of Medicine Houston Methodist Hospital and Texas Children's Hospital, Houston, TX, USA*

*Edited by:*

*Rafael Solana, University of Cordoba, Spain*

#### *Reviewed by:*

*Kamalakannan Rajasekaran, Blood Research Institute, USA Alejandra Pera, Instituto Maimonides de Investigaciones Biomédicas de Córdoba, Spain*

> *\*Correspondence: Katayoun Rezvani<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology*

> *Received: 24 September 2015 Accepted: 29 October 2015 Published: 17 November 2015*

#### *Citation:*

*Rezvani K and Rouce RH (2015) The Application of Natural Killer Cell Immunotherapy for the Treatment of Cancer. Front. Immunol. 6:578. doi: 10.3389/fimmu.2015.00578*

Natural killer (NK) cells are essential components of the innate immune system and play a critical role in host immunity against cancer. Recent progress in our understanding of NK cell immunobiology has paved the way for novel NK cell-based therapeutic strategies for the treatment of cancer. In this review, we will focus on recent advances in the field of NK cell immunotherapy, including augmentation of antibody-dependent cellular cytotoxicity, manipulation of receptor-mediated activation, and adoptive immunotherapy with *ex vivo*-expanded, chimeric antigen receptor (CAR)-engineered, or engager-modified NK cells. In contrast to T lymphocytes, donor NK cells do not attack non-hematopoietic tissues, suggesting that an NK-mediated antitumor effect can be achieved in the absence of graft-vs.-host disease. Despite reports of clinical efficacy, a number of factors limit the application of NK cell immunotherapy for the treatment of cancer, such as the failure of infused NK cells to expand and persist *in vivo*. Therefore, efforts to enhance the therapeutic benefit of NK cell-based immunotherapy by developing strategies to manipulate the NK cell product, host factors, and tumor targets are the subject of intense research. In the preclinical setting, genetic engineering of NK cells to express CARs to redirect their antitumor specificity has shown significant promise. Given the short lifespan and potent cytolytic function of mature NK cells, they are attractive candidate effector cells to express CARs for adoptive immunotherapies. Another innovative approach to redirect NK cytotoxicity towards tumor cells is to create either bispecific or trispecific antibodies, thus augmenting cytotoxicity against tumor-associated antigens. These are exciting times for the study of NK cells; with recent advances in the field of NK cell biology and translational research, it is likely that NK cell immunotherapy will move to the forefront of cancer immunotherapy over the next few years.

Keywords: natural killer cells, adoptive immunotherapy, CAR NK cells, ADCC, anti-KIR antibody, NK-92, transplantation

# INTRODUCTION

Natural killer (NK) cell-mediated cytotoxicity contributes to the innate immune response against various malignancies, including leukemia (1, 2). The antitumor effect of NK cells is a subject of intense investigation in the field of cancer immunotherapy. In this review, we will focus on recent advances in NK cell immunotherapy, including augmentation of antibody-dependent cytotoxicity, manipulation of receptor-mediated activation, and adoptive immunotherapy with *ex vivo*-expanded, chimeric antigen receptor (CAR)-engineered, or engager-modified NK cells.

# BIOLOGY OF NK CELLS RELEVANT TO ADOPTIVE IMMUNOTHERAPY

Natural killer cells are characterized by the lack of CD3/TCR molecules and by the expression of CD16 and CD56 surface antigens. Around 90% of circulating NK cells are CD56dim, characterized by their distinct ability to mediate cytotoxicity in response to target cell stimulation (3, 4). This subset includes the alloreactive NK cells that play a central role in targeting leukemia cells in the setting of allogeneic hematopoietic stem cell transplant (HSCT) (5). The remaining NK cells, predominantly housed in lymphoid organs, are CD56bright, and although less mature ("unlicensed") (3, 6, 7), they have a greater capability to secrete and respond to cytokines (8, 9). CD56bright and CD56dim NK cells are also distinguished by their differential expression of FcγRIII (CD16), an integral determinant of NK-mediated antibody-dependent cellular cytotoxicity (ADCC), with CD56dim NK cells expressing high levels of the receptor, while CD56bright NK cells are CD16 dim or negative (6). In contrast to T and B lymphocytes, NK cells do not express rearranged, antigenspecific receptors; rather, NK effector function is dictated by the integration of signals received through germ-line-encoded receptors that can recognize ligands on their cellular targets. Functionally, NK cell receptors are classified as activating or inhibitory. NK cell function, including cytotoxicity and cytokine release, is governed by a balance between signals received from inhibitory receptors, notably the killer Ig-like receptors (KIRs) and the heterodimeric C-type lectin receptor (NKG2A), and activating receptors, in particular the natural cytotoxicity receptors (NCRs) NKp46, NKp30, NKp44, and the C-type lectin-like activating immunoreceptor NKG2D (9).

The inhibitory KIRs (iKIRs) with known HLA ligands include KIR2DL2 and KIR2DL3, which recognize the HLA-C group 1-related alleles characterized by an asparagine residue at position 80 of the α-1 helix (HLA-CAsn80); KIR2DL1, which recognizes the HLA-C group 2-related alleles characterized by a lysine residue at position 80 (HLA-CLys80); and KIR3DL1, which recognizes the HLA-Bw4 alleles (9, 10). NK cells also express several activating receptors that are potentially specific for self-molecules. KIR2DS1 has been shown to interact with group 2 HLA-C molecules (HLA-C2), while KIR2DS2 was recently shown to recognize HLA-A\*11 (10, 11). Hence, these receptors require mechanisms to prevent inadvertent activation against normal tissues, processes referred to as "tolerance to self." Engagement of iKIR receptors by HLA class I leads to signals that block NK-cell triggering during effector responses. These receptors explain the "missing self " hypothesis, which postulates that NK cells survey tissues for normal levels of the ubiquitously expressed MHC class I molecules (12, 13). Upon cellular transformation or viral infection, surface MHC class I expression on the cell surface is often reduced or lost to evade recognition by antitumor T cells. When a mature NK cell encounters transformed cells lacking MHC class I, their inhibitory receptors are not engaged, and the unsuppressed activating signals, in turn, can trigger cytokine secretion and targeted attack of the virusinfected or transformed cell (13, 14). In parallel, cellular stress and DNA damage (occurring in cells during viral or malignant transformation) results in upregulation of "stress ligands" that can be recognized by activating NK receptors. Thus, human tumor cells that have lost self-MHC class I expression or bear "altered-self " stress-inducible proteins are ideal targets for NK recognition and killing (14–16). NK cells directly kill tumor cells through several mechanisms, including release of cytoplasmic granules containing perforin and granzyme (16–18), expression of tumor necrosis factor (TNF) family members, such as FasL or TNF-related apoptosis-inducing ligand (TRAIL), which induce tumor cell apoptosis by interacting with their respective receptors Fas and TRAIL receptor (TRAILR) (16–19) as well as ADCC (9).

# INTERACTION BETWEEN NATURAL KILLER CELLS AND OTHER IMMUNE SUBSETS

Increasing understanding of NK cell biology and their interaction with other cells of the immune system has led to several novel immunotherapeutic approaches as discussed in this review. NK cells produce cytokines that can exert regulatory control of downstream adaptive immune responses by influencing the magnitude of T cell responses, specifically T helper-1 (TH1) function (20). NK cell function, in turn, is regulated by cytokines, such as IL-2, IL-15, IL-12, and IL-18 (21), as well as by interactions with other cell types, such as dendritic cells, macrophages, and mesenchymal stromal cells (10, 22, 23). IL-15 has emerged as a pivotal cytokine required for NK cell development and maintenance. Whereas mice deficient in IL-2 (historically the cytokine of choice to expand and activate NK cells) have normal NK cells, IL-15-deficient mice lack NK cells (24).

Several cytokines are also known to inhibit NK cell activation and function, thus playing a crucial role in tumor escape from NK immune surveillance. Recently, considerable attention has been paid to the inhibitory effects of transforming growth factor-beta (TGF-β*)* and IL-10 on NK cell cytotoxicity (12, 25, 26). Several groups have shown that secretion of TGF-β by tumor cells results in downregulation of activating receptors, such as NKp30 and NKG2D, with resultant NK dysfunction (25, 26). Similarly, IL-10 production by acute myeloid leukemia (AML) blasts induces upregulation of NKG2A with significant impairment in NK function (3).

# MODULATION OF ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY

The CD56dim subset of NK cells expresses the Fcγ receptor CD16, through which NK cells mount ADCC, providing opportunities for its modulation to augment NK effector function (27, 28). In fact, a number of clinically approved therapeutic antibodies targeting tumor-associated antigens (such as rituximab or cetuximab) function at least partially through triggering NK cell-mediated ADCC. Several studies using mouse tumor models have established that efficient antibody–Fc receptor (FcR) interactions are essential for the efficacy of monoclonal antibody (mAb) therapy, a mainstay of cancer therapy (28, 29). Based on this premise, Romain et al. successfully engineered the Fc region of the IgG mAb, HuM195 targeting the AML leukemia antigen CD33, by introducing the triple mutation S293D/ A330L/I332E (DLE). Using timelapse imaging microscopy in nanowell grids (TIMING, a method of analyzing kinetics of thousands of NK cells and mAb-coated targets), they demonstrated that the DLE-HuM195 antibody increased both the quality and quantity of NK cell-mediated ADCC by recruiting NK cells to participate in cytotoxicity via CD16-mediated signaling. NK cells encountering DLE-HuM195-coated targets induced rapid target cell apoptosis by promoting conjugation to multiple target cells (leading to increased "serial killing" of targets), thus inducing apoptosis in twice the number of targets as the wild-type mAb (27).

Additional approaches under investigation to enhance NK cell-mediated ADCC include antibody engineering and therapeutic combination of antibodies predicted to have synergistic activity. For example, mogamulizumab (an anti-CCR4 mAb recently approved in Japan) is defucosylated to increase binding by FcγRIIIA and thereby enhances ADCC. Mogamulizumab successfully induced ADCC activity against CCR4-positive cell lines and inhibited the growth of EBV-positive NK-cell lymphomas in a murine xenograft model (30). These findings suggest that mogamulizumab may be a therapeutic option against EBV-associated T and NK-lymphoproliferative diseases (30). Obinutuzumab (GA101) is a novel type II glycoengineered mAb against CD20 with increased FcγRIII binding and ADCC activity. In contrast to rituximab, GA101 induces activation of NK cells irrespective of their inhibitory KIR expression, and its activity is not negatively affected by KIR/HLA interactions (31). These data show that modification of the Fc fragment to enhance NK-mediated ADCC can be an effective strategy to augment the efficacy of therapeutic mAbs (31).

Although enhanced NK-mediated ADCC occurs in the presence of certain mAbs, in the case of non-engineered mAbs (such as rituximab), this NK-mediated cytotoxicity is typically still under the jurisdiction of KIR-mediated inhibition. However, ADCC responses can be potentiated *in vitro* in the presence of antibodies that block NK cell inhibitory receptor interaction with MHC class I ligands (32). These include the use of anti-KIR Abs to block the interaction of iKIRs with their cognate HLA class I ligands. To exploit this pathway pharmacologically, a fully humanized anti-KIR mAb 1-7F9 (IPH2101) (33) with the ability to block KIR2DL1/L2/L3 and KIR2DS1/S2 was generated. *In vitro*, anti-KIR mAbs can augment NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts and autologous CD138+ multiple myeloma (MM) cells (34). Additionally, in a dose-escalation phase 1 clinical trial in elderly patients with AML, 1-7F9 mAb was reported to be safe and could block KIRs for prolonged periods (35). A recombinant version of this mAb with a stabilized hinge (lirilumab) was recently developed. Lirilumab is a fully humanized IgG4 anti-KIR2DL1, -L2, -L3, -S1, and -S2 mAb. The iKIRs targeted by lirilumab collectively recognize virtually all HLA-C alleles, and the blockade of the three KIR2DLs allows targeting of every patient without the need for prior HLA or KIR typing (33, 34). Furthermore, the combination of an anti-KIR mAb with the immunomodulatory drug lenalidomide was shown to potentiate ADCC and is being tested in a phase 1 clinical trial in patients with MM [NCT01217203 (35)]. A potential concern is related to how inhibitory KIR blockade may impact on the ability of NK cells to discriminate self, healthy cells from abnormal virally infected or cancerous cells. Preliminary *in vitro* data suggest that Ab blockade of iKIRs will preferentially augment the ADCC response, without increasing cytotoxicity against self healthy cells (32). It is reassuring that in the IPH2101 phase 1 studies, no alterations in the expression of major inhibitory or activating NK receptors or frequencies of circulating peripheral lymphocytes were reported, indicating that the Ab does not induce clinically significant targeting of normal cells by NK cells (35). Lin et al. recently reported on the application of an agonistic NK cell-targeted mAb to augment ADCC (36). Following FcR triggering during ADCC, expression of the activation marker CD137 is increased. Agonistic antibodies targeting CD137 have been reported to augment NK-cell function, including degranulation, secretion of IFN-γ, and antitumor cytotoxicity in *in vitro* and *in vivo* preclinical models of tumor (36–39). The combination of the agonistic anti-CD137 antibody with rituximab is currently being evaluated in a phase 1 trial in patients with lymphoma [NCT01307267 (35–37)].

