Natural killer cells and acute myeloid leukemia: promises and challenges

Abstract

Acute myeloid leukemia (AML) is considered as one of the most malignant conditions of the bone marrow. Over the past few decades, despite substantial progresses in the management of AML, relapse remission remains a major problem. Natural killer cells (NK cells) are known as a unique component of the innate immune system. Due to swift tumor detection, distinct cytotoxic action, and extensive immune interaction, NK cells have been used in various cancer settings for decades. It has been a growing knowledge of therapeutic magnitudes ranging from adoptive NK cell transfer to chimeric antigen receptor NK cells, aiming to achieve better therapeutic responses in patients with AML. In this article, the potentials of NK cells for treatment of AML are highlighted, and challenges for such therapeutic methods are discussed. In addition, the clinical application of NK cells, mainly in patients with AML, is pictured according to the existing evidence.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-022-03217-1.

Introduction

Acute myeloid leukemia (AML) is a malignant disorder of the bone marrow, characterized by clonal expansion of the progenitor cells with abnormal differentiation of the hematopoietic stem cells [1]. Caused by the propagation of the immature myeloid cells, AML is a form of cancer with the highest mortality and shortest 5-year survival rate (SR) among all leukemia subtypes. It is estimated that by the end of 2022, approximately 20,050 new adults will be diagnosed with AML, and 11,540 individuals will die [2].

Since the cytogenetic heterogeneity of AML has been recognized for about 40 years, it is considered a type of cancer with variable therapeutic responses [3, 4]. Despite the rapid diagnosis and directed therapy, which are vital for a better AML management, relapse rates are still high [5]. Many therapeutic methods have been conducted for years [6–8]; however, finding a proper approach to reach a better treatment for AML is still a great challenge and remained to be addressed.

Natural killer (NK) cells are innate lymphocytes of the immune system that monitor and recognize tumor cells by an unusual expression of cell surface markers [9]. First discovered in 1975, NK cells can lyse specific tumor cells without previous exposure, nor substantial damage to the patient’s tissues [10, 11].

Due to rapid tumor marker detection, fast anti-tumor action, and immune system response elevation, NK cells have been utilized to treat various types of cancer, AML in particular [12–14]. On the other hand, AML malignant cells can develop several mechanisms to escape from NK cells’ immunity [15, 16] and makes the therapeutic outcome even more challenging.

In this article, the potentials of NK cells for the treatment of AML are highlighted, and challenges for such therapeutic methods are discussed. In addition, the clinical application of NK cells, mainly in patients with AML, is pictured according to the existing evidence.

Natural killer cells

Overview

As a member of the innate lymphoid cells (ILCs) family, NK cells are large granular bone marrow-derived lymphocytes with prevailing cytotoxic capacity. The term “natural killer” is characterized by the natural ability to directly kill virally infected or cancerous cells, without any need for pre-activation or previous exposure [17, 18]. After B and T lymphocytes, NK cells represent the third-largest lymphocytes population in the peripheral blood circulation, yet unlike other lymphocytes can identify a wide range of tumor mediators [11, 19].

Since their scientific establishment in the 1970s, NK cells have been drawing much attention for their fascinating properties in cancer immunotherapy. NK cells are first discovered in the adult mice spleen via an unintended experiment aimed at distinguishing the T cell-associated cytotoxicity [10, 20] and then characterized as naturally occurring non-T lymphoid cells [21]. In the same year, their functionality was observed in humans, which introduced them as the non-thymus-derived lymphoid cells that can make impulsive lymphocyte-associated cytotoxicity [21, 22].

Before entering the peripheral blood circulation, NK cells are differentiated in the bone marrow, spleen, thymus, liver, and lymph nodes [23]. NK cell differentiation is a linear process of turning functionally suboptimal and highly proliferative immature organoids into a large granular population of effectors, and finally, terminally differentiated cytotoxic cells [9, 17]. The procedure is mediated by numerous intermediates along with a remarkable expression of specific cell markers [23, 24]. Despite T cells activity that mostly leads to host tissue damages, NK cells will die after their cytotoxic attack, which can be a privilege to be used as a therapeutic method [25].

NK cells are primarily categorized into two subtypes based on the CD56 and CD16 expression level. About 90% of the total NK cell population of the peripheral blood are capable of applying cytolytic activity, which are called CD56^dimCD16⁺ NK cells [26, 27]. The cytolytic capacity is mainly provided through the secretion of cytotoxic granules, such as granzymes and perforin. Following the NK cells’ activation, perforin is released and forms several pores in the tumor cell’s membrane. This process is followed by the secretion of granzymes, which are believed to deliver inside the cell through the disrupted membrane to induce cell apoptosis [27, 28]. The other 10% of the NK cell population in the peripheral blood, also known as CD56^brightCD16⁻, are largely presented in the lymph nodes to produce and release immunoregulatory cytokines. Interferon (IFN)-γ, interleukin (IL)-10, and tumor necrosis factor (TNF)-α/β are the examples of such cytokines [26, 29].

To recognize any modification of the host cells caused by malignant alterations or viral infections, two major types of polymorphic yet germline-encoded receptors are expressed on the NK cells’ surface [30]. The first type are activating receptors that are responsible for recognizing pathological ligands and interacting with signaling pathways, based on effector activities. Natural cytotoxicity receptors (NCRs), activating killer immunoglobulin-like receptors (KIRs), CD94/natural killer group 2C (NKG2C), NKG2D, and DNAX accessory molecule-1 (DNAM-1) are of the activating receptors [31, 32]. The second type, inhibitory receptors, are designed to identify major histocompatibility complex (MHC) class I molecules, leukocyte immunoglobulin-like receptors (LIRs), inhibitory KIRs, and NKG2A are considered as NK cell inhibitory receptors [31, 33] (Fig. 1).

Fig. 1.

