Beyond CAR-T cells: Natural killer cells immunotherapy

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Review

Beyond CAR-T cells: Natural killer cells immunotherapy夽 María Dolores Corral Sánchez a , Lucía Fernández Casanova b , Antonio Pérez-Martínez a,c,∗ a

Servicio de Hemato-Oncología Pediátrica, Hospital Universitario La Paz, Madrid, Spain Centro Nacional de Investigaciones Oncológicas, Madrid, Spain c Departamento de Pediatría, Facultad de Medicina, Universidad Autónoma de Madrid (UAM), Instituto de Investigación Sanitaria del Hospital Universitario La Paz (IdiPAZ), Madrid, Spain b

a r t i c l e

i n f o

Article history: Received 7 June 2019 Accepted 27 August 2019 Available online xxx Keywords: Immunotherapy Natural killer cells KIR receptors NKG2D CAR-T cells

a b s t r a c t Children and adolescents suffering from refractory leukaemia, relapse after stem cell transplantation, solid metastatic tumour or refractory to conventional treatments still condition a dismal prognosis. The critical role of the immune system in the immunosurveillance of cancer is becoming relevant with the development of new treatments such as the checkpoint inhibitor drugs and genetic modified T lymphocytes, tisagenlecleucel or axicabtagene ciloleucel. In addition, other immunotherapies are being developed such as cell therapy with Natural Killer (NK) lymphocytes. The rapid and potent cytotoxic activity of NK cells respecting healthy cells and the possibility of expansion, manipulating them and combining them with other treatments, make these cells a powerful therapeutic tool to be developed, with a very high safety profile. Furthermore, new strategies are being developed to increase the therapeutic benefit of NK cells such as genetic manipulation for the expression of chimeric antigen receptors. ˜ S.L.U. All rights reserved. © 2019 Elsevier Espana,

Más allá de las células CAR-T, inmunoterapia con linfocitos natural killer r e s u m e n Palabras clave: Inmunoterapia Células natural killer Receptores KIR NKG2D Células CAR-T

A pesar de la mejoría en el pronóstico del cáncer infantil, la recaída o la refractariedad a los tratamientos convencionales todavía condicionan un mal pronóstico. En el momento actual, la investigación en el área de la inmunoterapia, con medicamentos como los inhibidores de puntos críticos de control inmunitario y los linfocitos T modificados genéticamente, tisagenlecleucel o axicabtagene ciloleucel, están revolucionando el tratamiento del cáncer. En paralelo, se están desarrollando otras inmunoterapias como la terapia celular con linfocitos Natural Killer (NK). La rápida y potente actividad citotóxica de las células NK respetando las células sanas y la posibilidad de expandirlas, manipularlas y combinarlas con otros tratamientos, hacen de estas células una poderosa herramienta terapéutica a desarrollar, con un perfil de seguridad muy alto. Además, se están desarrollando nuevas estrategias para incrementar el beneficio terapéutico de estas células como la manipulación genética para la expresión de receptores de antígeno quiméricos. ˜ S.L.U. Todos los derechos reservados. © 2019 Elsevier Espana,

Introduction In recent years numerous advances have been made in acquiring knowledge regarding the role played by the immune system

夽 Please cite this article as: Corral Sánchez MD, Fernández Casanova L, PérezMartínez A. Más allá de las células CAR-T, inmunoterapia con linfocitos natural killer. Med Clin (Barc). 2019. https://doi.org/10.1016/j.medcli.2019.08.008 ∗ Corresponding author. E-mail address: [email protected] (A. Pérez-Martínez).

in the prevention and treatment of neoplasms.1 The greatest success reported so far is that of treatment with genetically modified T lymphocytes (TL) for the expression of chimeric antigen receptors (CAR), such as CD19, which is having a great impact on the prognosis of CD19 positive malignant hemopathies, such as acute lymphoblastic leukemia B (ALL-B) and diffuse large cell lymphoma. However, as we learn more about this therapeutic strategy, we are also starting to recognize its limitations and to propose other strategies that may be complementary or synergistic. These strategies could include immunotherapy with natural killer (NK) lymphocytes.