Other factors, such as specific CD16 polymorphisms and NKG2D engagement, can also influence ADCC, with certain polymorphisms (such as FcγRIIIa-V158F polymorphism) resulting in a stronger IgG binding (40). These findings are clinically relevant, as supported by the observation that patients with non-Hodgkin lymphoma (NHL) with the FcγRIIIa-V158F polymorphism experienced improved clinical response to rituximab (41, 42). In summary, several antibody combinations designed to boost ADCC have shown promising results in preclinical and early clinical trials, thus warranting further study of this strategy to enhance NK cell activity against tumor cells.

# ADOPTIVE TRANSFER OF AUTOLOGOUS NK CELLS

The early studies of adoptive NK cell therapy focused on enhancing the antitumor activity of endogenous NK cells (43). Initial trials of adoptive NK therapy in the autologous setting involved using CD56 beads to select NK cells from a leukapheresis product and subsequently infusing the bead-selected autologous NK cells into patients (43, 44). Infusions were followed by administration of systemic cytokines (most commonly IL-2) to provide additional *in vivo* stimulation and support their expansion. This strategy met with limited success due to a combination of factors (44). Although cytokine stimulation promoted NK cell activation and resulted in greater cytotoxicity against malignant targets *in vitro*, only limited *in vivo* antitumor activity was observed (43–45). Similar findings were observed when autologous NK cells and systemic IL-2 were given as consolidation treatment to patients with lymphoma who underwent autologous BMT (46). The poor clinical outcomes observed with adoptive transfer of *ex vivo* activated autologous NK cells followed by systemic IL-2 were attributed to three factors: (1) development of severe life-threatening side effects, such as vascular leak syndrome as a result of IL-2 therapy; (2) IL-2-induced expansion of regulatory T cells known to directly inhibit NK cell function and induce activation-induced cell death (47–49); and (3) lack of antitumor effect related to the inhibition of autologous NK cells by self-HLA molecules.

# EXPLOITING THE ALLOREACTIVITY OF ALLOGENEIC NK CELLS – ADOPTIVE IMMUNOTHERAPY AND BEYOND

An alternative strategy is to use allogeneic instead of autologous NK cells, thus taking advantage of the inherent alloreactivity afforded by the "missing self " concept (13). Over the past decade, adoptive transfer of *ex vivo*-activated or -expanded allogeneic NK cells has emerged as a promising immunotherapeutic strategy for cancer (24, 50–52). Allogeneic NK cells are less likely to be subject to the inhibitory response resulting from NK cell recognition of self-MHC molecules as seen with autologous NK cells. A number of studies have shown that infusion of haploidentical NK cells to exploit KIR/HLA alloreactivity is safe and can mediate impressive clinical activity in some patients with AML (50–52). In fact, algorithms have been developed to ensure selection of stem cell donors with the greatest potential for NK cell alloreactivity for allogeneic HSCT (50).

Promising results in the HSCT setting suggest that the application of this strategy in the non-transplant setting may be a plausible option. Miller et al. were among the first to show that adoptive transfer of *ex vivo-*expanded haploidentical NK cells after lymphodepleting chemotherapy is safe, and can result in expansion of NK cells *in vivo* without inducing graft-vs.-host disease (GVHD) (50). In a phase I dose-escalation trial, 43 patients with either hematologic malignancies (poor prognosis AML or Hodgkin lymphoma) or solid tumor (metastatic melanoma or renal cell carcinoma) received up to 2 × 107 cells/kg of haploidentical NK cells following either low intensity [low-dose cyclophosphamide (Cy) and methylprednisolone or fludarabine (Flu)] or high intensity regimens (Hi-Cy/Flu). All patients received subcutaneous IL-2 after NK cell infusion. Whereas adoptively infused NK cells persisted only transiently following low intensity regimens, AML patients who received the more intense Hi-Cy/Flu regimen had a marked rise in endogenous IL-15 associated with expansion of donor NK cells and induction of complete remission (CR) in five of 19 very high-risk patients. The superior NK expansion observed after high-dose compared to low-dose chemotherapy was attributed to a combination of factors including prevention of host T cell-mediated rejection and higher levels of cytokines, such as IL-15. These findings provided the first evidence that haploidentical NK cells are safe and can persist and expand *in vivo*, supporting the proof of concept that NK cells may be applied for the treatment of selected malignancies either alone or as an adjunct to HSCT (50).

Another pivotal pilot study, the NKAML trial (Pilot Study of Haploidentical NK Transplantation for AML), reported that infusion of KIR-HLA-mismatched donor NK cells can reduce the risk of relapse in childhood AML (51). Ten pediatric patients with favorable or intermediate risk AML in first CR were enrolled following completion of 4–5 cycles of chemotherapy. All patients received a low-dose conditioning regimen consisting of Cy/Flu prior to infusion of NK cells (median, 29 × 106 /kg NK cells) from a haploidentical donor, followed by six doses of IL-2. NK infusions were well tolerated with limited non-hematologic toxicity. All patients had transient engraftment of NK cells for a median of 10 days (range 2–189 days) with significant expansion of KIRmismatched NK cells. With a median follow-up of 964 days, all patients remained in remission, suggesting that donor-recipient HLA-mismatched NK cells may reduce the risk of relapse in childhood AML (51).

Other strategies currently under investigation include the infusion of KIR-ligand-mismatched haploidentical NK cells as part of the pre-HSCT conditioning regimen (NCT00402558), and NK cell infusion to prevent relapse or as therapy for minimal residual disease in patients after haploidentical HSCT (NCT01386619).

# ADOPTIVE NK CELL THERAPY IN SOLID MALIGNANCIES

Natural killer cell-based immunotherapies are also a promising therapeutic option for solid tumors. A number of studies have shown that the presence of intratumoral NK cells correlates with delayed tumor progression and improved outcomes (53–55). However, the successful application of NK cell-based therapies in the solid tumor setting poses a special challenge. In addition to the immune evasion strategies common to hematologic malignancies, such as secretion of immunosuppressive cytokines and downregulation of activating ligands (55–57), additional challenges specific to solid tumors exist; NK cells must not only traffic to sites of disease, but also penetrate the tumor capsule in order to exert their effector function. Furthermore, tumor targets must be inherently susceptible to NK-mediated cytotoxicity (58). Several groups have focused on strategies to alter the tumor microenvironment by targeting myeloid-derived suppressor cells or regulatory T cells (Treg) rather than the tumors themselves (58, 59). In fact, the prospect of combining NK cell-based immunotherapy with approaches to target the immunosuppressive tumor microenvironment or immune checkpoints, such as KIR blockade, is especially relevant to the treatment of solid tumors (55, 58). Several early phase clinical trials have demonstrated the feasibility of adoptive therapy with autologous or allogeneic *ex vivo* activated/expanded NK cells in patients with refractory solid malignancies [NCT01875601 (60)]; however, outside of the post-HSCT setting (namely in neuroblastoma), limited data on the clinical efficacy of NK cells in eradicating solid tumors exist. Currently, several trials are actively recruiting patients with refractory solid tumors for adoptive NK therapy (including NCT01807468, NCT02130869, and NCT0210089).

# THE IDEAL MANUFACTURING STRATEGY FOR *EX VIVO* ACTIVATION OF NK CELLS

Recent approaches to adoptive NK therapy focused on infusion of NK cells that have undergone a process of *ex vivo* cytokine activation and expansion (61). A number of cytokines (IL-2, IL-12, IL-15, IL-18, IL-21, and type I IFNs) have been studied to activate and expand NK cells *ex vivo* (62–65). The most extensively studied cytokine is IL-2 (62, 63). This is not surprising, considering IL-2 was the only cytokine available in clinical grade until recently. Nevertheless, NK cells expanded in the presence of IL-12, IL-15, and IL-18, either alone or in combination, have shown remarkable activity against tumor targets in experimental models and offer an attractive strategy for clinical expansion of NK cells (64, 65). IL-15, in particular, is appealing as it does not stimulate Tregs (65). IL-15 has been tested in preclinical models with promising results; however, very high doses were necessary to observe any meaningful *in vivo* antitumor effects, and toxicity of systemic cytokine administration and cytokine-induced NK-cell apoptosis remained major issues (65). Recently, Miller et al. compared the persistence and *in vivo* efficacy of adoptively infused freshly activated NK cells (FA-NK) and *ex vivo*-expanded NK cells (Ex-NK) in a xenotransplantation model. They showed that *in vivo* NK cell persistence is cytokine dependent, with IL-15 being superior to IL-2. They also reported that cryopreservation of FA-NK or Ex-NK was detrimental to NK cell function, and that culture conditions influence homing, persistence, and expansion of NK cells *in vivo* (66).

Although the results from the abovementioned trials proved that transient persistence of adoptively transferred NK cells obtained via apheresis is feasible and safe, the requirement of a willing, available donor precludes the widespread applicability of this approach. Hence, more recent efforts have focused on optimizing methods for *ex vivo* expansion of NK cells from peripheral blood mononuclear cells (PBMCs) collected by a simple blood draw, with a goal of producing large quantities of purified, functionally active NK cells for clinical use. These expansion strategies include the use of "feeder cells," such as monocytes in the form of irradiated PBMCs, EBV-transformed lymphoblastoid cell lines (EBV-LCLs) or gene-modified, irradiated K562 cells expressing membrane-bound IL-15 or IL-21 and 41BB ligand for costimulation (61, 66–69) in gas-permeable large-scale expansion flasks. These techniques have dramatically increased the yield and activation status of NK cells, potentially overcoming the need for leukapheresis. Because the feeder cells used in these manufacture methods are lethally irradiated prior to use in culture (leaving the remaining feeder cells to be lysed by the expanding NK cells), the risk of infusing viable feeder cells is negligible. However, a number of safeguards have also been incorporated that include monitoring the growth rate of feeder cells and testing for the presence of viable feeder cells at the end of the culture period. Clinical products are, therefore, only released if no viable gene-modified K562 cells or transformed LCLs are present, with strict cutoff values for contaminating B cells and monocytes at the end of the culture period as well (67).

Although these expansion methods can produce large numbers of functionally active NK cells, concomitant expansion of competing cells with immunosuppressive properties, such as Tregs remains problematic. Early studies reported that NK cell infusions from haploidentical donors are able to induce remissions in some patients with AML, but not others (50–52). Several groups, therefore, set out to explore factors that may contribute to the failure of NK expansion *in vivo*. Bachanova explored the effect of competition between Tregs and NK cells in 57 patients with refractory AML who received lymphodepleting chemotherapy followed by NK cell infusion and IL-2 administration [NCT00274846 and NCT01106950 (70)]. Fifteen patients also received the IL-2-diphtheria toxin fusion protein (IL2DT) to deplete Tregs prior to NK cell infusion. IL2DT treatment was associated with increased donor NK cell persistence and improved CR and disease-free survival at 6 months (33 vs. 5% in patients not receiving IL2DT; *P* < 0.01). In the IL2DT cohort, NK cell expansion correlated with higher post-chemotherapy serum IL-15 levels (*P* = 0.002) and effective peripheral blood (PB) Treg depletion (<5%) at day 7 (*P* < 0.01). This study shed light on the importance of optimizing the cytokine milieu to facilitate the *in vivo* expansion of adoptively transferred NK cells and identifying ways to abrogate the immunosuppressive elements, such as regulatory T cells.