Schematic representation of NK cells cytotoxic function

Properties

Cytotoxic function

The cytotoxic functionality of the NK cells is regulated by a compound array of activating and inhibitory receptors that carry stimulatory and suppressive signals, respectively [30]. Because of their natural ability to kill various cells, a particular type of regulation is defined to evade self-cell injuries (Fig. 1A). This feature is mainly acquired through encountering inhibitory receptors (KIRs, for example) with the host MHC class I molecules on the surface of the cells [34]. When NK cells meet the healthy cells, activating receptor functions will be hindered by inhibitory receptor signals; hence, host cells are less likely to be attacked by NK cells [35, 36].

Conversely, activating receptors are stimulated by the molecular changes in the surface of the normal cells. Due to the downregulation of MHC class I ligands by virally infected or cancerous cells, known as the “missing-self” phenomenon (Fig. 1B), the inhibitory response will be surpassed by the activating signals. The same process will happen when the expression of activating ligands is upregulated due to damage or stress to the healthy cells, also known as the induced “self-ligands” phenomenon (Fig. 1C) [37, 38]. As the result, NK cells immunity is provoked against non-self cells by secreting cytolytic granules, cytokines, and inducing death receptor-associated apoptosis, which in turn leads to massive cellular destruction [32, 39]. These mechanisms are encountered through diverse clinical experiments, which result in effective cytotoxic function of NK cells against various cancers, AML for example [40–42].

A. NK cells can recognize healthy cells through a dynamic balance of delivery signals via inhibitory and activating receptors. B. NK cells can detect downregulated MHC class I molecules as “missing-self” and release cytotoxic granules to lyse non-self cells. C. Stressed or damaged cells are recognized by activating receptors of NK cells due to the overexpression of the induced stimulatory “self-ligands.” D. NK cells can also elicit cell-mediated cytotoxicity through tumor antigen-specific (IgG) antibody binding to CD16 receptor, also known as antibody-dependent cell-mediated cytotoxicity (ADCC). Provided by Biorender.com.

There are other ways in which NK cells can contribute to the cytotoxic activities. Similar to direct tumor cell exposure (mostly via NKG2D and NCRs), neoplastic cells can also be killed by antibody-dependent cell-mediated cytotoxicity (ADCC) (Fig. 1D). This antibody-dependent process is mediated via CD32 (FcγRIIC) and mainly CD16 (FcγRIIIA) receptors, which are bind to a variety of antibodies (IgG, for example). After the Fc receptors engagement, cell death is induced through NK cell granulocytic apoptosis via death signaling cytokines [43]. NK cells activity can also be fortified by anticancer drugs such as dinaciclib [44] or other immune components such as activated dendritic cells (DCs) and macrophages through regulatory cytokines [14, 45], which are explained at Supplementary Fig. 2 in detail.

Effector function

Upon NK cell activation, both innate and acquired immunity arms will be impacted. The maturation and activation of macrophages, DCs, and T cells are boosted by NK cells through organization of cell receptor and secretion of cytokines, IFN-γ and TNF-α in particular [46, 47] (Supplementary Fig. 2). Cytokine secretion, particularly by CD56^brightCD16⁻ NK cells, holds great promises in effector mediation and cancer immunotherapy. For instance, IFN-γ is shown to mediate T_helper-IT_cytotoxic-I (Th1/Tc1) immune reaction, as well as preventing tumor cell’s proliferation [48]. The effector function of Th2/Tc2 is also mediated by IFN-γ, leading to attract other immune cells to collaborate against tumor cells growth [49].

When it comes to antileukemic activity, several cytokines are represented as effective tools. For example, a specific combination of IL-2 and IL-15 was reported to be ideal for the expansion and activation of the NK cells in patients with AML, which resulted in function recovery of NK cells [50]. However, the main problem of their antileukemic effect is mainly described as the short-term persistence of NK cells in the body. Meanwhile, a mixture of cytokines (IL-18, IL-15, IL-12, and type-I IFN) is shown to stimulate NK cells’ activity for weeks that results in significant amplification of antileukemic responses [51, 52] (Supplementary Fig. 2).

Upon priming by different soluble factors (IL-12, IL-15, IL-18, and type-I IFN), NK cells can boost the maturation and activation of DCs, macrophages, and T cells, via a combination of cytokines and cell surface receptors (IFN-γ and TNF-α). Besides, NK cells can also destroy immature DCs (iDC), activated T cells (CD4⁺), and hyper-activated macrophages. These NK cell-based regulatory functions are mainly held in check by the recognition of fundamentally expressed self-molecules via the inhibitory receptors, such as inhibitory KIRs and CD94/NKG2A. Provided by Biorender.com.

Despite unique abilities of NK cells to overcome cancer evasion, several cytokines such as IL-10 and transforming growth factor (TGF)-β are released from tumor cells, which results in NK cells dysfunction [53, 54]. While the combination of IL-10 and IL-15 is shown to increase the cytotoxicity of NK cells [55], secretion of IL-10 by regulatory T cells (T_regs) can negatively affect NK cells activity [56], which is further explained at Supplementary Fig. 3 [57–59]. Activating signals from NKG2D and NKp30 are also downregulated via TGF-β secretion [60], as a result, leading to facilitate tumor escape from the NK cells’ immune surveillance.

Cancer immune surveillance

A connection between tumor suppression and NK cells activity is well documented based on animal experiments, where the deficiency of the NK cells had resulted in a higher rate of tumor growth [61, 62]. The lack of NK cells functionality, which is determined via tumor infiltration, is also shown to be associated with worse outcomes of patients with cancer [63]. Therefore, relative observations of primary NK cells dysfunction and deficiency toward cancer incidence have suggested the role of NK cells in cancer immune surveillance [64]. The idea, which is originally embodied in Burnet and Thomas’s theory [65], has been considerably improved since its introduction, and through time, developed as the cancer immunoediting hypothesis [66].