˜ S.L.U. All rights reserved. 2387-0206/© 2019 Elsevier Espana,

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The cytotoxic activity of NK cells was first described in 1964 in laboratory animals, by Cudkowicz and Stimpfling.2 Later, in 1975, Herberman et al.3 described the NK population in humans as a cell population similar to lymphocytes, with spontaneous cytotoxic capacity against tumoral cells or cells infected by viruses. Karre et al. proposed a hypothesis to explain these findings, namely, the loss of human leukocyte antigen (HLA) class I expression in tumoral or infected cells.4 At the beginning of this century, the importance of the role of NK cells in the clinical context was first described, specifically in the field of hematopoietic cell transplantation (HCT). In this context, NK cells have demonstrated their importance in two aspects: on the one hand is their antileukemic effect mediated by the alloreactive NK cells mainly in myeloid hemopathies and with non-identical HLA donors,5 and on the other, as a post-transplant adoptive immunotherapy strategy, avoiding the risk of Graft versus Host Disease (GVHD) induced by the LT.6–8 Additionally, in recent years, treatment schemes with allogeneic NK cells have been developed, outside the context of HCT, obtained from healthy donors, both at baseline resting cells, and as ex vivo expanded and activated cells. Clinical and preclinical studies have demonstrated its safety and efficacy in various hematological tumors and in solid tumors.9,10 At the present time, there are more than 700 clinical trials that use NK cells as an adoptive immunotherapy for the treatment of various neoplasms registered in Clinicaltrials.gov. The majority only include adult patients. Approximately 180 of these trials are performed in the pediatric population. Table 1 shows some of the results. NK cell biology NK cells are lymphocytes of the innate immune system that function as the first line of defense against viral infections and tumor cells. They make up about 15–29% of the circulating lymphocytes. They trigger a spontaneous response without prior sensitization. They are characterized by expression of the phenotypic surface marker CD56 and the absence of the associated T lymphocyte receptor (CD56+ CD3−). They are produced in the bone marrow from the hematopoietic progenitor and they migrate for differentiation to the lymphatic tissues, spleen, liver, lungs and peripheral blood.1,7,8 Two subtypes of NK cells are distinguished according to the density of expression of the CD56 protein: NK bright and NK dim.1,7 The dim NK cells are characterized by the expression of the receptor of the constant fraction of the immunoglobulins (CD16) and the low expression of CD56, while bright NK cells have a high expression of CD56 and an absence of CD16 and of the killer

NK

Healthy cell

NK

Tumoral/ infected cell

NK

Tumoral/ infected cell

Missing self

Induced self

Figure 1. Missing self hypothesis: the decrease or absence of expression of HLA class I molecules in tumor or infected cells activates NK cells. Induced self hypothesis: tumor or infected cells express stress ligands that are recognized by the activating receptors of NK cells. Activating receptors are shown in red, inhibitors in orange, stress ligands in green, and HLA class I molecules in blue.