Although these data are encouraging, adoptive transfer of NK cells under good manufacturing practices (GMP) requires significant infrastructure and specialized processing equipment, thus limiting the availability and scalability of these NK cell therapies to a few specialized institutions (61). Nonetheless, the feasibility of centralized processing and safe delivery of *ex vivo*-manufactured NK cells for infusion at remote clinics have been demonstrated, suggesting that the practice might become more widespread as procedures are optimized (71). For example, in order to improve access to *ex vivo* activated NK cells and ease the burden associated with producing cellular products at individual treatment centers, the National Heart, Lung, and Blood Institute (NHLBI, Bethesda, MD, USA) sponsored the Production Assistance for Cellular Therapies (PACT) program. Using this approach, activated NK cells have been sent to other centers for infusion into patients (72, 73).

Since the initial reports of successful adoptive transfer of NK cells (50–52), many groups continue to perform extensive preclinical exploration of the ideal manufacturing strategy for *ex vivo* activation and expansion of NK cells. Several expansion methods optimized in the preclinical setting have been successfully scaled up for the clinic (61, 67–70). In addition to the six clinical trials of adoptive NK cell therapy for leukemia that have reported their data (48–52, 70), there are currently 12 active clinical trials enrolling patients with hematologic malignancies for NK cell adoptive therapy, a number that is steadily rising.

# ALTERNATIVE SOURCES OF NK CELLS FOR ADOPTIVE TRANSFER – IS CORD BLOOD THE ANSWER?

Although the majority of clinical studies of NK cell immunotherapy have used PB NK cells, several alternative sources of NK cells exist. These include bone marrow, human embryonic stem cells (hESCs), induced pluripotent stem cells [iPCSs (74, 75)], and umbilical cord blood (CB). While the generation of NK cells from hESCs or iPCS has been largely experimental to date, clinicalgrade generation and expansion of NK cells from CB-derived CD34+ cells has been successfully achieved (76).

Umbilical CB as a source for NK cells lends additional clinical advantages. CB contains a high percentage of NK cells (77, 78) and serves as an immediate "off-the-shelf " source of NK cells, with less stringent requirements for HLA matching, and lower risk of causing GVHD following infusion due to the naivety of the cord T cell repertoire (77, 78). Although no direct comparison of PB- and CB-derived NKs has been performed in the clinical setting, *in vitro* studies have identified a number of differences between CB and PB NK cells. CB NK cells form weaker conjugates with target cells due to the lower membrane expression of adhesion molecules on their surface (79, 80). CB NK cells also express higher levels of lectin-like inhibitory receptors (CD94/ NKG2A) and lower levels of KIRs, indicating an immature phenotype (81). CB NK cells are similarly sensitive to cytokines for *in vivo* expansion and persistence (82). However, it appears that the requirements for *in vitro* expansion of CB NK cells may be different to those required for PB NKs. CB NKs are less responsive to IL-2 stimulation, which may be related to the lower expression of IL-2Rα and reduced activation of the STAT5 signaling pathway as compared with PB NK cells (83). The combination of IL-15 and IL-18, however, can induce significant proliferation and cytokine production by CB NK cells, while the killing capacity of CB NK cells is significantly enhanced after stimulation with IL-15 (83). As with PB-derived NK cells, T-cell contamination is a concern, but can be ameliorated by CD3 depletion. T-cell contamination should be limited to <1–5 × 105 /kg (61) to minimize the risk of GVHD. In addition, CD56+ selection reduces B-cell contamination to <1%, which minimizes passenger B lymphocyte-mediated complications, such as EBV-related post-transplant lymphoproliferative disorder (PTLD) and acute hemolytic anemia.

More recently, efforts have focused on optimizing the largescale expansion of purified CB-derived NK cells. Shah et al. were the first to describe a strategy for expanding NK cells from cryopreserved CB units in which they employed K562-based artificial antigen-presenting cells (aAPCs) expressing membranebound IL-21 (clone 9.mbIL21) (77, 84). The clone 9.mbIL21 cell line is GMP-grade and expresses membrane-bound IL-21, 4-1BB ligand, CD64 (FcγRI), and CD86. After only 14 days of culture in a gas-permeable culture system, mean-fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for CD56+CD3− NK cells. aAPC-expanded CB-NK cells displayed a phenotype similar to that of expanded PB-NK cells and maintained strong expression of the transcription factors eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses and efficiently killed various MM targets *in vitro*. Finally, aAPC-expanded CB-NK cells showed significant *in vivo* activity against MM in a xenogenic mouse model. These findings highlight a clinically applicable strategy for the generation of highly functional CB-NK cells using an aAPC platform, which can be potentially extended to other hematologic malignancies and solid tumors (77). A number of phase I/II clinical trials are underway to test the feasibility and efficacy of CB-NK cell adoptive therapy in patients with hematologic malignancies (NCT01619761, NCT01729091 NCT02280525, NCT01914263, and NCT00412360) (summarized in **Table 1**).

# HUMAN NK CELL LINES AS A SOURCE OF NK IMMUNOTHERAPY

The adoptive transfer of NK cell lines has several theoretical advantages over the use of patient- or donor-derived NK cells. These are primarily related to the lack of expression of iKIRs, presumed lack of immunogenicity, ease of expansion and availability as an "off-the-shelf " product (85). Several human NK cell lines, such as NK-92 and KHYG-1, have been documented to exert antitumor activity in both preclinical and clinical settings (86–88). NK-92, the most extensively characterized NK-cell line, was established in 1994 from the PB of a male Caucasian patient with NHL. NK-92 cells are IL-2-dependent, harbor a CD2+CD56+CD57+ phenotype and exert potent *in vitro* cytotoxicity (86). Infusion of up to 1010 cells/m2 NK-92 cells into patients with advanced lung cancer and other advanced malignancies was well tolerated and the cells persisted for a minimum of 48 h with encouraging clinical responses (86, 88–91). However, potential limitations of using NK cell lines, such as NK-92 cells, include the requirement for irradiation to reduce the risk of engrafting cells with potential *in vivo* tumorigenicity, and the need for pre-infusion conditioning to avoid host rejection. Furthermore, infusion of allogeneic NK cell lines may induce T and B cell alloimmune responses, limiting their *in vivo* persistence and precluding multiple infusions. A number of studies are testing NK-92 cells (Neukoplast®) in patients with solid tumors, such as Merkel cell cancer and renal cell carcinoma, as well as in hematological malignancies (85).

While results from clinical studies of NK cell adoptive therapy are encouraging (48–52, 70), significant gaps remain in our understanding of the optimal conditions for NK cell infusion. Based on the pioneering work from Rosenberg et al. demonstrating the importance of lymphodepletion to support the expansion of tumor-infiltrating T cells (92) and given its emergence as a key determinant of efficacy with CAR therapy, several groups are actively investigating the ideal preparative regimen to promote the expansion and persistence of adoptively infused NK cells (53, 69, 70, 75). Available data support the use of high-dose Cy/ Flu regimen as the frontrunner, considering it is reasonably well tolerated and shown to support the *in vivo* expansion of NK cells

#### TABLE 1 | Published results of NK adoptive immunotherapy trials in hematologic malignancies.

*HR, high risk; haplo, haploidentical; LR, low risk; IR, intermediate risk; Hi-Cy/Flu, high-dose cyclophosphamide and fludarabine; CR, complete remission; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; MDS, myelodysplastic syndromes; SCT, hematopoietic stem cell transplant; HL, Hodgkin lymphoma; NHL, non-Hodgkin lymphoma; pedi, pediatric; CRi, complete remission with incomplete platelet recovery; IL2DT, IL-2-diphtheria fusion protein.*

*aNK cells infused outside of the setting of hematopoietic cell transplantation.*

(51, 70). IL-15 is an ideal candidate cytokine for the expansion of

, especially since it does not promote expansion

of regulatory T cells (66), which have been shown to suppress NK cell effector function in IL-2-based trials (69, 70). In a recent

carcinoma, rhIL-15 was shown to activate NK cells, monocytes, phase 1 study in patients with metastatic melanoma or renal cell

, and CD8 T cells (93). However, as an intravenous bolus dose,

rhIL-15 proved too difficult to administer because of significant

93). Based on these promising data, alternative

NK cells

γδ

clinical toxicities (

CH

the specificity of T cells against leukemia (

an anti-CD19-BB-

with acute lymphoblastic leukemia (

the risk of GVHD. T cell-depleted allogeneic NK cells, by con activated T cells from an allogeneic source are likely to increase -

malignancies treated with haploidentical NK cells (

for patients with cancer.

and, provide an excellent source of off-the-shelf cellular therapy cells provide attractive candidate effector cells to express CARs their shorter lifespan and potent cytolytic function, mature NK

The feasibility of genetically engineering NK cells to express CARs has been shown in the preclinical setting (97, 98). Primary

engineered to express CARs against a number of targets includ human NK cells, as well as NK-92 cells, have been successfully ing CD19, CD20, CD244, and HER2 (97). CAR-transduced

NK cells mediate efficient

*in vitro* and

targets (97, 98) although to date, no clinical data of CAR NK cell

expression of a receptor containing CD3

molecules (anti-CD19-BB-

ζ) in human NK cells after mRNA

reported adequate transfection efficiency 24 h after electropora electroporation using a clinical-grade electroporator. The authors -

tion, with median anti-CD19-BB-

anti-CD19-BB-

purified and 61.3% in expanded NK cells. NK cells expressing ζ secreted IFN-γ in response to CD19-positive

transfect expanded NK cells, achieving excellent receptor expres retroviral transduction. A large-scale protocol was developed to after mRNA transfection were comparable to those achieved by target cells. Interestingly, the levels of CAR expression in NK cells -

sion and considerable cytotoxicity of CAR-transduced NK cells

in xenograft models of B-cell leukemia (

(

cytotoxic against a variety of hematologic and solid malignancies NKG2D ligands on the surface of tumor cells, rendering NK more strategy is the development of CAR-modified NK cells that target

100). NK cells have also been successfully engineered to target

99). Another interesting

ζ expression of 40.3% in freshly

therapy have been reported. Shimasaki et al. recently tested the ζ and 4-1BB signaling

*in vivo* killing of tumor

50

–52). Given

murine models, as well as in patients with leukemia and solid trast, should not cause GVHD, as predicted by observations in

95, 96); however, infusions of

94). Recently, use of

ζ receptor transduced into autologous or allo

geneic T cells produced dramatic clinical responses in patients

Chimeric antigen receptors have been used extensively to redirect

clinical trial for AML (NCT01385423).

dosing strategies are being investigated, including continuous IMERIC ANTIGEN RECEPTOR-MODIFIED NK CELLS

infusion of donor NK cells are currently being tested in a phase I intravenous infusions. To this effect, systemic IL-15 along with

*in vivo*

(51, 70). IL-15 is an ideal candidate cytokine for the expansion of NK cells *in vivo*, especially since it does not promote expansion of regulatory T cells (66), which have been shown to suppress NK cell effector function in IL-2-based trials (69, 70). In a recent phase 1 study in patients with metastatic melanoma or renal cell carcinoma, rhIL-15 was shown to activate NK cells, monocytes, γδ, and CD8 T cells (93). However, as an intravenous bolus dose, rhIL-15 proved too difficult to administer because of significant clinical toxicities (93). Based on these promising data, alternative dosing strategies are being investigated, including continuous intravenous infusions. To this effect, systemic IL-15 along with infusion of donor NK cells are currently being tested in a phase I clinical trial for AML (NCT01385423).

# CHIMERIC ANTIGEN RECEPTOR-MODIFIED NK CELLS

Chimeric antigen receptors have been used extensively to redirect the specificity of T cells against leukemia (94). Recently, use of an anti-CD19-BB-ζ receptor transduced into autologous or allogeneic T cells produced dramatic clinical responses in patients with acute lymphoblastic leukemia (95, 96); however, infusions of activated T cells from an allogeneic source are likely to increase the risk of GVHD. T cell-depleted allogeneic NK cells, by contrast, should not cause GVHD, as predicted by observations in murine models, as well as in patients with leukemia and solid malignancies treated with haploidentical NK cells (50–52). Given their shorter lifespan and potent cytolytic function, mature NK cells provide attractive candidate effector cells to express CARs and, provide an excellent source of off-the-shelf cellular therapy for patients with cancer.