The theory of immunoediting is designed to describe the three major phases of cancer immunity [67]. In the first phase, also known as elimination, tumor cells will be detected and removed by the immune system. Due to releasing cytotoxic granules and inducing death receptors, circulating cancerous cells are eliminated by NK cells to prevent possible metastasis [68]. The process is finalized when all of the tumor cells are eradicated; however, if cancerous cells are not completely removed, cancer immunoediting will be shifted into the equilibrium phase [69]. During this phase, tumor growth is generally controlled via both adaptive and innate immune systems, while tumor cells, AML cells for example, are provided with an exquisite opportunity for acquiring DNA mutations and heterogeneity within cancer microenvironment [70, 71]. As this dynamic balance goes on, tumor cells will be developed to suppress, avoid, or resist anti-tumor immunity. These cells will be selected, proliferated, and finally, colonized as progressive tumors to escape from the NK cells’ immune surveillance in the final phase, which is also known as the tumoral escape [72, 73].

Cytotoxic T lymphocytes (CTLs), the predominant lymphocytes around tumor cells, can be attracted via chemokines and cytokines secreted by NK cells. Meanwhile, tumor cells are detected and destroyed by CTLs through a complex MHC class I identification [74]. As an answer, the remained tumor cells, including AML blasts, can downregulate the MHC class I ligands to hide from CTLs’ inflammatory response [75, 76]. As mentioned earlier, NK cells’ activating signals are repressed by inhibitory responses through a self-MHC-I suppression mechanism. When MHC-I is downregulated, activating responses will override the inhibitory signals, and therefore, tumor cells will be identified and destroyed by NK cells [37, 77]. NK cells are also designed to detect and kill cancer stem-like cells (CSCs), which are relatively accountable for chemotherapy-resistant malignancies and uncontrolled tumor growth [78].

Acute myeloid leukemia

As the most common subtype of acute leukemia in adults, AML is considered a clinically heterogeneous disorder of the bone marrow. While there are remarkable progresses in approving new drugs or therapeutic methods for AML treatment, the outcomes are still far from expectations [5, 13]. Despite cytogenetic heterogeneity of AML, the malignant cells can be recognized, eliminated, or remained in a dynamic equilibrium by both innate and adaptive immune arms [79]. AML blasts are predisposed to be attacked by T lymphocytes and NK cells via expression of MHC class I molecules, leukemia-associated antigens (LAAs), or NK cell activating ligands [80]. Nevertheless, AML can predominantly evade NK cells’ immune surveillance through several mechanisms focused on antileukemic dysfunction of the immune responses [81, 82]. An overview of such mechanisms is given comprehensively via Supplementary Fig. 3.

NK cell dysfunction

The antileukemic immune response of NK cells is provided by direct cytotoxicity or activation of other immune components [83, 84]. In AML, however, impaired killing capacity of NK cells is found due to altered expression of both ligands and receptors (Supplementary Fig. 3). Since the function of the NK cells is tightly regulated by the inhibitory and activating signals, the imbalanced expression of receptors will lead to the NK cells dysfunction [81, 85, 86].

Activating receptors downregulation

Expression of NCRs such as NKp46, NKp44, and NKp30 is shown to be downregulated in NK cells of the majority of the AML patients [87, 88]. Besides, DNAM-1 expression is diminished on the surface of the NK cells in AML patients compared with the control group [89]. Along with NKG2D, other activating receptors are also represented to detect and kill leukemic target cells [90, 91]. However, among diagnosed AML patients the ones with a lower amount of NCRs were found to live shorter than others, suggesting the pivotal role of activating receptors regulation in AML-associated responses [92].

Inhibitory receptor upregulation

Along with activating receptors, high expression of various inhibitory receptors is shown to play a significant role in AML evasion from NK cells’ immune surveillance [93, 94]. The association between KIR inhibitory receptors and AML immune escape is controvertibly demonstrated with several studies in the haploidentical hematopoietic stem cell transplant (HSCT) setting. Several studies have suggested a mismatch association between recipients’ leukocyte ligands and donor KIRs, which results in relapse rates reduction of AML patients [95–97]. In the same setting, CD94/NKG2A upregulation is also indicated to be linked with lower NK cell cytotoxicity against AML blasts [98], all of which represent the regulatory role of inhibitory receptors concerning with AML immune escape.

AML immunosuppression

As mentioned above, downregulation of activating receptors as well as upregulation of inhibitory receptors can lead to cytotoxic dysregulation of NK cells (Supplementary Fig. 3). NK cells’ antileukemic responses can also be compromised through various AML receptor–ligand interactions or immunosuppressive factors secretion [86, 99], which are explained here in detail.

Receptor–ligand interactions

Due to an absence or reduction of ligand expression for activating receptors, AML cells can develop a chronic stimulus system to escape from NK cells attack. Shedding NKG2D ligands by AML cells can result in reduction of NK cells’ activating responses and, consequently, impair cytotoxic functionalities [100, 101]. Downregulation of DNAM-I on NK cells besides lower expression of DNAM-I ligands on the leukemic blasts further supports such interactions [102].

Increased expression of inhibitory ligands on AML, CD137L and glucocorticoid-induced TNFR-related protein–ligand (GITRL) for example, is also shown to be associated with receptor-ligand interactions. Both CD137L and GITRL overexpression are found to impair NK cells’ killing capacity or IFN-γ secretion, through either direct GITR ligation on the NK cells or indirect IL-10 secretion by AML blasts [103, 104]. Upregulation of CD200 on the surface of AML cells is another evidence to decline antileukemic responses of NK cells [105], altogether leading to a poorer prognosis for AML patients.

Immunosuppressive factor secretion

Aside from interactions between activating/inhibitory ligands and NK cell receptors, AML cells can also evade NK cells’ immune surveillance through secretion of specific immunosuppressive factors [106]. By using apoptosis resistance facilities, AML cells are capable of hiding from CD95 and TNF-based killing pathways, which are basically designed for NK cells to attack and kill malignant blasts [107, 108].