immunoglobulin-like receptors (KIR). These phenotypic differences bestow on them functional differences. Thus, the dim NK cells are responsible for antibody-dependent cellular cytotoxicity (ADCC), through the CD16 receptor, and they mediate the immediate innate immune response against tumoral or infected cells. The bright NK cells have immunoregulatory properties due to the production of cytokines such as interferon gamma (IFN-©), granulocyte colony stimulating factor and tumor necrosis factor alpha, among others, in addition to having greater proliferation capacity. In healthy individuals, about 90% of the NK cells of the peripheral blood and spleen are dim NK cells, and most of the NK cells in the lymph nodes are bright NK cells.1 However, it has been shown that cancer patients have a higher percentage of bright NK cells in peripheral blood.11 NK cell receptors and antitumor activity The activity of the NK cells in the immunological synapse is regulated by activating and inhibitory signals that act through various receptors that recognize ligands in the target cells. Under physiological conditions, there is a balance between activating and inhibiting signals.1 The inhibitory receptors identify the cells themselves and transmit signals to induce cell tolerance and prevent autoreactivity. The activating signal is mediated primarily through the NKG2D receptor, which recognizes and binds to the MIC/A, MIC/B and ULBP 1–6 ligands, which are overexpressed in cells subjected to stressful stimuli or with DNA damage. Other activating receptors are the natural cytotoxicity receptors NKp30, NKp46 and NKp44, which bind to ligands such as B7-H6 (tumor cells), BAT3, hemagglutinin and neuraminidase (viral molecules). The DNAM-1 activating receptor (CD226) is a costimulary adhesion molecule that binds to the CD112 or Nectin-2 and CD155 ligands (poliovirus receptor) that are expressed in different cell types, including infected or transformed cells, and it acts in synergy with other activating receptors. They are also activators of the CD16 receptor, which mediates the ADCC, and KIR activators, mainly the KIR2DS1 and KIR3DS1, which bind to the HLA-C2 and Bw4 groups, respectively. The inhibitory signal is mediated primarily through KIR inhibitors (iKIR), which recognize and activate in the absence of HLA class I molecules. Another important inhibitor receptor is NKG2A, which recognizes HLA-E12 and competes with other activating signals, such as NKG2D. Recently, NKG2A-HLA-E interactions have been described as a new cancer immune checkpoint.13 The activation of NK cells after binding the activating receptors to their ligands remains subject to the inhibition signals received through iKIR. Therefore, there is a hierarchy that is maintained in all the receptors known to date, except in the case of the NKG2D activating receptor when the binding to its ligands is capable of overcoming the inhibitory signals provided by the KIRs.14 The binding of the HLA class I molecules to the iKIR generates a signal that inhibits the NK cells, and therefore the healthy tissues that express ubiquitous HLA class I molecules are tolerated. However, the decrease or absence of expression of HLA class I molecules, a situation that occurs when the healthy cells become tumorous or when they are infected by viruses, would activate ¨ the NK cells, which would eliminate them by recognizing missing¨ self(missing-self hypothesis).15 In parallel, cellular stress and DNA damage in infected or transformed cells mediate the expression of s¨ tress ligandst¨ hat are recognized by NK cell activating receptors (induced-self hypothesis).16 Fig. 1 shows these mechanisms of action. Immunotherapy with NK cells In 1980, Professor Rosenberg’s group demonstrated that incubation of human or mouse lymphocytes in media with IL-2 resulted

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Table 1 The most relevant clinical trials based on immunotherapy with NK cells, which show results. Identification

Trial name

N

Administered treatment

Results

NCT00582816

Reduced intensity haploidentical transplantation with NK cell Infusion for pediatric acute leukemia and high risk solid tumors

6

Methylprednisolone, ATG, cyclosporine, Flu, melphalan, thiotepa, and rituximab + haploidentical HCT + NK cell infusion

Biological variables (GVHD) GVHD 3/6 Grade iii skin, could not be analyzed 2/6 Grade iv gastrointestinal, 1/6 Grade iii gastrointestinal

HLA-nonidentical stem cell and 16 Cy, Flu, thiotepa, natural killer cell melphalan, and transplantation for children OKT3 + haploidentical HCT less than two years of age with with TL purging + NK cell high risk hematologic infusion on day +7. Dose of malignancies NK cells: 38.9 × 106 /kg (9.8–102.5)

Not enough data to assess response

Infections 5/6 Metabolic disorders 5/6 Neurological: seizures (2/6), leukoencephalopathy (1/6) GVHD No severe GVHD, a case of chronic limited GVHD

Brandon Triplett, MD

Graft failures 1/16

St. Jude Hospital

Post-HCT lymphoproliferative 1/16 Infections 9/16 Seizures 1/16 Gastrointestinal 2/16 Infections 1/6

Phase II

NCT00526292

Katherine Hsu, MD Memorial Sloan Kettering Cancer Center Phase II NCT01106950

HLA haploidentical natural killer cell infusion for treatment of relapsed or persistent leukemia following allogeneic hematopoietic stem cell transplant

6

Flu/Cy + NK cell infusion

01 patient complete remission and 5 progressed

Adoptive transfer of haploidentical natural killer cells to treat refractory or relapsed AML

15 Flu, Cy and denileucine diftitox + NK cell infusion + IL-2

Survival 50% at 1 year

Three patients died from HCT complications

Died of progression 3/6

53% complete remission 4 weeks after the last dose

Cardiac disorders 2/15

Disease-free survival: 33% at 6 months

Infections 6/15 Renal disorders 3/15

Haploidentical donor NK cell adoptive therapy and double T cell depleted umbilical cord blood transplantation with post-transplant IL-2 immune therapy for refractory acute myeloid leukemia

