Immune evasion and therapeutic opportunities based on natural killer cells

Abstract

Natural killer (NK) cells can elicit an immune response against malignantly transformed cells without recognizing antigens, and they also exhibit cytotoxic effects and immune surveillance functions in tumor immunotherapy. Although several studies have shown the promising antitumor effects of NK cells in immunotherapy, their function is often limited in the tumor microenvironment because tumor cells can easily escape NK cell-induced death. Thus, for efficient tumor immunotherapy, the mechanism by which tumor cells escape NK cell-induced cytotoxicity must be fully understood. Various novel molecules and checkpoint receptors that mediate the disruption of NK cells in the tumor microenvironment have been discovered. In this review, we analyze and detail the major activating and inhibitory receptors on the surface of NK cells to delineate the mechanism by which tumor cells suppress NKG2D ligand expression and increase tumor receptor and inhibitory receptor expression [NKG2A, programmed cell death 1 (PD-1), and T-cell immunoglobulin and immunoreceptor tyrosine inhibitory motif (TIGIT)] on the NK cell surface, and thus inhibit NK cell activity. We also reviewed the current status of treatments based on these surface molecules. By comparing the therapeutic effects related to the treatment status and bypass mechanisms, we attempt to identify optimal single or combined treatments to suggest new treatment strategies for tumor immunotherapy.

Introduction

Tumor immunotherapy has revolutionized the field of tumor treatment. The use of immune checkpoint blockade (ICB) has been a major breakthrough in tumor immunotherapy; however, it has achieved success in cancer treatment for only a fraction of patients (1). Moreover, its application is limited in clinical practice, thus hindering the broad use of immunotherapy. Immune evasion contributes significantly to tumor progression and metastasis (2). The mechanisms of immune evasion must be investigated to identify novel targets, provide a deeper understanding of the mechanisms underlying ICB resistance, and help develop therapeutic strategies for overcoming the current immunotherapy bottleneck.

Chimeric antigen receptor (CAR) T-cell therapies are cutting edge immunotherapy techniques; however, they have been associated with adverse events, such as neurotoxicity and cytokine release syndrome (3). Moreover, CAR-T cells are expensive and complex to produce because they are manufactured specifically for each patient. Natural killer (NK) cells have similar biological properties to T-cells but are not truly antigen-specific and respond early to tumor-related threats; therefore, research interest in NK cells as candidates for immunotherapy has grown exponentially (4). NK cells, which are innate immune cells, kill virus-infected and malignant cells without recognizing antigens, and they present immune surveillance and cytotoxic functions in tumor immunotherapy (5). Hence CAR-NK cell therapies have rapidly attracted the attention of immunologists. Clinical trials using cord blood-derived CAR-NK cells have demonstrated clinical efficacy (6). Moreover, the use of CAR-NK cells in vivo does not induce the development of adverse events associated with CAR-T cells (6). These encouraging results have laid a foundation for the application of NK cells in future immunotherapy strategies.

Tumors present several mechanisms of evading the immune system’s surveillance, including activation of immune checkpoints that can restrict NK cell function. Although many NK cell-based clinical trials are ongoing, the results have not always been satisfactory. These antitumor strategies need to overcome the challenge of decline in NK cell activity associated with mechanisms that bypass the immune system (7). The activation of NK cells is relatively straightforward, and the overall mechanism of immune escape might not involve the complex antigen presentation of T-cells (1). Therefore, summarizing the mechanism of NK cell-based immune evasion might provide insights for personalized treatment and more reasonable and effective combined immunotherapy. Currently, tumor immunotherapy approaches include administration of single or combination target drugs or adoptive immunotherapy. Therefore, an analysis from the perspective of NK cell-based immune evasion can facilitate the development of improved treatment methods.