Multiple mechanisms contribute to the AML immune escape from NK cells either through NK cells’ alteration, AML target cells’ expression, or via third-party immune cells’ contribution. NK cells of patients with AML often show modifications in receptor phenotypes compared to the NK cells of healthy group. Shedding of ligands and low expression of NK cells activating receptors are also impair AML cells recognition. Besides, secretion of specific immunosuppressive factors by AML cells can directly affect NK cells cytotoxicity and also induce T_regs, which in turn diminish NK cells’ activating responses. The later effect may be further increased due to suppression of DCs function and reduction of immature dendritic cells (iDC) destruction by NK cells, which can further induce activity of T_regs. IS, immunosuppressive; AR, activating receptor; IR, inhibitory receptor. Provided by Biorender.com.

Other immune cells contribution

In addition to NK cell dysfunction and AML-induced immunosuppression, AML immune escape is also facilitated through various interactions between AML cells, NK cells, and other immune system components (Supplementary Fig. 3).

Dendritic cells (DCs)

As mentioned earlier, immature dendritic cells (iDCs) are destroyed by NK cells to limit the subsequent unwanted inflammation. Impaired destruction of iDCs by NK cells is vastly demonstrated in AML patients, which can further induce toleration against tumor antigens presented by iDCs [14, 109]. During the development of leukemia in mice, stimulation of DCs activity by T cells was also suppressed due to a poorer cytotoxicity of NK cells [110].

Regulatory T cells (T_regs)

The number of T_regs, which is associated with inhibition of NK cells cytotoxicity, was demonstrated to be higher in AML patients than in healthy controls or patients under complete remission (CR) [111, 112]. T_regs of leukemic samples were shown to be more suppressive toward NK cells and responder T cells through either direct cell interaction or soluble factor secretion, such as IL-10 and TGF-β1 [112, 113]. Besides, in an animal experiment, the reduction of T_regs resulted in more cytotoxic activity of NK cells [114]. These findings together with previous results indicate the importance of constant investigation on the interactions between NK cells and AML within the immune system to have a clearer understanding of disease development and better clinical achievements.

NK cells against AML

A robust rationale for investigating NK cells as antileukemic agents is established through explicit data from in vitro experiments, animal models, and clinical trials [12, 115, 116]. The combination of signal regulation in NK cells activity and underlying AML characteristics of the immune escape capacity provides essential clues on how to harness maximum potentials of NK cells against AML [13, 117]. However, when it comes to clinical applications, various laboratory procedures and comprehensive clinical circumstances need to be executed to meet the sophisticated regulatory requirements.

Adoptive NK cell transfer

Autologous NK cells

As a solution to enhance NK cells’ immune surveillance, cell lineages from patients’ peripheral blood can be expanded ex vivo and transduced back to their own body as autologous NK cell [118, 119]. Due to the use of the patient’s own blood as a convenience source of cells, independency from immunosuppressive therapies, and low possibility of graft versus host disease (GvHD), autologous NK cell transfer is known as the first major effort of using NK cells for cancer immunotherapy [40, 120–122] (Fig. 2A).

Fig. 2.

Adoptive NK cell transfer for AML patients. A In autologous NK cell transfer, endogenous NK cells are needed to be harvested from patient’s own peripheral blood. For this purpose, peripheral blood mononuclear cells (PBMC) of patient is isolated, and T cells are depleted. Purified NK cells are then expanded and proliferated in vitro to infuse to patient’s own blood. B. In allogeneic NK cell transfer, however, NK cells are isolated from PBMC of a healthy HLA-matched or haploidentical donor. Following T cell depletion and in vitro NK cell expansion, allogeneic NK cells will be infused to the AML patients. Provided by Biorender.com

Despite the considerable in vivo expansion of infused cells, a significant therapeutic outcome is often failed, owed to the inhibitory effects by self-human leukocyte antigen (HLA) ligands in some type of tumors, AML in particular [123, 124]. Furthermore, since autologous NK cells are attained from profoundly pre-treated patients, the expansion and the functional capacities are less likely to be as equal as expectations [123].

Various strategies are being developed to overcome current checkpoints of autologous NK cell linages. Lirilumab, as an anti-KIR antibody, is established to block inhibitory signals by avoiding interactions with MHC class I ligands, leading to upregulation of NK cells’ killing capacity [125]. Moreover, the use of combination therapy, consisting of autologous NK cells and various anti-tumor agents, is shown to enhance therapeutic responses against tumors [126].

On the other hand, many clinical applications of autologous NK cells within the cancer settings are yielded disappointing results. In a study designed to pre-activate NK cells with IL-2, despite a considerable in vitro cytolytic activity and in vivo persistence of NK cells, no significant clinical responses was perceived. However, NK cells’ persistent mediated ADCC without in vitro cytokine reactivation suggest that combination of monoclonal antibodies with autologous adoptive NK cell transfer deserves more evaluation [124]. Similar to the AML setting, patients who received unmodified NK cells have shown a poorer outcome than others with alloreactive NK cell transfer [124, 127], which provides a strong basis for shifting from autologous NK cells to allogeneic NK cell transfer.

Allogeneic NK cells

In this method, NK cells are acquired from healthy HLA-matched or haploidentical (partly matched) donors, expanded ex vivo similar to autologous expansion methods, and undergone exclusion of T cells to prevent possible GvHD [128] (Fig. 2B). Adoptive allogeneic NK cell transfer can also be promoted from umbilical cord blood (UCB) through pre-activation and expansion with IL-2 to be performed in both HSCT and non-HSCT settings [123, 129].