2

Flu/Cy and TBI + umbilical cord infusion + IL-2 (total 12 doses)

Michael Verneris, MD Masonic Cancer Center, University of Minnesota Phase II NCT00187096

One patient died before day +100

Metabolic disorders 6/6

Jeffrey S. Miller, MD Masonic Cancer Center, University of Minnesota Phase II

NCT00871689

Survival

Graft failures 2/6 primary and 1/6 late

Kenneth DeSantes, MD Wisconsin, United States Phase I–II

NCT00145626

Adverse effects

01 patient remission on day +100

Intracranial hemorrhage 1/15 Respiratory distress 3/15 Leukoencephalopathy 01 patient

Death 2/2

01 patient graft failure and recurrence of underlying disease

Pilot study of haplo-identical natural killer cell transplantation for acute myeloid leukemia

25 From day −1 IL-2 is administered until 2 weeks are completed. The patients were divided into 2 groups:

Duration of NK cell grafting: 10 days.

Infections 2/25

Survival at 2 years: 100, 0 and 45% in each group, respectively

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Table 1 (Continued) Identification

Trial name

N

J. Rubnitz, MD St. Hospital Jude

Phase II

NCT02259348

CD45A-depleted haploidentical 6 hematopoietic progenitor cell and natural killer cell transplantation for hematologic malignancies relapsed or refractory despite prior transplantation

Administered treatment

Results

Adverse effects

a. Group 1 (AML in full remission): Flu/Cy b. Group 2 (Refractory or relapsing AML or with increased minimal residual disease) i. Group 2A: Flu/Cy ii. Group 2B: clofarabine + etoposide + Cy Flu/Cy + IL-2 + NK cells + ATG, rituximab, thiotepa and melphalan + HCT with purging of TL and of CD45RA on day 1. G-CSF from day +6

Chimerism peak reached 7% Donor NK cells detectable on day +28: 29% (7–30%)

Liver dysfunction 1/25

02 patients relapsed per year

Infections 6/6

Brandon Triplett, MD St. Jude Phase II

NCT01621477

Survival

2/6 patients survived without disease 12 months after treatment

Seizures 2/6

T-cell replete haploidentical 17 Clofarabine, cytarabine, donor hematopoietic stem cell busulfan, plerixafor, plus natural killer (NK) cell cyclophosphamide, rabbit transplantation in patients ATG + HCT with hematologic malignancies relapsed or refractory despite previous allogeneic transplant

10 patients relapsed before the year

Brandon Triplett, MD St. Jude Phase II

Pulmonary hemorrhage 1/6 Liver failure 1/6 Renal failure 1/6 Respiratory distress 4/6 GVHD: 3/6 acute grade iii, 1/6 acute grade iv GVHD 8/17 (3 grade iii and 7/17 patients were 01 grade iv). alive one year after HCT, 4 of them disease free

Infections 5/17

Two HCT-related deaths at 100 days

Seizures 1/17 Kidney failure 1/17 Respiratory distress 7/17

ATG: antithymocyte globulin; Cy: cyclophosphamide; GVHD: Graft versus Host Disease; Flu: fludarabine; G-CSF: granulocyte colony stimulating factor; HLA: human leukocyte antigen; TBI: Total body irradiation; IL-2: interleukin 2; AML: acute myeloblastic leukemia; TL: T lymphocytes; NK: natural killer; OKT3: muromonab CD3; HCT: hematopoietic cell transplantation.

in the generation of lymphoid cells capable of lysing tumor cells. These cells were called lymphocyte activated killers (LAK).17 LAK cells showed antitumor activity when injected in vivo in animal models.18 After this, several clinical trials were conducted in patients with solid and hematological tumors.19 Autologous NK cells The first clinical trials in humans using NK cells were based on cell selection using CD56+ lymphocyte immunomagnetic methods from the patient’s apheresis, its infusion and subsequent systemic administration of cytokines to stimulate their proliferationin vivo, essentially IL-2.1 Three important limitations were described with this method: the toxicity of the systemic administration of cytokines (capillary hyperpermeability syndrome), due to high doses of IL-2; the expansion of TL regulators that limit the antitumor effect of the NK cells, as a result of the administration of low doses of IL-2, and the inhibition of autologous NK cells in vivo by recognizing HLA class I in the patient.1 To overcome these limitations other strategies have been developed, such as the use of allogeneic NK cells or the ex vivo expansion and activation of autologous NK cells with cell lines20 or cytokines. Recently, the infusion of ex vivo expanded and activated autologous NK cells with the K562-mbIL15-41BBL and IL-2 cell line, in patients with refractory or relapsed multiple myeloma, in combination with chemotherapy has been described. The infusions were well tolerated and the therapeutic efficacy was relevant.21