The feasibility of genetically engineering NK cells to express CARs has been shown in the preclinical setting (97, 98). Primary human NK cells, as well as NK-92 cells, have been successfully engineered to express CARs against a number of targets including CD19, CD20, CD244, and HER2 (97). CAR-transduced NK cells mediate efficient *in vitro* and *in vivo* killing of tumor targets (97, 98) although to date, no clinical data of CAR NK cell therapy have been reported. Shimasaki et al. recently tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after mRNA electroporation using a clinical-grade electroporator. The authors reported adequate transfection efficiency 24 h after electroporation, with median anti-CD19-BB-ζ expression of 40.3% in freshly purified and 61.3% in expanded NK cells. NK cells expressing anti-CD19-BB-ζ secreted IFN-γ in response to CD19-positive target cells. Interestingly, the levels of CAR expression in NK cells after mRNA transfection were comparable to those achieved by retroviral transduction. A large-scale protocol was developed to transfect expanded NK cells, achieving excellent receptor expression and considerable cytotoxicity of CAR-transduced NK cells in xenograft models of B-cell leukemia (99). Another interesting strategy is the development of CAR-modified NK cells that target NKG2D ligands on the surface of tumor cells, rendering NK more cytotoxic against a variety of hematologic and solid malignancies (100). NK cells have also been successfully engineered to target antigens on a variety of solid tumors. For example, an NK-CAR targeting the ganglioside GD2 (present on neuroblastoma cells) has been tested in preclinical studies (101, 102). GD2 is also expressed on breast cancer stem cells, thus raising the potential for its widespread use as a target for immunotherapy (103). Additional antigens targeted by NK CARs include HER2 (overexpressed in a number of solid tumors), CD138, and CS1 (overexpressed in MM) (104, 105).

Although these data support the use of CAR engineering to redirect the specificity of NK cells to augment their cytotoxicity, a number of challenges remain. These include the relative difficulty in expressing exogenous genes in primary human NK cells and the need to expand NK cells in culture to achieve adequate numbers for clinical studies of immunotherapy. To counteract this difficulty, some groups have expressed CARs in the human NK-like cell line NK-92, in an attempt to engineer a uniformly cytolytic effector cell population (106). As previously mentioned, NK-92 cells can be easily expanded in culture and their safety has been shown in phase I clinical trials in human subjects. Thus, CAR-expressing NK-92 cells may offer a practical source of cells for NK cell-based immunotherapeutic trials. In order to prevent the risk of engrafting cells with potential *in vivo* tumorigenicity, however, NK-92 cells must be irradiated prior to infusion, which may in turn significantly impact their *in vivo* persistence and long-term antitumor efficacy. Although limited *in vivo* persistence could prove beneficial once the alloreactive NK cells have eradicated the tumors, a number of studies of adoptive therapy with NK cells and CAR-modified T cells have reported the importance of cell persistence in inducing long-term antitumor response (50, 95, 96).

As with CAR-modified T cell therapy, a number of variables can affect the activation, antitumor efficacy, and persistence of CAR-NK cells. Second and third generation CAR constructs incorporating additional costimulatory domains (e.g., CD28, OX-40, or 4-1BB) have been shown to enhance both *in vitro* and *in vivo* activation, and the persistence of CAR T cells. Further studies exploring the optimal vector, construct and transduction method are necessary to identify the "perfect NK CAR."

# SAFETY CONCERNS RELATED TO ADOPTIVE TRANSFER OF CAR-MODIFIED NK CELLS

When considering the use of CAR-modified effector cells, one must take into account their safety profile. Many of the same concerns raised with CAR-modified T cells may be relevant to CAR-NK cells. These include on-target/off-tumor effects, GVHD, cytokine release syndrome, tumor lysis syndrome, and toxicity to normal tissues due to limited selectivity of the target antigen (107–109). Thus, the necessity of equipping CAR-modified NK cells with a "safety switch" or suicide gene is an important question to explore. While mature allogeneic CAR-engineered NK cells are expected to be short lived, data on the persistence of more immature NK cells, such as those derived from CB, are lacking. Interestingly, a recent study reported that IL15/4-1BBLactivated NK cells infused early after T-depleted allogeneic stem

TABL

Reference Burns et al. (43)

aMiller

et al. (50)

aRubnitz and

Fresh-activated NK (FA-NK)

*N* = 10

LR/IR AML (pedi)

Haplo-related donors

Haplo-related HSCT donors

Pre-SCT conditioning

regimen (Bu/Flu/thymo)

(from CD34+ fraction)

Hi-Cy/Flu

Inaba (51)

Yoon et al. (49)

aCurti and

CD56+ selected NKs

AML-CR and

Haplo-related donors

Hi-Cy/Flu

5 × 106 cells/kg followed by

6/13 (46%) remain in CR

IL-2 × 6 doses

relapsed (adult)

*N* = 13

Ruggeri (52)

Stern et al. (48)

NKs

Klingemann and

Apheresis-mobilized CD56

HL, NHL, MM

Haplo donors

None

1 × 105–2 × 107 cells/kg

remission

1° endpoint safety/feasibility; 7/13 in

*N* = 13

Grodman (71)

aBachanova (70)

Choi et al. (116)

*protein.*

*aNK cells infused outside of the setting of hematopoietic cell transplantation.*

Apheresis-mobilized, *ex vivo*

*N* = 41

Haplo donors

Bu/Flu/ATG *HR, high risk; haplo, haploidentical; LR, low risk; IR, intermediate risk; Hi-Cy/Flu, high-dose cyclophosphamide and fludarabine; CR, complete remission; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; MDS,*

*myelodysplastic syndromes; SCT, hematopoietic stem cell transplant; HL, Hodgkin lymphoma; NHL, non-Hodgkin lymphoma; pedi, pediatric; CRi, complete remission with incomplete platelet recovery; IL2DT, IL-2-diphtheria fusion*

Median 1 × 108 cells/kg

Reduced leukemia progression 46 vs. 74%

IL-15/21 induced NK cells

Treg depletion

NK infusion w/IL-2 ± IL2DT

AML

Haplo donors

Hi-Cy/Flu

Mean 2.6 ± 1.5 × 107 cells/

IL-2 alone: 9/42 (21%) CR/CRi

IL2DT: 8/15 CR/CRi (53%)

kg

*N* *N*

= 15 (+IL2DT)

= 42 (IL-2 alone)

selection

1–3 doses positively selected

ALL, AML (adult and

Haplo donors

Pre-SCT conditioning

Median 1.2 × 107 cells/kg

4/16 (25%) alive

regimen

pedi)

*N* = 15

IL-7/15/21 *ex vivo* cultured NKs

 HR ALL/AML/MDS

(adults)

*N* = 14

*Ex vivo* IL-2 activated

Relapsed lymphoma

Autologous

None

autologous NKs or bolus IL-2

IL-2 activated NK cells

HR AML (adults)

Haplo-related donors

Hi-Cy/Flu

1 × 106–2 × 107 cells/kg

5/19 (26%) CR

followed by 14 days IL-2

Median 29 × 106 cells/kg

10/10 (100%) CR at 964 days

followed by IL-2 × 6 doses

Median 2.2 × 106 cells/kg

1° endpoint safety/feasibility; (no toxicity;

low-grade GVHD); 4/14 (28%) alive and well

*N* = 19

*N* = 29

Approach

E 1 | Published results of NK adoptive immunotherapy trials in hematologic malignancies.

Disease

NK source of cells

Conditioning regimen

Dose of cells 4 × 107–8 × 107 cells/kg

1° endpoint safety/feasibility; no change in

outcome compared to historical controls

Outcome

cell transplantation in patients not receiving immunosuppressive prophylaxis could contribute to acute GVHD (110). To this effect, the insertion of a suicide safety switch system, as employed with CAR-modified T cells (111, 112), can provide an efficient means for depletion of these cells if needed. Inducible suicide systems have safely and effectively eradicated GVHD in patients receiving adoptively transferred T cells without causing deleterious effects (112). However, these systems have not been extensively studied in NK cells, and in the absence of clinical data on the *in vivo* persistence of CAR-modified NK cells, the necessity of a suicide switch in this setting remains unknown.

Despite the growing wealth of preclinical experience with CAR-engineered NK cells, to date, only two clinical studies (both targeting CD19+ malignancies using a retroviral transduced anti-CD19-BB-ζ NK-CAR) have obtained regulatory approval: one is a recently completed pediatric study at St. Jude Children's Research Hospital, where haploidentical NK cells modified with anti-CD19- BB-ζ CAR were infused into patients with B-ALL (ClinicalTrials. gov.NCT00995137) and the other is an ongoing study at the National University Hospital in Singapore (ClinicalTrials.gov. NCT01974479) using IL-2-activated haploidentical CAR-modified NK cells in pediatric and adult patients with refractory B-ALL (99). The results of these studies have not been reported to date.

# BISPECIFIC AND TRISPECIFIC ENGAGERS

An innovative immunoglobulin-based strategy to redirect NK cytotoxicity towards tumor cells is to create either bispecific or trispecific antibodies (BiKE, TriKE) (113). BiKEs are constructed by joining a single-chain Fv against CD16 and a single-chain Fv against a tumor-associated antigen (BiKE), or two tumorassociated antigens (TriKE). Gleason et al. showed that bispecific (bscFv) CD16/CD19 and trispecific (tscFv) CD16/CD19/CD22 engagers directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and cytokine production against various CD19-expressing B cell lines. The same group also developed and tested a CD16 × 33 BiKE in refractory

AML and demonstrated that the potent killing by NK cells could overcome the inhibitory effect of KIR signaling (113, 114).

Notably, activated NK cells lose CD16 (FcRγIII) and CD62L through a metalloprotease called ADAM17, which is expressed on NK cells, which may in turn impact on the efficacy of Fc-mediated cytotoxicity (115). Romee et al. recently showed that selective inhibition of ADAM17 enhances CD16-mediated NK cell function by preserving CD16 on the NK cell surface, thus enhancing ADCC (115). Additionally, Fc-induced production of cytokines by NK cells exposed to rituximab-coated B cell targets can be further enhanced by ADAM17 inhibition. These findings support a role for targeting ADAM17 to prevent CD16 shedding and to improve the efficacy of therapeutic mAbs. The same group subsequently discovered that ADAM17 inhibition enhances CD16 × 33 BiKE responses against primary AML targets (114).

## NK CELLS – WHAT DOES THE FUTURE HOLD?

Recent advances in the understanding of NK cell immunobiology have paved the way for novel and innovative anti-cancer therapies. Here, we have discussed a representation of these novel immunotherapeutic strategies to potentiate NK cell function and enhance antitumor activity including ADCC-inducing mAbs, *ex vivo* activated or genetically modified NK cells and bi- or trispecific engagers (**Figure 1**).

Although experience has shown that adoptive immunotherapy with allogeneic NK cells may be more efficacious than

## REFERENCES

with autologous NK cells, to date, their long-term antitumor benefits have been modest (3). Expansion and persistence of NK cells following infusion appear to be the main determinants of clinical response (50–52, 70), thus underscoring the importance of identifying ways to enhance their persistence and antitumor activity. It is likely that the combination of high-dose lymphodepleting chemotherapy with additional modifications (such as Treg depletion, *in vivo* administration of cytokines, such as IL-15 or enhancement of CD16-mediated antigen targeting) may maximize NK persistence and efficacy.

In addition, the possibility of third-party "off-the-shelf " products with partially HLA-matched NK cells from CB, thirdparty donors, or NK cell lines allow the advantage of unlimited sources of cells to improve the practicality of cell therapy. With increasing focus on genetically modifying NK cells to redirect their specificity or engager-modified NK cells, it is likely that NK cells will move to the forefront of cancer therapy over the next few years.

# AUTHOR CONTRIBUTIONS

RR and KR wrote and edited the manuscript.