In 2002, the first evidence of allogeneic NK cell transplant was elucidated by Ruggeri et al. in the HSCT setting, where AML diagnosed patients who received mismatched KIR and HLA-C allogeneic bone marrow transplants were remarkably expressed lower relapse rates [130]. Due to alloreactive cytotoxicity pre-cultured in IL-2 for 2 days, results suggested the mediation role of alloantigen-specific NK cells against AML blasts in mice (survival rate of 100%) without the risk of GvHD [131]. A few years later, haploidentical NK cell infusions outside the HSCT setting were revealed to induce complete remissions in 5 of 19 poor prognosis diagnosed AML patients. This remission rate was significantly higher (CR, 75%) when KIR–ligand mismatched donors where applied [132]. These findings are also replicated in additional cohort studies [133, 134]. Besides, the current reports of the recent clinical trials on the application of adoptive NK cells for AML patients in both HSCT and non-HSCT settings are shown at Table 1.

Table 1.

Ongoing clinical trials of adoptive NK cell therapy in AML

Identifier	Phase	Conditions	NK cells’ source	Other interventions	Outcome measures
Hematopoietic Stem Cell Transplant (HSCT) setting
NCT05247957	I	AML	CAR-NK cell	–	DLT, MTD, LFS
NCT05272293	I/II	AML	Expanded haploidentical NK cells	K562-mbIL21-41BBL	LFS, OS, Persistence of infused NK cells, Number of T, B, and activated NK cells
NCT05256277	I	AML	Cytokine-induced memory-like NK cells	CT101a	ORR, OS, DOR, TTP, LFS, safety
NCT04632316	I/II	AML	PBMC-derived NK cell	Cyclophosphamide-Fludarabine (Cy/Flu)	AE, MRD, Response, Survival (EFS), QOL
NCT01904136	I/II	AML, MDS, CML	PBMC-derived NK cell	Cyclophosphamide, Fludarabine, Melphalan, Mycophenolate Mofetil, Tacrolimus	MTD, AE, Survival (OS and RFS)
NCT01823198	I/II	High-risk AML or MDS	PBMC-derived NK cell	IL-2, Busulfan, Fludarabine Phosphate	NK cell dose, Survival (OS and DFS), AE (GvHD and Toxicity)
NCT04166929	II	AML, MDS	PBMC-derived NK cell	–	Relapse
NCT03300492	I/II	AML, MDS	PBMC-derived NK cell	NK-DLI	AE, Survival (PFS), Response, NK cell dose
NCT02809092	I/II	R/R AML	PBMC-derived NK cell	IL-21	MTD, Response, NK cell expansion
NCT04836390	II	AML	PBMC-derived NK cell	IL-21	Survival (RFS), Success rate, AE (GvHD)
NCT01619761	I	AML, MDS, et al	UCB-derived HSPC-NK cell	Cyclophosphamide, Fludarabine Phosphate, Lenalidomide, Melphalan, Mycophenolate Mofetil, Tacrolimus	AE (GvHD and TRM), Survival (OS and DFS)
NCT02727803	II	AML, MDS, et al	UCB-derived HSPC-NK cell	Busulfan, Clofarabine, Cyclophosphamide, Fludarabine Phosphate, Melphalan, Rituximab	Survival (PFS and OS), AE (GvHD, Infection, and TRM)
NCT04024761	I	Relapsed AML, MDS, or MPN	Cytokine Induced Memory-like NK Cell	IL-2, Fludarabine, Cyclophosphamide	AE (IR, IS, and GVHD), Response, Survival (LFS and OS)
NCT03068819	I/II	Relapsed AML	Cytokine Induced Memory-like NK Cell	CD3 + T Cell Product Infusion	Survival (OS and LFS), Response, AE (GvHD)
NCT02782546	II	R/R AML	Cytokine Induced Memory-like NK Cell	ALT-803, Tacrolimus, Mycophenolate mofetil, G-CSF	Survival (LFS and OS), Response
Non-Hematopoietic Stem Cell Transplant (non-HSCT) setting
NCT04209712	Early Phase I	AML with MRD	PBMC-derived NK cell	IL-2	MRD, AE
NCT02890758	I	AML, MDS, et al	PBMC-derived NK cell	ALT-803	MTD, AE (GvHD), in vivo NK cell levels, Survival (OS and RFS), Response, DOR
NCT04221971	I	R/R AML	PBMC-derived NK cell	Chemotherapy	AE, Response, NK cells’ metabolism, migration and reconstruction, Recovery, Relapse
NCT03955848	N/A	AML in Remission	PBMC-derived NK cell	IL-2	Survival (RFS and OS)
NCT04220684	I	R/R AML or MDS	PBMC-derived NK cell	IL-21, Cytarabine Hydrochloride, Fludarabine	MTD, AE, Response, Survival (LFS and RFS), DOR, patients proceeding transplantation, Recovery
NCT04327037	I	AML	PBMC-derived NK cell	IL-2	AE, Days of persistence of NK cells, Recovery
NCT04347616	I/II	R/R AML	UCB-derived HSPC-NK cell	IL-2	MRD, Life span and Functional activity of NK cells, Plasma cytokine concentrations
NCT04310592	I	AML	Placental-derived HSPC-NK cell	CYNK-001	MTD, AE, MRD, TTP, Response, Survival (OS and PFS)
NCT01898793	I/II	R/R AML or MDS	Cytokine Induced Memory-like NK Cell	ALT-803, IL-2, Fludarabine, Cyclophosphamide,	MT/TD, Response, AE, DOR, TTP, Survival (DFS and OS)
NCT04354025	II	R/R AML	Cytokine Induced Memory-like NK Cell	IL-2, Fludarabine, Ara-C, G-CSF	Patients able to proceed to SCT, Response, MRD, AE, DOR, TTP, Survival (DFS and OS)