Other clinical trials based on the use of autologous NK cells include patients with solid tumors such as glioma.20 A strategy to increase the cytotoxic activity of the NK cells has been the development of anti-KIR monoclonal antibodies, which bind with great affinity to the iKIRs, blocking their binding to the HLA class I molecule. This therapeutic option has been explored in adult patients with acute myeloid leukemia (AML).22

Allogeneic NK cells After characterization of the KIRs, immunotherapy with NK cells focused on the use of allogeneic NKs from healthy donors to exploit the inherent alloreactivity of NK cells,1 in the context of the HCT. The first clinical trial that demonstrated the safety of the infusion of ex vivo allogeneic NK cells expanded with IL2, outside the context of the HCT, was published in 2005 by Miller et al.23 It included 43 patients with metastatic renal carcinoma, metastatic melanoma and AML. NK cells were obtained from peripheral blood apheresis of haploidentical donors. Prior to the infusion, lymphoblastic chemotherapy was administered. No cases of GVHD were observed. A transient response was achieved in 30% of patients with AML. Subsequently, other clinical trials have been published using haploidentical NK cells with KIR-HLA disparity, demonstrating a discrete antitumor activity in both the treatment of leukemia and that of solid tumors, and with a high safety profile in all cases.24

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Other strategies that are currently being investigated include the infusion of NK cells to prevent relapse or as a treatment for minimal residual disease in patients after HCT.25 NK cell lines (ex vivo expanded) Various NK cell lines, such as NK92 and KHYG-1, have demonstrated antitumor efficacy in preclinical and clinical studies.26 The NK92 cell line is currently the only cell line approved by the Food and Drug Administration and the European Medicines Agency for use in clinical trials. It is characterized by the expression of activating receptors and the absence of KIR.1,7,27 Published studies include that of Arai et al., in patients with melanoma and metastatic renal carcinoma,28 and that of Williams et al. in patients with hematologic tumors in relapse after an autologous transplant.29 A good treatment tolerance was observed, with some case of partial response.26 The fundamental advantage of these cell lines is that they can be easily maintained in vitro and expanded under the Good Manufacturing Practices (GMP). In addition, their antitumoral efficacy can be enhanced by the expression of CAR in the NK cells. Among the main limitations for their use are the cost and the need for irradiation to avoid their expansion in vivo. In addition, its infusion could induce the responses of TL and B lymphocytes, limiting their ability to expand and persist in vivo, which would require multiple infusions so as to observe clinical effects.1,27 Use of NK cells in clinical practice Currently, cell therapy with NK is not approved outside of clinical trials. Optimization of donor selection, source selection and production process could impact its use in clinical practice. Donor selection The importance of donor selection for haploidentical HCT was first documented in 2002 by Ruggeri et al.,30 who demonstrated that the NK-cell alloreactivity decreased rejection, improved performance and induced a graft effect against leukemia. Allorreactivity of the NK cells is determined by the analysis of the KIR ligands in the donor and the recipient, and of iKIRs in the donor. The importance of KIR activators in the same context has led to the need to establish the KIR typification as a transcendent process. The KIR family is located on chromosome 19p13.4.8 Academically they are divided into 2 large haplotypes: KIR-A and KIR-B (Fig. 2). The distribution of haplotypes varies according to race and geographic location. In addition, the genetic content is highly polymorphic. The KIR-A haplotype contains only one activating receptor, KIR2DS4, while the KIR-B haplotype contains 2 or more activating receptors, in addition to one or more specific B gene: KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5, KIR2DL2 and KIR2DL5.30 There are studies that show that the donors with KIR-B haplotype and those with a high content of haplotype B genes confer a lower risk of relapse in adult patients with AML.31 Recently, the European Society for Blood and Marrow Transplantation has established its recommendations in the choice of a haploidentical donor including these immunological variables, especially in grafts of haploidentical donors purged in LT,32 so that the optimal donor is selected according to the KIR-B content. Source selection NK cells can be obtained from various sources: peripheral blood, umbilical cord, bone marrow and embryonic cells. Using periph-