### FUNDING

The authors' research efforts are funded in part by the Lymphoma Research Foundation (RHR), Lymphoma SPORE (RHR), MDACC Leukemia SPORE Grant CA (KR), and SINF (KR).

monocyte-derived dendritic cells. *Blood* (2008) **112**(5):1776–83. doi:10.1182/ blood-2008-02-135871

and responsiveness to IL-2 but not cytolytic activity. *J Immunol* (2002) **169**(8):4230–6. doi:10.4049/jimmunol.169.8.4230

antigen receptors compares favorably with antibody-dependent cellular cytotoxicity. *OncoImmunology* (2013) **2**(10):e26527. doi:10.4161/onci.26527

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Rezvani and Rouce. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# **Utilizing chimeric antigen receptors to direct natural killer cell activity**

*David L. Hermanson 1,2 and Dan S. Kaufman 1,2 \**

*1 Department of Medicine, Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, MN, USA 2 Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA*

Natural killer (NK) cells represent an attractive lymphocyte population for cancer immunotherapy due to their ability to lyse tumor targets without prior sensitization and without need for human leukocyte antigens-matching. Chimeric antigen receptors (CARs) are able to enhance lymphocyte targeting and activation toward diverse malignancies. CARs consist of an external recognition domain (typically a small chain variable fragment) directed at a specific tumor antigen that is linked with one or more intracellular signaling domains that mediate lymphocyte activation. Most CAR studies have focused on their expression in T cells. However, use of CARs in NK cells is starting to gain traction because they provide a method to redirect these cells more specifically to target refractory cancers. CAR-mediated anti-tumor activity has been demonstrated using NK cell lines, as well as NK cells isolated from peripheral blood, and NK cells produced from human pluripotent stem cells. This review will outline the CAR constructs that have been reported in NK cells with a focus on comparing the use of different signaling domains in combination with other co-activating domains.

#### *Edited by:*

*Francisco Borrego, Cruces University Hospital, Spain*

#### *Reviewed by:*

*Evelyn Ullrich, Goethe University Frankfurt, Germany Cristina Eguizabal, Basque Center for Transfusion and Human Tissues, Spain*

#### *\*Correspondence:*

*Dan S. Kaufman, Department of Medicine, Stem Cell Institute, University of Minnesota, 420 Delaware Street SE, MMC 480, Minneapolis, MN 55455, USA<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology*

> *Received: 23 March 2015 Accepted: 08 April 2015 Published: 28 April 2015*

#### *Citation:*

*Hermanson DL and Kaufman DS (2015) Utilizing chimeric antigen receptors to direct natural killer cell activity. Front. Immunol. 6:195. doi: 10.3389/fimmu.2015.00195* **Keywords: chimeric antigen receptors, natural killer cells, cancer immunotherapy, NK-92 cells, induced pluripotent stem cells**

# **Introduction**

Natural killer (NK) cells are an important component of the innate immune system due to their ability to lyse infected or malignant cells without prior sensitization and without human leukocyte antigens (HLA)-restriction (1). They play an important role in immune surveillance and early control of many malignancies. NK cells recognize infected or transformed cells through multiple cell surface receptors including NKG2D, CD16 [the receptor that mediates antibodydependent cellular cytotoxicity (ADCC)], and natural cytotoxicity receptors (NCRs) such as NKp44, NKp46, and NKp30 (2). These receptors activate signaling adapter proteins such as DAP10, DAP12, and CD3ζ, which contain immuno-tyrosine activation motifs (ITAMs) that initiate the release of cytolytic granules containing perforin and granzymes, as well as mediate production and release of cytokines and chemokines such as IFN-γ and TNF-α (3). Importantly, NK cellmediated cytotoxicity does not rely on the presentation of self HLA. Therefore, NK cells hold significant clinical interest as a cell-based therapy for cancer because of their ability to be used in an allogeneic setting and potentially provide an off-the-shelf cellular product. Clinical trials using NK cells obtained from haploidentical donors demonstrate long-term remissions in patients with refractory acute myelogenous leukemia (4). Trials against solid tumors such as breast cancer and ovarian cancer have also demonstrated efficacy (5). NK cell lines (NK-92 cells) (6) and NK cells derived from umbilical cord blood (7) have also been tested in clinical trials (NCT01729091, NCT02280525).

Chimeric antigen receptors (CARs) are engineered proteins designed to activate lymphocytes, particularly T cells, upon target recognition. CARs contain a single-chain variable fragment (scFv) fused to a variety of possible intracellular signaling domain(s). The scFv is designed to target antigens either overexpressed or unique to tumor cells. The signaling domain initially tested was the ζ chain of the T cell receptor complex CD3 in first generation CARs (8). Second generation CARs employ co-activating proteins such as CD28, CD137 (4-1BB), or CD134 (OX40) in combination with CD3ζ to increase T cell activation and proliferation (9, 10). Finally, CAR constructs incorporate multiple co-activation domains and CD3ζ in the third generation CARs (11). The clinical success of anti-CD19 CAR-expressing T cells for treatment of Bcell malignancies has fueled the design and evaluation of CARs for T cell therapy toward other antigens and malignancies (12). While development of T cell-CAR-based therapies seems to be revolutionizing tumor immunotherapy, one major obstacle with this approach is the need to collect and utilize autologous cells. A second concern with the use of T cells is their long-term persistence, resulting in chronic on-target-off-tumor effects such as B-cell aplasia with the anti-CD19 CARs being used currently in clinical trials.

Natural killer cells provide an alternative to the use of T cells for adoptive immunotherapy since they do not require HLA matching, so can be used as allogeneic effector cells (13). Clinical trials of adoptively transferred allogeneic NK cells demonstrate these cells can survive in patients for several weeks to months (4, 13). Additionally, expression of CARs in NK cells may allow these cells to more effectively kill solid tumors that are often resistant to NK cell-mediated activity compared to hematologic malignancies (especially acute myelogenous leukemia) that are typically more NK cell-sensitive (4, 5). As such, CAR-expressing NK cells have gained significant interest to provide a targeted, allogeneic, "universal" cell population for treatment of refractory malignancies. This review will focus on the CAR constructs and activating domains that have been reported to be used in NK cell lines (such as NK-92 cells) and peripheral blood (PB) NK cells. Additional work using NK cells produced from human pluripotent stem cells is also discussed.

## **Chimeric Antigen Receptors Used in NK Cell Lines**

Natural killer cell lines have been utilized to evaluate CARs targeting several different antigens. By far, the most commonly studied NK cell line has been the NK-92 cell line, which has been previously (6) and is currently being used in clinical trials (NCT00900809 and NCT00990717). Other NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells (14). Working with NK cell lines has several advantages such as providing a more homogeneous cell population compared to NK cells isolated from PB. In addition, the NK-92 cells have been well-defined and there is no need to perform any isolation of the NK cells from donors. However, NK cell lines also have distinct disadvantages. NK-92 cells lack the expression of several typical NK cell activating receptors such as CD16, NKp44, and NKp46 (15, 16). Also, NK-92 cells are tumor cell lines with multiple cytogenetic

#### **TABLE 1 | CAR constructs utilized in NK cell lines (NK-92)**.

abnormalities (17) and are latently infected with Epstein–Barr virus (18). Therefore, for safety purposes, these cells must be irradiated prior to infusion.

The majority of studies to express CARs in NK-92 cells have used first generation CAR constructs that contain CD3ζ as their sole signaling domain. The scFvs of these CARs have targeted CD20 (19–21), CD19 (20–22), ErbB2 (HER2) (23–25), GD2 (26), and CD138 (27) (**Table 1**). In addition to directly targeting cell surface proteins, CARs can also recognize HLA-peptide complexes such as HLA-A2 expressing the melanoma-associated gp100 peptide (28). CARs directed toward CD19 and CD20 are designed to target B-cell malignancies and have also been studied extensively in T cells (29). The only other difference in the anti-CD19 or anti-CD20 CAR constructs used in NK-92 cells is the transmembrane region. One study used the CD3ζ transmembrane sequence (19) while another used the CD8 transmembrane sequence (22). However, without a direct comparison it is unknown if one construct is superior. Another study used an HLA-A2 transmembrane region coupled to a CD3ζ signaling domain (28), suggesting the transmembrane region may be easily altered without impacting CAR expression and functionality. Interestingly, comparison of CAR transfected NK-92 cells with ADCC function using NK-92 cells engineered to express CD16 found that the anti-CD20 CAR engineered cells lysed primary CLL cells more effectively than NK-92 cells acting through ADCC using rituximab (21). This study suggests that even first generation CARs may be an improvement over ADCC-mediated anti-tumor activity by NK-92 cells. It is important to note that NK-92 cells require transfection of CD16 in order to perform ADCC. This leaves open the possibility that PB-NK cells may still be better equipped to perform ADCC better than NK-92 cells.

Solid tumor antigens can also be targeted by first generation CAR constructs expressed in NK-92 cells. An anti-ErbB2 CAR construct against HER2-positive breast, ovarian, and squamous cell carcinoma cell lines mediated improved killing ability of NK-92 cells (23). Additionally, this study showed a reduction in tumor growth using ErbB2-expressing NIH 3.3 cells mixed with NK-92s in a subcutaneous mouse model (23). An anti-GD2 CAR using just the CD3ζ transmembrane and signaling domains was able to target primary glioblastoma cells as well as GD2-positive melanoma and breast carcinomas (26). NK-92 cells can also be targeted against multiple myeloma (MM) using an anti-CD138 CAR with only CD3ζ as a signaling domain (27). Notably, mice bearing a subcutaneous tumor treated with CAR-expressing NK-92 cells survived significantly longer than NK-92 cell alone in a CD138-positive tumor model; whereas, when a CD138-negative MM tumor was used no difference was detected (27). These data clearly demonstrate that first generation CARs are an effective means to induce target cell lysis in NK-92 cells both *in vitro* and in mouse models; however, many of the tumor models are subcutaneous, which may fail to properly recapitulate the complete tumor environment or NK cell trafficking issues.

Second generation CARs expressing a second signaling domain in conjunction with CD3ζ vastly improves the overall activity CAR-expressing T cells (9). This has generated interest in using second generation CARs in NK cells. Similar to first generation CARs, several different scFvs have been used with second generation CARs including EpCAM for multiple carcinomas including breast and ovarian cancer (30), an HLA-A2 EBNA3C complex for Epstein–Barr virus (31), CS1 for MM (32), and ErbB2 for HER2 positive cancers (24, 25). The most common second generation CAR utilized in NK-92 cells pairs the CD28 intracellular domain with CD3ζ (**Table 1**). Notably, NK cells do not naturally express CD28 (35); therefore, the effect that this domain has in NK cells is unclear. Other second generation CARs combine CD137 (4-1BB) intracellular domain with CD3ζ. Similar to first generation CARs, all of the constructs lead to antigen specific killing of target cells, displaying the diverse set of tumor antigens CARs can target. Comparison of an ErbB2 scFv fused with CD3ζ alone, CD28/CD3ζ, or CD137/CD3ζ tested head-tohead against breast cancer cells found that both of the second generation constructs improved killing compared to the first generation CARs (25). Specifically, the CD28/CD3ζ had 65% target lysis in ErbB2-positive MDA-MB453 while the CD137/CD3ζ lysed 62% and CD3ζ alone killed 51% (25). Another modification in their construct design was the modification of a cysteine to a serine in the CD8α signaling peptide used, which the authors suggest improves surface expression of the CAR in NK-92 cells. Finally, CD28/CD3ζ was compared to DAP12 alone using an anti-PSCA CAR in YTS NK cells for prostate cancer (34). In 293T cell lines engineered to express PSCA, a significant increase in cell killing was observed with the DAP12 containing CAR compared to the CD28/CD3ζ CAR, suggesting DAP12 may provide a better signaling domain than CD3ζ (34).

# **Chimeric Antigen Receptor use in Peripheral Blood NK Cells**

Chimeric antigen receptors have also been evaluated in PB-NK cells, which can be isolated from donors through simple blood draws or by apheresis if larger numbers of cells are needed. In contrast to NK-92 cells, activated PB-NK cells express a wider range of activating receptors, such as CD16, NKp44, and NKp46 as well as KIRs, which play an important role in NK cell licensing (36). In addition, PB-NK cells can be given without irradiating the cells so have the ability to expand *in vivo*, which has been correlated with effectiveness in trials involving AML (4). A greater variety of CAR constructs have been used and directly compared in PB-NK cells targeting CD19 (37–39), CD20 (33), or ErbB2 (40, 41) (**Table 2**). Imai et al. describe the use of two first generation anti-CD19 CARs, CD3ζ, or DAP10 signaling, and one second generation CAR with CD137 and CD3ζ. Compared to CD3ζ, DAP10 induced a much weaker response in PB-NK cells, and addition of the CD137 domain to the CAR resulted in augmented killing of RS4:11 and 380 (ALL) cell lines (37). Another study compared CD3ζ or 2B4 alone, 2B4 combined with CD3ζ, and a CD137/CD3ζ anti-CD19 CAR and tested them against the leukemic cell line REH. *In vitro* studies demonstrated the 2B4 alone CAR was slightly less active compared to CD3ζ alone. Comparing the second generation CARs, both were significantly better than CD3ζ alone while similar activity was observed in the 2B4/CD3ζ and CD137/CD3ζ CARs (38). When this work was extended to an anti-GD2 CAR for neuroblastoma with just the CD3ζ and 2B4/CD3ζ endodomains, again the 2B4/CD3ζ was significantly better than CD3ζ alone (38). Another study compared CD3ζ alone with a CD28/CD3ζ CAR using ErbB2 as a target.