AE adverse event; AML acute myeloid leukemia; CML chronic myeloid leukemia; DFS disease-free survival; DLT dose-limiting toxicity; DOR duration of response; EFS event-free survival; GvHD graft versus host disease; HSPC hematopoietic stem and progenitor cell; IL interleukin; IR immune reactions; IS immune suppressants; LFS leukemia free survival; MDS myelodysplastic syndrome; MPN myeloproliferative neoplasm; MRD minimal residual disease; MTD maximum tolerated doses; MT/TD maximal tolerated or tested dose; N/A not applicable; NK natural killer cell; ORR overall response rate; OS overall survival; PBMC peripheral blood mononuclear cell; PFS progression-free survival; QOL quality of life; RFS relapse-free survival; R/R relapsed/refractory; SCT stem cell therapy; TRM treatment-related mortality; TTP time to progression; UCB umbilical cord blood

Similar to autologous NK cell transfer, allogeneic NK cell-based therapy is associated with the challenges of timely ex vivo expansion, low clinical-grade activation, and lack of in vivo persistence [135, 136]. A combination of cytokines can play an essential role in the activation (IL-18 and IL-21), proliferation (IL-2 and IL-15), and effector function (IFN-γ and TNF-α) of NK cells [46, 50, 137]. IL-15 super agonist complex, ALT-803, is identified as a safe agent at the first inhuman phase I study focusing on elderly AML patients who relapsed after the HSCT [138]. The act of using ALT-803 for AML patients after HSCT as a promising relapse prophylactic agent is also under investigation (NCT02989844).

Furthermore, engineered AML cells with the encoding gene to express IL-15 or IL-12 are constructed to decrease the toxicity of systemic administration of cytokines [139, 140]. A phase I clinical trial is recruiting to test the safety and tolerability of a novel treatment based on modification of AML cells to express IL-12 in a non-HSCT setting (NCT02483312). In brief, the importance of applying genetic modification methods on facing challenges of AML-based therapy seems almost inevitable

Genetic modifications

Increasing activation and proliferation

Different methods have been investigated to enhance both activity and proliferation of NK cells. Anti-tumor activity of NK cells can be amplified through upregulation of activating receptors, which results in increasing sensitivity to tumor activating ligands [141, 142]. NKG2D, as one of the main NK cell activating receptors, is physiologically associated with DNAX-activating protein (DAP)-10 in humans and DAP10/12 in mice [143, 144]. When NKG2D ligand (NKG2D-L) on AML cells are recognized by NKG2D receptors on NK cells, cytotoxic capacity of NK cells will be activated, cytokine release will be induced, and cellular proliferation will be promoted via the promotion and stabilization of NKG2D expression by DAP10 [145].

Through NKG2D/CD3ζ/DAP10 signaling pathway, a wide range of tumor cells including leukemic blasts are predisposed to substantial cytotoxic activity of NK cells, while self-hematopoietic cells remain almost intact. Despite the hampering potential of tumor cells to inhibit activating ligands, NKG2D-L is less likely to be shed by AML cells due to the high expression of NKG2D receptors on NK cells [146, 147]. The result of an unexpected targeting potential of NK cells toward myeloid-derived suppressor cells (MDSCs) via NKG2D/CD3ζ receptors is also indicated in another study [148].

Lower expression of inhibitory NK cell receptors has been tested as another genetic-based method to activate the anti-tumor capacity of NK cells. As mentioned earlier, AML cells can develop several suppressive mechanisms to evade NK cells’ immune surveillance [15]. As a response to IFN-γ secretion, highly expressed HLA-E by AML blasts can also lead to impaired cytolytic capacity of the NK cells [98]. CD94-NKG2D receptors, which are designed for inhibitory signaling transduction by HLA-E ligands, can be targeted via anti-NKG2A expression blocker proteins. As a result, cytolytic capacity of NK cells will be augmented due to lower expression of NKG2A surface receptors [149].

Explicit pieces of evidence are indicated the association between population of cytotoxic NK cells and antileukemic capacity in various AML settings [150–153]. NK cell expansion can be promoted via cytokine-encoding genes such as membrane-bound IL-15 (mbIL-15). Through an animal experiment, mbIL-15 expansion is shown to sustain the propagation, as well as the lifetime of infused NK cells [154]. These studies collected with previous results provide a robust rationale toward the pivotal role of enhancing NK cells activation and proliferation to defeat AML-based immunosuppression.

Chimeric antigen receptors (CAR) NK cells

The immune response of the NK cells against AML is tightly bounded with signaling balance between transferred NK cells and associated target cell ligands [115, 155]. Since the introduction of CAR-T cells, a successful therapy against lymphoid and leukemic disorder subtypes, lots of attention has been appealed to the cytolytic enhancement capacity of chimeric-based techniques [156–158]. Despite several promising results at the refractory stage [159, 160], adverse effects such as cytokine release syndrome (CRS) remained to be resolved for CAR-T cell therapy against AML [161–163]. Instead, NK cells are considered as promising alternatives to T cells due to their shorter lifespan, favorable cytotoxic profile, and lower manufacturing costs [12, 164, 165].

NK cells can be readdressed with CAR-based methods toward tumor cell surface ligands. Since some of the cell markers of AML are shared with the hematopoietic stem cells (HSCs), collecting optimal choices of leukemia-specific markers that can be specifically identified and attacked by CAR-NK cells is a huge obstacle [166, 167]. Derived from NK cell lines (NK-92 cells) or UBC, CAR-NK cells can be used as an efficient “off-the-shelf” product [168]. The substantial benefit of using off-the-shelf CAR-NK cells in refractory/relapsed CD19⁺ leukemia supports their application for further clinical trials [169].