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eral blood mononuclear cell collection by leukapheresis is the most widely used source in practice. Peripheral blood The NK cells obtained by leukapheresis are not usually mobilized, since the administration of the granulocyte colony stimulating factor has a detrimental effect on the activation and proliferation capacity of the NK cells. The methodology for selecting NK lymphocytes includes both immunomagnetic methods and expansion methods in culture plates or bioreactors.9,33–35 Umbilical cord Umbilical cord blood has some advantages, such as the high content of NK cells and the lower risk of GVHD due to the immaturity of its TL. However, cord-derived NK cells have an immature phenotype (determined by a greater expression of inhibitory receptors such as NKG2A and a lower KIR expression) and not such a strong binding to the target cells, due to a lower expression of adhesion molecules.36 Recently, large-scale production of NK cells from umbilical cord has been achieved from cryopreserved cord units, co-cultivated with the genetically modified K562 line to express IL-21, 41BB, CD64 and CD86 on their surface.37 The NK cells cultured in this manner are capable of expanding while active against different lines of multiple myeloma. Phase i/ii clinical trials are testing its viability and efficacy in various hematological malignancies (NCT01619761, NCT01729091, NCT02280525). Umbilical cord blood CD34+ cells can also be selected using immunomagnetic methods and can then be generated in static culture media or in bioreactors. Large-scale production by these methods was used in a phase i trial, with elderly patients with AML.38 Cells obtained in this way express high levels of the NKG2D receptor, thus being effective against leukemic cells. Bone marrow Due to the logistical complexity of obtaining bone marrow stem cells compared to apheresis, there is little clinical experience in the production of NK cells from this source.39 Embryonic stem cells NK cells derived from embryonic stem cells or induced pluripotent stem cells have been produced on a clinical scale.40 There are different protocols capable of producing and expanding phenotypically mature and cytotoxic NK cells from embryonic stem cells or induced pluripotent stem cells, including culture with murine stromal cells and various cytokines for 30 days, or the use of an embryonic body, followed by culture with different cells presenting artificial antigens (APC) and cytokines.41 Production of NK cells for clinical use The amount of infused NK cells and their degree of activation are important for optimizing the antitumor efficacy, and even more so when there is a lack of alloreactivity. Therefore, the expansion and activation is necessary. Different protocols have been developed, such as the stimulation with soluble cytokines (IL-2, IL-15, lL-18, IL-21) or by co-cultivation with APC (cells genetically engineered to express antigens). Adapting both strategies to the clinical scale has allowed the production of a large number of functional NK cells under GCP conditions, for use in clinical trials.34,35 The immunomagnetic selection of NK cells and cytokine stimulation NK cells are obtained by peripheral blood leukapheresis in a closed aseptic system. The process is carried out by immunomagnetic selection in the CliniMACS System or Prodigy® (Miltenyi) in 2 sequential steps: first is the TL purging and then the NK cells are

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Tel A

Cen A

3DL3

3DL3

2DL3

2DS2

2DP1

2DL2

2DL5B

2DL1

2DS3/5

3DP1

2DL1

Cen B

3DP1

2DL4

2DL4

3DL1

3DS1

2DS4

2DL5A

2DS3/5

3DL2

2DS1

3DL2

Tel B

Figure 2. KIR-A and KIR-B haplotypes. The distribution of the different genes provides the possibility of more than 80 KIR haplotypes.