#### **TABLE 2 | CAR constructs utilized in PB-NK cells**.

While no direct lysis experiment was performed, similar levels of INF-γ production were observed in PB-NK cells engineered with just CD3ζ or CD28/CD3ζ (41). While different measures were used, the finding that CD28/CD3ζ does not improve activity in PB-NK cells whereas the same construct was found to be more active in NK-92 suggests there may be differences in CAR activation of PB-NK and NK-92 cells.

One unique approach to CAR creation was to use the ectodomain of NKG2D, an NK cell activation receptor, and link it directly to CD3ζ (42). This approach utilizes natural NKG2D ligands commonly overexpressed on malignant cells to active the CAR. Further, NKG2D associates with DAP10 providing a secondary signaling molecule. Indeed, co-expression of DAP10 with the NKG2D/CD3ζ CAR increased surface expression. This CAR was tested against multiple cell lines derived from several malignancies with the best responses demonstrated against ALL, osteosarcoma, prostate carcinoma, and rhabdomyosarcoma (42).

A third source of NK cells suitable for CAR expression are NK cells derived from human pluripotent stem cells – both induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs) (43–47). These NK cells display a similar phenotype to PB-NK cells (43, 44, 48), and hESC/iPSC-NK cells can be grown on a clinical scale (48). iPSC-derived NK cells engineered with a CD4/CD3ζ CAR are able to inhibit HIV replication (49). In these studies, the CAR was expressed in the iPSC cells, which were then differentiated into CAR-expressing iPSC-NK cells. The CD4/CD3ζ iPSC-NK cells were shown to suppress the *in vitro* replication of HIV, providing a platform from which to work for the further development of CAR positive iPSC-NK cells. iPSCderived NK cells combine the best of PB-NK and NK-92 cells since the cells express NKp44, NKp46, and KIRs, are a homogeneous population with no evidence of undifferentiated iPSCs or T cells in the expanded NK cell population. Additionally, CARs can be easily expressed in hESC and/or iPSC-derived NK cells using nonviral gene transfer methods (49, 50). This is in contrast to PB-NK cells that are much more challenging to achieve high levels of stable CAR expression.

# **Outlook**

As the interest in using CARs in not only T cells (10) but also in NK cells continues to grow, there are still a number of questions that remain to be answered. Perhaps most important is what CAR constructs mediate optimal anti-tumor (or anti-viral) activity. Limited studies in NK-92 cells and in PB-NK cells directly compare first and second generation CARs. Second generation CARs in PB-NK cells are generally more active than first generation CARs. Additionally, the use of CD3ζ seems better than DAP10 as the signaling domain (37, 38). In NK-92 cells, DAP12 outperformed a CD28/CD3ζ CAR, but it remains unclear if NK-92 cells provide a good model for how CARs may function in PB-NK cells or hESC/iPSC-derived NK cells. Since NK cells do not naturally express CD28 (35, 51), it is not clear if CD28 is functioning in CAR-expressing NK cells. Different CAR constructs may be required to provide optimal NK cell activation depending on the tumor type or target antigen. More direct comparisons using various intracellular signaling domains and scFvs are needed to best resolve these questions.

Additional research is also needed to determine whether use of an NK cell line (such as NK-92 cells), PB-NK cells, or iPSC-NK cells will provide the best overall benefit. Both 4-1BBL/IL-15 (52) and mbIL-21 (53) artificial antigen presenting cells (aAPCs) can be used to expand PB-NK or iPSC-NK cells (48). Therefore, production of enough NK cells from these sources for clinical use is not a problem. However, it remains to be determined if one aAPC leads to an improved population for adoptive transfer, and the methods to engineer PB-NK cells still need to be further improved. iPSC-NK cells represent an attractive population of cells for NK-CAR therapy because once engineered the iPSC line can be maintained indefinitely and provide an almost limitless supply of NK cells. In addition, careful monitoring of the insertion site of the CAR can be achieved. Finally, NK cell lines provide another alternative but in general express fewer natural NK cell receptors and must be irradiated prior to infusion, which limits *in vivo* expansion and persistence of NK cells.

The method for CAR incorporation provides another important consideration. To get stable expression of CARs, retro- and lentivirus methods have dominated. However, following transduction of NK-92 cells a selection step is usually required to get a pure CAR-expressing population. In PB-NK cells, the efficiencies of gene transfer were at best 69% (37) and ranged as low as 13–24% (38) with most reporting around a 50% transduction efficiency. One way around this issue is the possibility of expressing the CAR in iPSCs and subsequent differentiation into mature NK cells (49), which is done via nucleofection with transposon and avoids the hazards of viral methods. Another consideration is whether the use of suicide systems, such as Cas9 or thymidine kinase (TK), will need to be put in place if unexpected toxicities arise despite the expectation that CAR-expressing NK cells will only circulate for a few weeks (14).

Despite the questions that remain, the ability to engineer NK cells with CARs holds great promise as a novel cellular immunotherapy against refractory malignancies and potentially chronic infectious diseases. The success of T cell-CARs in cases of ALL and CLL has revolutionized the prospects for cell-based immunotherapy. CAR-NK cells can build upon this success to provide important benefits as CAR-based therapy expands. Notably, NK cells can provide a homogenous, offthe-shelf, standardized product that can be used in as an allogeneic product to treat patients. Therefore, this process does not need to be done on a patient-specific basis, as with current T cell-CAR-based therapies. The ability to more potently direct NK cell-mediated cytotoxicity against refractory tumors through the expression of CARs can continue to revolutionize cancer treatment.

## **Author Contributions**

DH performed the review of the literature and wrote the manuscript. DK wrote and edited the manuscript.

# **Acknowledgments**

DH Ph.D., would like to acknowledge the Cancer Research Institute for support through the Irvington Postdoctoral Fellowship award.

# **References**

antigen receptors compares favorably with antibody-dependent cellular cytotoxicity. *Oncoimmunology* (2013) **2**:e26527. doi:10.4161/onci.26527

positive carcinomas. *Proc Natl Acad Sci U S A* (2008) **105**:17481–6. doi:10.1073/ pnas.0804788105

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Hermanson and Kaufman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Genetic manipulation of NK cells for cancer immunotherapy: techniques and clinical implications

#### *Mattias Carlsten\* and Richard W. Childs*

*Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA*

Given their rapid and efficient capacity to recognize and kill tumor cells, natural killer (NK) cells represent a unique immune cell to genetically reprogram in an effort to improve the outcome of cell-based cancer immunotherapy. However, technical and biological challenges associated with gene delivery into NK cells have significantly tempered this approach. Recent advances in viral transduction and electroporation have now allowed detailed characterization of genetically modified NK cells and provided a better understanding for how these cells can be utilized in the clinic to optimize their capacity to induce tumor regression *in vivo*. Improving NK cell persistence *in vivo* via autocrine IL-2 and IL-15 stimulation, enhancing tumor targeting by silencing inhibitory NK cell receptors such as NKG2A, and redirecting tumor killing via chimeric antigen receptors, all represent approaches that hold promise in preclinical studies. This review focuses on available methods for genetic reprograming of NK cells and the advantages and challenges associated with each method. It also gives an overview of strategies for genetic reprograming of NK cells that have been evaluated to date and an outlook on how these strategies may be best utilized in clinical protocols. With the recent advances in our understanding of the complex biological networks that regulate the ability of NK cells to target and kill tumors *in vivo*, we foresee genetic engineering as an obligatory pathway required to exploit the full potential of NK-cell based immunotherapy in the clinic.

 They were initially identified based on their ability to lyse tumor cells without a need for priming (2–5). NK cells are now known to play an important role in host immunity against both cancers and certain viral infections (6–8).

NK cells can mediate cytotoxicity via multiple distinct mechanisms. Degranulation is the most studied cytotoxicity pathway, where NK cells release cytotoxic granules upon contact with the target. Cytotoxicity via this pathway is dictated by a balance of signals from an array of germline encoded activation and inhibitory cell surface receptors. Most activation receptors need simultaneous co-stimulation by other activation receptors to trigger NK cell cytotoxicity (9). One exception from this rule is the Fc receptor CD16, which alone can trigger NK cell degranulation against antibody-coated target cells via antibody-dependent cellular cytotoxicity (ADCC) (9). Other routes by which NK cells can kill targets are the death receptor pathways TRAIL/TRAIL-R and

#### *Edited by:*

*Francisco Borrego, Cruces University Hospital, Spain*

#### *Reviewed by:*

*Claudia Rossig, University Children's Hospital Münster, Germany Antonio Di Stasi, University of Alabama at Birmingham, USA*

#### *\*Correspondence:*

 *Mattias Carlsten, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive (MSC 1230), Building 10-CRC/3E-5332, Bethesda, MD 20892, USA<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology*

> *Received: 19 March 2015 Accepted: 13 May 2015 Published: 10 June 2015*

#### *Citation:*

*Carlsten M and Childs RW (2015) Genetic manipulation of NK cells for cancer immunotherapy: techniques and clinical implications. Front. Immunol. 6:266. doi: 10.3389/fimmu.2015.00266*

Fas/FasL. Instead of triggering release of cytotoxic granules, death receptor pathways prompt apoptosis via caspase activation in target cells.

More than a decade has passed since initial reports established the anti-tumor potential of NK cells in patients with cancer. These studies showed that haplo-identical donor NK cells could prevent relapse in acute myeloid leukemia (AML) following hematopoietic stem cell transplantation (HSCT) and that adoptively infused mature donor NK cells could induce remission in AML patients (6, 10). Despite this revelation, doubts remain about the true therapeutic potential of NK cells in cancer immunotherapy. In contrast to therapy utilizing T cells, enthusiasm for NK cell-based immunotherapy has been tempered by uncertainties about their *in vivo* persistence, and doubts regarding their ability to migrate to tumor tissues following adoptive infusions. Although recent data have shown CMV reactivation reduces the risk for AML relapse following HSCT (11) potentially caused by CMV-induced NK cells cross-reacting with AML cells, NK cells, unlike T-cells, lack antigen specificity, further tempering enthusiasm for their use as immune effectors in cellular therapy.

Genetic manipulation of NK cells to improve their persistence, cytotoxicity, tumor targeting capacity, and ability to home to disease sites *in vivo* holds potential to advance the efficacy of NK cell-based cancer immunotherapy. However, until relatively recently, the genetic manipulation of NK cells has proven to be challenging. Viral transduction, successfully used for T cells, has been associated with low levels of transgene expression and unfavorable effects on cell viability when used with NK cells. Recent optimization of viral transduction and the establishment of electroporation technologies for efficient gene transfection have revived the enthusiasm for studies evaluating genetic modification of NK cells.  One example of how this technique can be utilized is to introduce genes into NK cells coding for gamma-cytokines (IL-2 and IL-15) to induce independence from the obligate need of exogenous cytokines for proper *in vivo* persistence and expansion post infusion. This and similar strategies may further improve the efficacy of NK cell-based immunotherapy, as tumor regression following adoptive NK cell infusions in AML patients has been reported to be dependent on their ability to expand *in vivo* (6), while being limited by regulatory T cells also mobilized following exogenous cytokine administration (12, 13). The introduction of chimeric antigen receptors (CARs) and the down-regulation of inhibitory NK cell receptors such as NKG2A are additional examples of specific genetic manipulations that can be utilized to improve the outcome of adoptive NK cell immunotherapy.

Given their rapid and efficient method of recognizing tumor cells, NK cells represent a unique immune cell to genetically reprogram in an effort to improve the outcome of cell-based cancer immunotherapy. This review focuses on methods for introducing transgenes into NK cells and the advantages and limitations of such strategies. It also gives an overview of strategies for genetic reprograming of NK cells that have been evaluated to date and an outlook on how these specific strategies may be best utilized in clinic to maximize the anti-tumor potential of NK-cell based immunotherapy.