CAR-NK cells have several advantages; however, still some problems such as loss of targeted antigen, a hostile tumor microenvironment, and tumor heterogeneity remained to be addressed to maximize the CAR-NK related AML immunotherapy [170]. Further research has described a Cas9/ribonucleoprotein-based complexes method for gene targeting in NK cells. Using CRISPR/Cas9 gene editing, targeted genes have been inserted into a safe-harbor locus in combination with an adeno-associated virus-mediated gene delivery approach was used to create primary CD33 CAR-NK cells with enhanced anti-AML activity [171]. CD33 is expressed on a restricted group of terminally differentiated cell lines and is not found on primitive stem cells. It is also detected on AML cells as well as leukemia stem cells (LSCs) of around 90% of patients, therefore, can be a promising target receptor for CAR-NK cells [171–174]. In addition, a targeting effect of specific human NK cell lines toward CD33⁺ AML blasts [175], as well as the first inhuman report on the safety of CD33 CAR-NK-92 cells infusion are discovered in patients with AML [176]. CD4 is another AML-based ligand, which is not highly expressed on the hematopoietic cells. To support this notion, the role of CD4⁺ AML blasts elimination is investigated as CD4/CD28/4-1BB/CD3ζ CAR-NK-92 cells [177].

Currently, the application of CAR-NK cells is extensively studied in various cancer settings [178–180], but harnessing their potential as a promising therapeutic method toward AML needs to be further investigated, once their application is relatively limited to the preclinical studies [181]. However, the knowledge acquired from CAR-T cell and CAR-NK cell therapy is worth to be applied to CAR-NK-based therapies for AML patients in the future.

Antibodies

Antibodies targeting tumor-associated antigens

As an attractive method of cancer immunotherapy, antibodies targeting tumor-associated antigens playing a great part in the induction of ADCC by NK cells. The outcomes for unconjugated antibodies are generally poor when used alone [182, 183]; however, the effects could be improved by engineering Fc parts of the antibodies to enhance binding to CD16 or integrating with other therapeutic methods [184, 185]. Preclinical studies have investigated the efficiency of new Fc-optimized antibodies directing against various possible antigens such as IL-1 receptor accessory protein (IL1RAP), CD33, CD133, and CD157 [186–189]. Besides, new antibodies regimens combined with NK cell transfer methods have shown promising outcomes, and therefore, these valuable strategies could be used in upcoming clinical trials [190, 191].

Antibody–radio conjugates (ARCs) and antibody–drug conjugates (ADCs) are favorable strategies to augment the antibodies' potency, while they could have better clinical outcomes on AML patients [192–194]. The combination of antineoplastic calicheamicin agent with anti-CD33 antibody and gemtuzumab ozogamicin (GO), is currently the only ADC antibody approved by the Food and Drug Administration (FDA) for the treatment of R/R CD33⁺ and newly diagnosed AML patients [195, 196]. Preliminary findings of different ADCs targeting CD33, CD37, leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4), C-type lectin-like molecule 1 (CLL-1), and FLT3 highlight further clinical potential of using antibodies targeting tumor-associated antigens for the treatment of AML [197–201].

Antibodies targeting NK cell inhibitory receptors

Inhibitory receptors of NK cells play a critical role in the immune escape of tumor cells, making them great targets for cancer immunotherapy. Over the last decades, the number of identified NK cells inhibitory receptors has been increasing. Aside from MHC-I inhibitory LIRs, KIRs, and CD94/NKG2A receptors, other NK cell immune checkpoints have shown to cause cellular dysfunction [202]. Besides the benefits of KIR–ligand mismatch between recipients and donors in improving the results of HSCT, pharmacologic blockage of KIR via anti-KIR antibodies can potentially prevent the interaction of KIR-HLA-C and augment the function of NK cells. IPH-2101 and IPH-2102 (lirilumab) are antibodies that target KIR2D and have reported to be safe in elderly AML patients in their first CR [203, 204]; however, in a phase II study, the leukemia-free survival (LFS) of lirilumab did not compare satisfactorily to the placebo [205]. The combination of azacitidine with lirilumab also did not exhibit substantial development in R/R AML patients in terms of median survival (overall survival, OS 4.2 months) or response rate (overall response rate, ORR 14%). Thus, due to unsatisfactory results, the relevant clinical trial was terminated (NCT02399917) [206]. Besides, blockade of LIR-1 or NKG2A have resulted in amplified cytotoxicity of NK cells against AML blasts, suggesting a necessary option of the combination of anti-NKG2A, anti-KIR, anti-LIR-1 antibodies for a better effectiveness in patients with AML [207, 208].

BiKE and TriKE

As the recombinant factors of single-chain variable fragments (scFv), Bi-specific killer cell engager (BiKE) and tri-specific killer cell engager (TriKE), function as immunologic synapses within NK cells and tumor cells. Maintaining the specificity of the antibodies, they minimize the size of the antibodies to increase their distribution. CD16-binded BiKE and TriKE can activate NK cells through specified CD16 signaling pathway against tumor cells with targeted antigens in such an effective manner [209]. Scientists designed a novel BiKE (CD16*33 BiKE) that binds to both CD16 and CD33. CD16*33 BiKE specifically triggers cytotoxicity and cytokine release of NK cells when combined with CD33⁺ and primary AML cell lines. Besides, NK cells’ effector function were significantly enhanced when co-cultured with a disintegrin and metalloprotease-17 (ADAM-17) inhibitor to prevent shedding of CD16 [210]. The same research group later designed a TriKE by integrating a modified human IL-15 into the CD16*33 BiKE, which provided a signal for proliferation and activation of NK cells [211]. A phase I/II clinical trial of using CD16*33*IL-15 TriKE (GTB-3550) for CD33⁺ R/R AML treatment is under investigation (NCT03214666).

Through linking anti-CD16 to either scFv alongside the same antigen (CD16*33*33 TriKE, for example) or scFv alongside two different antigens (CD16*33*123 TriKE, for example), TriKEs displayed a greater binding affinity and NK cell cytotoxicity toward AML malignant cells compared to BiKEs [212, 213]. Since CD33 is largely expressed on the surface of healthy myeloid cells, NKG2DLs have become promising targets. Compared to the engineered NKG2D-Fc protein, CD16*NKG2D BiKE displayed an increased attraction to CD16 while induced larger leukemia cell killing capacity [214]. Moreover, CD16*CLL-1*IL-15 TriKE presented a robust activity of NK cells against AML both in vivo and in vitro [215]. Altogether, these molecules became potential candidates for AML personalized immunotherapy based on clinical and preclinical findings.