selected, to obtain a practically pure population.33 This is obtained by using magnetic labeling with the murine monoclonal antibodies CD3 and CD56 conjugated with iron-dextran particles, by means of a purging program in the case of the TLs, followed by an enrichment program for selection of the NK cells. This cellular product can be infused without further manipulation, or after it is expanded in culture media or in bioreactors using different cytokines (IL-2, IL-15, IL-12, IL-18, IL-21 or IFN-γ) either alone or in combination. Most published protocols use IL-2. An attempt has been made to optimize NK cell expansion with the help of other cytokines such as IL-15, improving viability and product expansion. The use of IL-15 in vitro increases the expression of activating receptors, thereby improving the cytotoxicity of the final product, avoiding cellular senescence of the product. The IL-2 and IL-15 cytokines have different effects on NK cells. In addition, the toxicities of both cytokines are different. IL-15 has a lower risk of capillary leak syndrome than IL-2. These data show that IL-15 may be a good candidate for use in immunotherapy, but although it may have some advantages, it seems that lengthy stimulation with IL-15 could have a leukemogenic effect, through activating the JAK/STAT pathway, especially STAT3 and STAT5. Therefore, it should be used with caution.42 Recently, Miller et al. published a study43 in which recombinant IL-15 was administered subcutaneously in ascending doses to 19 adult patients with refractory solid tumors. The treatment was well tolerated except in 3 patients, to whom it was suspended due to adverse effects. Other groups in the context of early clinical trials used stimulated NK cells for 12–18 h ex vivo with IL-15, with a good toxicity profile and acceptable efficacy. This method increases the expression of activating receptors and of activation markers with respect to the baseline NK cells.9,34

Activation and expansion system with APC Several expansion methods have been described ex vivo with different APCs. One of the methods most used is that developed by Professor Campana’s group in 2009.33 This method uses the genetically modified K562 leukemic cell line to express in its IL15 membrane and the CD137 ligand (41BBL), which is a potent costimulatory molecule (K562-mb15-41BBL line). These cells naturally lack HLA on their surface, which adds to the activation of NK cells using the missing self mechanism. Their co-culture with mononuclear cells in the presence of IL-2 causes expansion and activation of NK cells, with an increase in their cytotoxicity (Fig. 3). Mononuclear cells obtained by peripheral blood leukapheresis are incubated in a culture medium by adding IL-2. In this way, expansions of up to 2–3 logarithms can be achieved, with a high antitumoral activity and a high purity of NK cells after 14–21 days of culture.33–35 In addition, these activated and expanded NK cells (NKAE) present a genetic expression profile that includes the coding of cytokines and their receptors, adhesion molecules and molecules involved in the stimulation of TL. Several clinical trials have shown that the safety profile of this cellular product is promising, and its infusion is generally well tolerated.34

A potential disadvantage of the NKAE method is that the prolonged culture time can cause senescence in the NK cells, reducing their expandability in vivo after infusion to patients. This effect is due to telomere shortening caused by high replicative activity and genomic instability caused by high doses of IL-15.44 One way to avoid this obstacle could be the use of the modified cell line K562-mbIL21-41BBL, developed by Somanchi and Lee in 2016.45 This cell line allows an elevated expansion of NK cells with activated phenotype and greater cytotoxic capacity than the baseline NK maintaining the length of their telomeres. Several clinical trials are underway with this method (NCT01787474, NCT02809092, NCT03348033) and optimal safety and efficacy results have already been published.46 Genetic modification of NK cells and combination with other treatments to increase their clinical efficacy Genetically modified NK cells: CAR-NK Similar to therapy with CAR-T cells, it is possible to transfer genes to the NK cells of chimeric receptors of different tumorspecific antigens. The specificities found in clinical trials are CD19, CD22, CD33, PSMA, Her2, CD7, mesothelin, GD2 and NKG2D.47,48 As an advantage over the CAR-T cells, the NK cells may be safer, since they do not produce GVHD and they lack the ability to expand clonally, which would reduce the likelihood of developing and prolonging the cytokine release syndrome. In addition, they can eliminate tumor cells through various receptors, which would reduce the escape capacity by decreased expression of the target antigen. However, its reduced half-life may be a disadvantage, since a certain amount of persistence is necessary to provide long-term antitumor effects. Another disadvantage is the difficulty of genetically manipulating the NK cells, although electroporation techniques and optimization of lentiviral transduction protocols are giving good results.49,50 Currently, there are several clinical trials with CAR-NK registered in Clinicaltrials.gov. The first published results of these studies demonstrate its safety.51 Preclinically, in a neuroblastoma mouse model,52 a study was made of CAR-NK cells with NKG2D specificity, which explored their ability to eliminate the immunosuppressive tumor microenvironment, improving the efficacy of subsequent treatment with CAR-T GD-2 cells. Bindings by bi-specific and tri-specific antibodies An innovative strategy to increase the cytotoxicity of NK cells is the use of bi-specific and tri-specific antibodies (BiKEs and TriKEs) that bind NK cells to tumor cells.53 The BiKEs are constructed by binding a single-chain variable fragment (scFv) directed against CD16 and an scFv directed against a tumor-associated antigen. In 2012 Miller et al. showed that BiKEs and TriKEs CD16/CD19 and CD16/CD19/CD2253 activated the NK cells through CD16, significantly increasing the cytotoxic activity and the cytokine production