# Methods and Challenges with Genetic Manipulation of NK Cells: Viral Transduction Versus Transfection

Genetic manipulation of T cells has successfully been used in both preclinical and clinical research (14). In contrast, studies on genetically engineered NK cells have historically been limited by poor efficacy of transgene delivery and substantial procedure-associated NK cell apoptosis. In this section, we discuss available approaches for gene delivery into NK cells, characterizing how each approach developed over time while highlighting the positive and negative aspects of each method (**Box 1**).

#### Viral Transduction

The reduced efficacy of viral transduction of NK cells compared to T cells may in part be related to the innate properties that characterize NK cells. Innate immune receptors, such as pattern recognition receptors that recognize foreign genomic material, are likely involved in triggering apoptosis of NK cells following viral transduction (15). Best results from studies of viral transduction of NK cells have been achieved using either NK cell lines or primary NK cells that have undergone expansion *ex vivo* (**Table 1**). In contrast, viral transduction of primary resting human NK cells typically

*a Only those studies that have reported transgene expression following genetic manipulation of NK cell are reported in this table. n.r., not reported.*

results in substantially lower transduction efficiencies. Most studies on viral transduction of NK cells have utilized retro- and lentiviral vectors. Although adenoviral- and vaccinia virus vectors have been utilized for transduction of NK cells, their use has been limited and they will not be discussed further in this review.

*Retroviral vectors* were the first viral vectors used to genetically modify NK cells. The first report on retroviral transduction of NK cells was published in the late 1990s and focused on genetic manipulation of the NK cell line NK-92 (16). This study reported a transduction efficacy of only 2–3%. Optimization of retroviral transduction approaches over the past decade has resulted in higher transduction efficiencies, especially when used with human NK cells that have undergone *ex vivo* expansion (**Table 1**). A recent report showed that retroviral transduction of *ex vivo* expanded NK cells with genes coding for either IL-15 or membrane bound IL-15 (mbIL-15) resulted in an average 69 and 71% transduction efficiency, respectively (25). Although retroviral transduction of NK cells has been reported to not alter the function, phenotype, and proliferative capacity of NK cells (20, 23), their viability following retroviral transduction has rarely been reported. A significant deleterious impact on the viability of primary NK cells undergoing retroviral transduction may preclude utilizing this approach in a clinical setting. Further, retroviral transduction also requires active cell division, impeding the use of this method with primary non-activated NK cells. This limitation is less important when retroviral transduction is utilized with NK cell lines such as NK-92 that have continuous and unlimited proliferation capacity. However, as discussed later in this review, it is important to note that this NK cell line does have phenotypic and function differences from primary human NK cells, which may have therapeutic implications for clinical therapy.

#### Lentiviral Vectors

More recently, studies evaluating transduction of NK cells using lentiviral vectors have been pursued. In contrast to transduction with retro- and adenoviral vectors, lentiviral vectors can incorporate transgenes into the genome of non-dividing cells. Further, lentiviral vectors allow for gene modification of NK cells without altering their phenotypic and functional properties as occurs following stimulation with i.e., cytokines. The first report on the successful use of lentiviral vectors for genetic modification of NK cells was performed in primary murine NK cells (42), with subsequent studies establishing that lentiviral transduction of human NK cells could also be achieved (**Table 1**). Although most studies have reported lentiviral transduction of NK cell lines with efficiencies of 15–40% (27, 28), the efficiency highly varies from only a few percent to nearly 100%, and in some cases, multiple rounds of transduction are required (26, 29). Recent data indicate that transduction efficiencies of primary human NK cells can be increased by drug-induced inhibition of intracellular innate immune receptors in NK cells (15). Unfortunately, and similar to studies utilizing retroviral transduction, the viability of NK cells after lentiviral transduction has rarely been reported. Using an optimized protocol, our lab has achieved a maximum transgene expression in up to 60% of *ex vivo* expanded NK cells 3 days after lentiviral transduction with GFP without incurring any deleterious effects on NK cell viability, phenotype, or function (Personal communication, R. Childs).

In summary, viral transduction of NK cells results in variable transduction efficacies and may require multiple rounds of transduction and/or post transduction cell enrichment to achieve acceptable transgene expression. Further, viral associated cell death and the need for post-transduction enrichment may compromise the clinical utility of this approach. Finally, although the risk may be low, the possibility of viral-induced insertional mutagenesis and immunogenicity (43, 44) occurring post transduction must be considered when utilizing this methodology in the clinic. Nevertheless, viral transduction of NK cells does achieve stable transgene expression which, depending on how the NK cell is being genetically modified, might be required to induce a durable and long-term clinical response.

#### Transfection

Compared to viral transduction, transfection of NK cells appears to be associated with lower degrees of apoptosis, less inter-individual and inter-experimental variability, with transgene delivery efficiency being completely independent of cellular division. In most cases, this approach results in a more rapid albeit transient expression of the transgene as compared to viral transduction where genes must first be incorporated into the cellular genome before expression can occur. Gene transfer using transfection can be achieved by either *electroporation* (including *nucleofection*) or *lipofection*. Since the latter has been used only in a few studies (45), this review will focus on strategies utilizing the electroporation approach.

Electroporation is a method where genetic material is delivered into cells following a short electric pulse that temporarily induces small pores in the cell membrane, allowing charged molecules such as DNA and RNA to move into the cell. This technology was first used with NK cell lines in the late 1990s (32, 36–38, 46, 47) and more recently has been used to genetically manipulate primary NK cells to express CARs (35, 39, 48) or cytokines for autocrine growth stimulation (49). With technological advances and the use of mRNA instead of cDNA, transfection efficiencies have increased dramatically, reaching up to 90% or more while having only a minimal deleterious effect on cell viability (**Table 1**). Remarkably, using mRNA electroporation, transfection efficiencies of 80–90% can be achieved in not only *ex vivo* expanded cells but also in primary resting (non-cytokine activated) human NK cells (40). Despite this remarkable advance, a detailed characterization on the effects of electroporation on the phenotype, function, and proliferative capacity of NK cells following electroporation has yet to be published.

As electroporation does not involve viral vectors, its use in the preclinical and clinical setting is associated with less regulatory issues. Also, as indicated above, electroporation most often leads to transient transgene expression, which may be viewed favorably from a safety perspective when new transgenes with unknown potential toxicities are being explored in early clinical trials. Regimens that use DNA electroporation technology have been employed to generate stable transgene expressing cells. Although the efficacy of this approach is typically lower than that achieved with viral transduction, it may be improved if combined with targeted integration techniques that avoid random integration in inactive heterochromatin regions. Such strategies also reduce the risk for off-target effects, including gene silencing due to random integration in active genes and integration in hot-spots that may trigger malignant transformation. With advantages in design of guiding RNAs and by having better on-target specificity compared to other gene editing technologies such as Zink-Finger nuclease (ZFN) and the transcription activator-like effector nucleases (TALEN) technologies, the recently developed clustered regularly interspaced short palindromic repeats (CRISPR) technique has rapidly become a popular tool for targeted gene integration (50). The CRISPR/ Cas9 system induces permanent modifications at specific sites of the genome via double-strand breaks (DSBs), and can be used to integrate new genes at specific sites via homology-directed recombination (50). Although only moderate degrees of genome integration are currently being achieved with this technique today, the CRISPR/ Cas9 system could be used to produce stably transduced NK cells by gene editing of primary NK cells prior to their *ex vivo* expansion.

# Gene Modification Strategies Aimed at Improving the Efficacy of NK Cell-Based Cancer Immunotherapy

With new advances in the field, genetic manipulation of NK cells has opened up possibilities to study many different pathways involved in NK cell tumor targeting and the ability to genetically modify NK cells to improve their tumor cytotoxicity. Here, we will discuss reported gene modification strategies that can improve *in vivo* persistence and expansion, tumor tissue migration, and the tumor targeting capacity of adoptively infused NK cells (**Figure 1**, **Table 2** and **Box 2**).

#### Strategies to Improve Persistence and Expansion of Infused NK Cells

*In vivo* persistence and expansion of infused NK cells have been shown critical for inducing tumor regression following adoptive NK cell infusion (6). Using retroviral transduction of the IL-2 gene into NK-92 cells, Nagashima et al. were able to propagate this NK cell line for up to 5 months *in vitro* without the addition of exogenous cytokines (16). Further, IL-2-expressing NK-92 cells where shown to also have enhanced tumor cytotoxicity compared to non-transduced parental NK-92 cells that were stimulated with exogenous IL-2. In line with these *in vitro* findings, these genetically modified cells showed improved *in vivo* persistence and anti-tumor responses when infused into tumor-bearing mice. Similar data with IL-2 gene delivery in expanded NK cells were reported by Konstantinidis et al. (51). As observed with IL-2 transduced NK-92 cells, retroviral transduction of *ex vivo* expanded NK cells with the mbIL15 gene also dramatically increased their survival *in vitro*; median cell recovery was 85% for mbIL-15 NK cells after 7 days in culture without IL-2, whereas mock-transduced NK cells were

FIGURE 1 | Schematic overview of how genetic manipulation can be can be used to improve the efficacy of NK cell-based cancer immunotherapy in the clinic. Genetic engineering of NK cells to promote persistence and expansion by autocrine cytokine stimulation, migration to the tumor tissue via introduction of receptors involved in cellular homing (i.e., chemokine receptors and adhesion molecules), as well as bolstering their anti-tumor cytotoxicity via introduction of CARs or activating NK cell receptors (aNKRs) or via silencing of inhibitory NK cell receptors (iNKRs), protection from suppressive cytokines in the tumor environment, and boosted function via autocrine cytokine stimulation.

TABLE 2 | Overview of strategies evaluated for improving the anti-tumor efficacy of primary human NK cells and NK cell lines *in vitro* and in preclinical animal models.

*RV, retroviral transduction; LV, lentiviral transduction; EP, electroporation; ADCC, antibody-dependent cellular cytotoxicity; HA-CD16, high-affinity CD16; DNT*β*RII, double negative TGF-*β *RII.*

# Box 2 | Examples of NK Cell Modalities to Gene Manipulate for Improved Clinical Efficacy.

essentially undetectable (25). Hence, the strategy of introducing genes coding for gamma-cytokines to improve *in vivo* NK cell persistence and expansion following infusion independent of exogenous cytokine administration appears promising.

#### Strategies to Enhance Migration of Infused NK Cells

Proper tumor tissue homing of infused NK cells is a prerequisite for their ability to induce tumor regression. However, studies characterizing the *in vivo* migration capacity of adoptively infused NK cells have been largely overlooked (60). Recent evidence suggests non-expanded and expanded NK cells have different migration patterns when infused into animal models (61). Moreover, using trogocytosis to transfer premade cell surface molecules from a feeder cell line to NK cells, Somanshi et al. have shown that migration of infused NK cells can be redirected by equipping them with the lymph node homing receptor CCR7 (62). Despite these data, no study has so far used gene modification techniques to actively direct infused NK cells to selected organs. Based on data from Somanshi et al., we have been able to use mRNA transfection to genetically engineer NK cells with the CCR7 receptor to improve their migration toward one of its ligands CCL19 (Carlsten M., Manuscript in preparation, April 2015). Other strategies may involve utilizing chemokine receptors, such as CXCR3 to improve NK cell migration to inflamed tissues, such as those infiltrated with metastatic tumors (63).

#### Strategies to Increase Tumor Cytotoxicity by Infused NK Cells

The majority of reports on expression of transgenes in NK cells have characterized the effects of CARs in NK cell lines, expanded NK cells, and primary non-expanded NK cells (**Table 2**). CARs are engineered receptors that have the extracellular specificity of an antibody combined with potent intracellular signaling adaptors such as CD3ζ, CD28 and/or 4-1BB. Importantly, these receptors do not require stimulation through co-receptors to trigger robust anti-tumor cytotoxicity. The recent breakthrough success of anti-CD19 CAR T cell therapy in patients with B cell malignancies has stimulated the research community to develop and investigate a wide array of CARs against multiple different epitopes expressed on numerous tumor types (64). Several of these CARs have been explored in NK cells (**Table 2**). CD19 and CD20 specific CARs against B cell malignancies (39–41, 53, 54), and CARs targeting CD33 on leukemia cells (38), CS1 and CD138 on myeloma cells (24, 48, 55), GD2 on neuroblastoma cells (23, 56), Her2/Neu and erbB2 on breast cancer cells (22, 35), carcinoembryonic antigen (CEA) on colon cancers (36), EpCAM on epithelial tumors (29), GPA7 on melanoma (59), NKG2D ligand on leukemia and solid tumors, and TRAIL-R1 on various tumor targets (58) have all been shown to have the capacity to redirect NK cell cytotoxicity against their target antigens. The majority of these studies have used viral vectors to transduce CARs into the NK cell, albeit electroporation has also been used in a few studies (**Table 2**).