Other therapeutic methods

Cytokines

As mentioned earlier, Cytokines play an essential role in proliferation, activation, and function of NK cells. Ex vivo stimulation with 50 ng/mL of IL-15 or 10 ng/mL of IL-2 was reported to be ideal for expansion and enabling NK cells of AML patients via promoting the function of activating receptors such as NKG2D, NKp46, and NKp30 [50, 216]. On the other hand, IL-2 alone may not be clinically effective as a monotherapy for AML patients [217–219]; however, in combination with histamine di-hydrochloride, IL-2 has been reported to be effective as a maintenance therapy in patients with AML, resulting in leukemia-free survival improvement [220, 221]. The mechanism of this method may partly be due to a remarkable expansion of CD56^bright subpopulations of NK cells [222].

In order to increase proliferation and expansion of NK cells, cytokines have been extensively used in the context of NK cell transfer as a “priming or arming” process. However, their short-lasting effect and short-term persistence within patients’ blood might limit their clinical application. Therefore, despite inhibitory KIR-KIR ligand interactions, a mixture of cytokines (IL-18, IL-15, and IL-12) are presented as an amplified antileukemia response and re-stimulation form several weeks to few months [51, 52, 223]. These cytokine-induced memory like (CIML) NK cells along with adaptive immune properties provide a promising method to improve adoptive NK cell transfer efficiency. The first inhuman clinical trial of CIML NK cells transfer in elderly AML patients revealed an effective remission (ORR = 67%) without the cause of GvHD, CRS, or neurotoxicity. Conversely, patients outcome were negatively related to CD8α⁺ NK cells frequency and NKG2A expansion on CIML NK cells within AML patients [116, 224]. Further preliminary data also encourage scientists with more ongoing clinical trials of using CIML NK cells transfer for R/R AML patients (NCT02782546, NCT04354025, NCT03068819, and NCT01898793) [225, 226].

Drugs (with immunomodulatory function)

To improve NK cells function against AML, many anti-tumor drugs with immunomodulatory properties have been designed in the past years. Since AML malignant cells can resist NK cell-mediated killing capacity (through either changing their surface ligand expression or immunosuppressive factor secretion), drug that can restore ligand expressions of AML cells can make them more vulnerable to NK cell attacks [227]. Azacitidine and decitabine, as hypomethylating agents, can upregulate NKG2DL expression on AML cell surface, resulting in improved NK cell immunity via the immune-mediated recognition by engagement of NKG2D-NKG2DL [228]. Simultaneously, they can upregulate the expression of tissue inhibitor of metalloproteinases-3 (TIMP3), an inhibitor of ADAM17, which can decrease soluble NKG2DLs shedding from AML cells [229]. Differentiation-promoting drugs (all-trans-retinoic acids, bryostatin 1, and vitamin D3) [228], histone deacetylase inhibitors (valproic acid and trichostatin A) [230, 231], and hydroxyurea [232] all have shown to potentially upregulate NKG2DLs expression on AML cells. On the other hand, dinaciclib-treated AML cells are linked to lowering inhibitory NK ligand (HLA-E) expression on AML cells, resulting to induce anti-tumor immunity of NK cells. Moreover, pomalidomide and lenalidomide can exert antileukemic effects directly or through NK cell-based immune-modulatory activities besides to HLA class I downregulation on AML blasts [44, 233].

The combination therapy including the above-mentioned drugs are widely used in both clinical practice and clinical trials for AML. Natural compounds such as casticin [234], safrole [235], ouabain [236], and α-phellandrene [237] can also promote NK cells functionality against AML blasts. Moreover, novel immunomodulatory agents were suggested in several researches, providing a therapeutic implication in patients with AML. For example, vascular endothelial growth factor receptor (VEGFR)-3 antagonist re-established NK cells killing cytotoxicity with increasing IFN-γ level [238, 239]. Also, through galunisertib, a TGF-β receptor kinase inhibitor, therapeutic efficiency of adoptive NK cell transfer can be improved significantly [240]. With the clarification of anti-tumor drugs’ mechanisms, merging pharmacologic methods with other NK cell-related immunotherapies can potentially strengthen the safety and efficiency of clinical outcomes for patients with AML.

Conclusion and future perspective

NK cells represented a promising immunotherapy strategy that is equipped with rapid tumor marker detection, fast anti-tumor action, and immune response elevation capacities. Despite numerous immune escape mechanisms, a pivotal role of NK cells in AML immune surveillance has been visualized owed to their explicit cytolytic capacity and distinguished effector function. From adoptive NK cell transfer, cytokines and immunomodulatory drugs to antibodies and genetically modified methods, NK cells have notably paved the road to a better management of AML.

Several challenges are remained to augment NK cell potentials in the setting of AML immunotherapy. Blocking inhibitory receptors, eliminating T_regs activities, and neutralizing immunosuppressive cytokines are some of the existing issues. Providing essential cytokines and growth factors, which are required for activation, propagation, and persistence of NK cells are another challenges remained to be addressed. Finally, to extend current therapeutic methods, a better understanding of NK cells dysfunction mechanisms and immunotherapy-based methods against AML are required to the alternative therapies in the future.

Supplementary Information

Below is the link to the electronic supplementary material.

Author’s contribution

SR conceptualized the title, collected the data, designed the figures, and prepared the first draft of the manuscript. NY edited and revised the manuscript and finalized the draft. NR critically revised the manuscript, edited and finalized the draft, and supervised the project. All the authors have read and approved the final draft of the manuscript.

Funding

The authors did not receive fund, grant, and support from any organization for the submitted work.

Data availability

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Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

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