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Day 0

2

Day 7 NKT 4.12

NK 9.75

2

10

10

101

101

0

10

CD56 2

10

T 68.5

Others 17.6 100

101

102

NKT 3.52

NK 61.8

0

10

103

NK 16.5

NKT 7.11

Others 10.0

T 66.4

100

2

101

102

103

NK 89.9

NKT 2.03

Others 5.45

T 2.65

10

101

100

7

101

T 15.8

Others 18.9 100

101

102

100

100

103

Day 14

CD3

101

102

103

Day 21

Figure 3. Progressive increase in the percentage of cells with NK phenotype (CD56+ CD3−) during coculture. The phenotype of the different cell populations was determined by flow cytometry. A progressive increase is observed in the proportion of CD56+ CD3− with purging of the rest of the cell lines, mainly from day 14 of culture.

compared to LLA-B cell lines. TriKEs CD16/IL15/CD33 for AML have also been developed which induce expansion.54

Drugs that increase the antitumor capacity Different drugs, such as proteasome inhibitors (bortezomib) or histone deacetylase inhibitors (HDACI), such as vorinostat, enhance the recognition of target cells. Bortezomib, for example, increases the expression of death receptors such as Fas and TRAIL-R2/DR5 and it induces apoptosis of the target cell through Fas/FasL and TRAIL/DR5 interactions.55 However, the HDACIs can reduce the cytotoxicity of NK cells, depending on the dose.56

NK cells memory The NK cells with a capacity of memory have been described in the context of cytomegalovirus (CMV) reactivations. These NK cells are phenotypically characterized by the expression of CD57 and CD94/NKG2C,57,58 and they are able to expand more rapidly and respond more forcefully to successive exposures to CMV. This memory capacity can be induced by culturing NK cells with IL12, IL-15 and IL-18. Preclinical in vitro studies have shown that cytokine-induced memory NK cells produce a greater amount of IFN-γ and show a greater cytotoxic capacity against the K562 myeloid leukemia cell line and against AML blasts,59,60 as well as tumor growth control and increased survival in in vivo studies in animal models. Several clinical trials are currently selecting patients to test the safety and efficacy of these cells. The antitumor and memory capacity of this subpopulation of NK cells could make them ideal candidates for their therapeutic use and for their genetic modification through the expression of CAR-NK.

Conclusions During the last decade, there has been significant progress in the clinical use of NK cells. The development of KIR typing methods for donor selection and the optimization of NK cell expansion methods have served as a platform for new clinical trials. In addition, the possibility of automating these large-scale production processes has allowed the use of NK cells to be a cost-effective treatment. Several studies have already demonstrated the safety and plausibility of treatment with NK cells in cancer patients. Trials published so far show that infusions of NK cells are well tolerated, without serious adverse effects, and that they do not induce intense GVHD. In addition, a beneficial therapeutic effect has been observed, although this response is short timewise, so this cell therapy could be a bridge treatment for other consolidation strategies. There are currently numerous clinical trials being conducted with various types of tumors, especially AML, as well as with solid tumors such as melanomas and carcinomas. The limitations for its systematic use in clinical practice include the expense and work involved in the isolation, expansion and activation procedures, it requires trained personnel and it must be performed under conditions of GCP for human use, which entails costs and the need of specially designed laboratories. Despite this, the potential of NK cells through the development of CAR-NK and memory NK cells will allow new strategies to be explored in combination with both conventional chemotherapy protocols and immunotherapies, including the CAR-T strategy. Financing This work has been funded in part by the Carlos III Health Institute and co-financed by the European Regional Development Fund (ERDF), grant (FIS) PI18/01301.

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