Based on clinical data showing superior response rates in rituximab-treated lymphoma patients homozygous for the highaffinity CD16-158V polymorphism (HA-CD16) compared to those who carry the low-affinity CD16-158F (LA-CD16) polymorphism (65, 66), several groups have recently addressed whether introduction of the HA-CD16 gene into NK cells lacking this polymorphism can be used as a strategy to augment ADCC against tumors. This approach has appeal as only a minority of patients is homozygous for HA-CD16 (67). Moreover, in contrast to CAR NK cells, infusions of NK cells genetically modified to express HA-CD16 may be used to improve the outcome of virtually any malignancy for which there is an FDA approved IgG1 antibody, without the expectation for any severe off target side-effects. *In vitro* experiments conducted by Binyamin and colleagues showed significantly improved cytotoxicity against a rituximab-coated B lymphoma cell line following stable transduction of the CD16 negative NK-92 cell line with HA-CD16 compared to NK-92 cells were equipped with LA-CD16 (19). Recently, our group explored a similar approach, where *ex vivo* expanded NK cells from CD16-158F/F (LA-CD16) donors were found to have substantially augmented ADCC following electroporation with mRNA coding for the HA-CD16 (68). These data suggest the addition of the HA-CD16 gene to patient NK cells that already express endogenous CD16 can be used to augment their ability to induce ADCC, and that this approach could be used as a strategy to improve the efficacy of antibody-based therapies for cancer patients.

Introduction of genes that render NK cells insensitive to suppressive cytokines such as TGF-β, thereby preserving their cytotoxicity, has also been studied. Yang et al. generated an NK-92 cell line resistant to the suppressive effects of TGF-β by genetically modifying them to express the dominant negative mutant form of TGF-β type II receptor (DNTβRII) on their surface (34). Adoptive transfer of these TGF-β insensitive NK-92 cells in lung cancer-bearing mice was associated with increased levels of IFN-γ released from the infused cells and resulted in increased survival rates compared to mice treated with wild-type NK-92.

Genetic reprograming of NK cells may also be directed to achieve specific protein silencing with the aim of improving tumor targeting by circumventing NK cell inhibitory signals induced upon interaction with tumor cells. Initial studies have focused on the use of shRNA technology for this purpose. In this context, shRNAs expressed inside cells are processed by the Dicer endonuclease complex to generate double-stranded small interfering RNAs that prevent translation of their target mRNAs (69), shRNAs have been used successfully to knock-down expression of the HLA-E-binding inhibitory NK cell receptor NKG2A (31). Using an inducible vector in IL-2 activated NK cells, Figueiredo et al. observed a 40% increased killing capacity against the HLA-E expressing cell line K562 HLA-E. Using a similar approach with the NK cell line NKL, our group observed increased killing capacity of HLA-E expressing 721.221 cells *in vitro* and in a preclinical mouse model (70). Further details on protocols for shRNA-mediated protein silencing in NK cells can be found in Purdy et al. (71). To date, studies utilizing CRISPR, ZFN, or TALEN to genetically modify NK cells to silence their inhibitory receptors for the same purpose of enhancing the anti-tumor capacity of NK cells have not yet been reported.

In conclusion, an array of gene modification strategies for NK cells has now been reported. Several of them hold promise for improving clinical responses of NK cell-based cancer immunotherapy. However, to date, few have been translated into clinical studies. The following section will discuss how these strategies can be incorporated in clinical NK cell cancer immunotherapy.

# Considerations for the Development of Clinical Protocols using Genetically Engineered NK Cells

Challenges associated with genetic manipulation of NK cells have significantly delayed the debut of this strategy in clinical cancer therapy. While recently initiated trials (NCT00995137 and NCT01974479) exploiting the role for CAR19-expressing *ex vivo* expanded NK cells in patients with B cell malignancies will give us a first insight into the potential of this approach; further optimization of clinical compliant methods for genetic modifications of NK cells is needed to exploit the full clinical potential of this approach. Moreover, additional research on the multiple aspects of NK cell tumor targeting that could be modified with this technique is needed. Although clinical responses following infusion of NK cells may be further improved by simply augmenting their tumor targeting capacity, studies evaluating the potential of this technology to improve the persistence of infused cells as well as avenues to promote proper NK cell migration and homing to the tumor tissue are also warranted (**Figure 1**).

Genetic engineering of NK cells to make them cytokine independent and thereby improve persistence, while boosting their cytotoxic capacity, may be one avenue to further explore. The advantage with this approach would be that exogenous cytokines would be unnecessary following NK cell infusion, which may reduce the risk of mobilizing regulatory T cells that directly suppress NK cell cytotoxicity (13). Challenges with taking this approach to a clinical context include the risk of inducing a cytokine release syndrome due to massive and unregulated NK cell proliferation. This approach also comes with the potential risk of inducing malignant transformation of the NK cells due to permanent autocrine growth stimulation, as have been observed for IL-2 engineered T cells (72). However, such scenarios may be avoided if genes coding for IL-2 or IL-15 are only temporarily introduced via mRNA electroporation of NK cells. Should stable transgene expression be required to induce proper tumor regression, an alternative strategy to prevent runaway NK cell proliferation would be to introduce an inducible suicide gene in the modified cells (73).

Migration to the tumor tissue is another aspect governing proper tumor targeting. This aspect has been largely overlooked and could potentially improve clinical outcome if infused NK cells are redirected to the tumor site instead of circulating nonspecifically into mostly non-tumor-bearing tissues. No studies aimed at improving the *in vivo* homing of infused gene engineered NK cells have yet been published.

As discussed above, numerous strategies for redirecting or boosting NK cell tumor killing *in vitro* have been explored. Introduction of CARs represent the most studied and developed approach that has recently reached clinical evaluation (**Table 2**). Expression of the high-affinity CD16 may soon also be tested in a clinical setting as this approach can be combined with already clinically available monoclonal antibodies that target an array of antigens expressed on a variety of different tumor types. Bolstering NK cell cytotoxicity via autocrine cytokine stimulation or via silencing of inhibitory NK cell receptors will likely require additional evaluation in preclinical animal models before they can be incorporated in clinical protocols. Once all these strategies are fully characterized pre-clinically, they may be combined to further improve the full anti-tumor potential of adoptively transferred NK cells. For instance, introduction of a CAR while simultaneously silencing the NKG2A inhibitory receptor may represent one such future approach. One can also consider adding autocrine cytokine stimulation to further improve cytotoxicity while simultaneously supporting their *in vivo* persistence. As NK cell degranulation is regulated by a balance of activating and inhibitory signals from well-defined cell surface receptors, it may also be possible to add CARs or other activation receptors together with selected receptors that mediate inhibition via ligands that are expressed on normal tissues (and not tumor cells), thereby giving genetically reprogramed NK cells an additional layer of target specificity. However, many additional preclinical studies will be needed before these approaches can reach clinic.

The choice of method for genetic reprograming of NK cells is another important factor that needs to be considered when taking genetically engineered NK cells to clinical evaluation. Viral transduction has the advantage of stable expression; however, as mentioned above, viral transduction of NK cells, especially primary cells, does not always lead to a satisfactory level of transgene expression and may require multiple rounds of transduction followed by selection of transgene positive cells. Moreover, proper expression of transgenes induced by viral transduction can take days, which may be of disadvantage since the lifespan of an NK cell may be relatively short following adoptive transfer (i.e., weeks). Future studies are warranted to better understand if multiple infusions of transfected NK cells can compensate transient transgene expression or if stable transgene expression is a prerequisite for inducing clinical responses following adoptive transfer of genetically engineered NK cells. Studies are also needed to fully understand the lifespan of NK cells, particularly those that have undergone *ex vivo* manipulation.

The optimal method for genetic manipulation of NK cells to be used in a clinical trial may also depend on what NK cell preparation is used (**Box 3**). The advantage with NK cell lines is that they can be utilized as an off-the-shelf product stably transduced to express the gene or genes of interest. They may also be long-lived if given the proper cytokine support. However, the downside with using NK cell lines, like NK-92, is the requirement for irradiation (10 Gy) prior to infusion to avoid the risk of engrafting cells that are potentially tumorigenic *in vivo* (74). Moreover, patients treated with infusions of NK cell lines would also need moderate to high level of preconditioning to suppress host immunity to avoid rejection of these allogeneic cells. Moreover, infusion of allogeneic cells can raise humoral immunity and lead to adaptive T-cell immune responses specifically against alloantigens, precluding repeated infusions even with the use of preconditioning. Similar allo-reactivity can be induced with the use of primary allogeneic NK cell infusions. The use of autologous NK cells circumvents these risks and precludes the need for preconditioning. The potential draw back with using autologous NK cells is that efficient tumor targeting can be prevented by inhibitory KIR interactions with self-HLA. A potential advantage with using an NK cell line versus primary NK cells is that large numbers of NK cells from the NK cell line can be infused, whereas the number of primary cells available for infusion are typically much more limited. However, this limitation has recently been circumvented by a number of highly efficient methods to expand primary NK cells *ex vivo* for clinical infusion (60). Ideally, infusion of autologous gene-modified NK cells can be used to avoid the rejection risk and the prerequisite for preconditioning. One approach to overcome limitations of autologous NK cell inactivation via self-HLA is to genetically modify these effectors to silence inhibitory self-HLA binding receptors, such as NKG2A and KIRs, which alone or in combination with for instance CARs, can improve the tumor targeting capacity of NK cells in the autologous setting.

# Concluding Remarks

Anti-tumor antibodies and CAR T cells have established immunotherapy as a viable treatment option for patients with cancer. Given their rapid and efficient method of recognizing tumor cells, NK cells represent a unique immune cell to genetically reprogram in an effort to improve the outcome of cell-based cancer immunotherapy. However, technical and biological challenges associated with gene delivery into NK cells have significantly tempered this approach. Viral transduction of NK cells initially resulted in low transgene delivery efficiencies that often required multiple rounds of transduction and/or cellular enrichment to achieve acceptable numbers of transgene expressing cells. Nevertheless, recent improvements in retro- and lentiviral transduction of NK cells have led to a flurry of preclinical studies on gene engineered NK cells. A number of studies have also shown that NK cells can be genetically reprogramed using mRNA electroporation. In contrast to viral transduction, this approach offers high transfection efficiencies without compromising their viability and does not require high-level biosafety laboratories. Although promising preclinical data on mRNA electroporated NK cells have emerged recently, concerns have been raised regarding the clinical utility of this approach as it only results in transient transgene expression.

Recently initiated clinical trials will soon give insight into the potential effectiveness of cell-based cancer immunotherapy strategies that utilize genetically modified NK cells. Nevertheless, further optimization of both viral transduction and electroporation of NK cells is still needed before this approach can be fully exploited in the clinic.

# Acknowledgments

The authors wish to acknowledge all the members of the NHLBI Laboratory of Transplantation Immunotherapy, the NHLBI

### References

Division of Intramural Research (DIR), the Swedish Research Council, the Dean R. O'Neill and Edward Rancic Memorial Cancer Research Fellowship for their many contributions and support for original research described in this manuscript.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Carlsten and Childs. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Natural Killer Cells for Immunotherapy – Advantages of the NK-92 Cell Line over Blood NK Cells

*Hans Klingemann\*, Laurent Boissel and Frances Toneguzzo*

*NantKwest, Inc., Culver City, CA, USA*

Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient's blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects.

#### *Edited by:*

*Francisco Borrego, BioCruces Health Research Institute – Cruces University Hospital, Spain*

#### *Reviewed by:*

*Mar Vales-Gomez, Consejo Superior de Investigaciones Cientificas, Spain Jacki Kornbluth, Saint Louis University School of Medicine, USA*

*\*Correspondence:*

*Hans Klingemann<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology*

> *Received: 29 September 2015 Accepted: 23 February 2016 Published: 14 March 2016*

#### *Citation:*

*Klingemann H, Boissel L and Toneguzzo F (2016) Natural Killer Cells for Immunotherapy – Advantages of the NK-92 Cell Line over Blood NK Cells. Front. Immunol. 7:91. doi: 10.3389/fimmu.2016.00091*

Keywords: NK-92 cells, immunotherapy, cancer therapy, ADCC, cellular cytotoxicity