The balance between the various activating and inhibitory receptor-mediated signals in NK cells determines the suppression or induction of NK cell function (8). NKG2D and NKG2A are representative NK cell activating and inhibitor receptors, respectively. Jhunjhunwala et al. conducted an in-depth analysis on immune evasion and suggested that NK cell-based immune evasion is represented by downregulated NK cell activity accompanied by changes in histocompatibility leukocyte antigen E (HLA-E) expression, NKG2D downregulation by tumor cell shedding of major histocompatibility complex class I (MHC I) polypeptide-related sequence A and B (MICA/B), and immune checkpoint upregulation, such as programmed cell death 1 (PD-1) and T-cell immunoglobulin and immunoreceptor tyrosine inhibitory motif (TIGIT) (1). Here, we provide an overview of the mechanisms underlying immune evasion and describe the current status of therapies and treatment options based on these mechanisms.

Mechanism of NK cell-based immune evasion

Mechanism based on NKG2D-NKG2DL axis

NKG2D is an essential activating receptor expressed on the surface of NK cells and many T-cells. The YXXM (Tyr.X.X.Meth) sequence in the cytoplasmic domain of DAP10 recruits growth factor receptor binding protein 2 (GRB2) and phosphatidylinositol 3-kinase (PI3K) (9). Activation of these two signaling pathways leads to an increased concentration of intracellular calcium and restructuring of the actin cytoskeleton in NK cells. Eventually, immune synapses form among tumor cells and NK cells (10), with NK cells acting via several key pathways that mediate the death of target cells.

Although NKG2D ligands are overexpressed in tumor cells, they are generally not expressed in normal tissues (11); however, they are upregulated following DNA damage, inflammation, and malignant transformation. Therefore, NKG2D ligands are promising targets for adoptive immunotherapy using NK cells and cytotoxic T-cells, such as NKG2D-based CARs (12). In addition, NKG2D-expressing CAR-NK cells exhibit significantly increased cytotoxicity, suggesting their clinical therapeutic potential (13,14). In a phase I clinical trial, the safety of NKG2D-expressing CAR-T cells has also been demonstrated (15).

Soluble MIC leads to endocytosis and degradation of NKG2D

Tumors present multiple strategies to subvert the biological function of NKG2D and evade the immune system. The first is the proteolytic shedding of the ligand [Figure 1 shows protein (16-18) functioning on this site], which results in the downregulation or degradation of NKG2D (Figure 2). After proteolytic shedding, MICA is released into the microenvironment. The released soluble MICA (sMICA) binds to NKG2D, leading to its endocytosis and degradation (19). Some tumor cells release large amounts of sMICA, which impairs NK cell functions and leads to tumor progression (20).

Figure 1.

Mechanism of immune evasion through NKG2D-NKG2DL axis and therapeutic strategies. Mechanism of immune evasion (left panel): Proteolytic release of MICA/B of tumor cells leads to the downregulation or degradation of NKG2D. Its release is regulated by MMPs, members of the ADAM family, and ERp5, and the site of proteolytic shedding is located in the extracellular α3-domain of MICA. sMICs present in tumor-secreted exosomes might also lead to the downregulation and degradation of the ligand. TGF-β in the TME inhibits NK cell functions by downregulating NKG2D expression. MICA/MICB polymorphisms can also impair the recognition and binding of NKG2D. Therapeutic strategy (right panel): 1) Specific antibodies targeting the MICA α3 domain can decrease the loss of MICA/B on the surface of tumor cells; 2) Antibodies induced by a new tumor vaccine targeting the MICA/B protein can increase the numbers of MICA/B by inhibiting protein hydrolysis and shedding; 3) A CAR subpopulation, which contains the transmembrane domain of NKG2D, can increase the expression of NKG2D and mediate strong antigen-specific NK cell signaling; and 4) The soluble ligands can be removed from the plasma of patients to recover NK cell functions. MICA/B, major histocompatibility complex class I (MHC I) polypeptide-related sequence A and B; sMIC, soluble MIC; TGF, transforming growth factor; TME, tumor microenvironment; NK, natural killer.

Figure 2.

Primary mechanism of immune evasion of tumors based on NK cells. (A) NKG2D is an activating receptor of NK cells. Proteolytic shedding of MICA/B from the tumor cell surface leads to the downregulation or degradation of NKG2D. TGF-β in the TME also inhibits NK cell functions by downregulating NKG2D expression; (B) TGF-β in the TME inhibits NK cell functions by upregulating the expression of NKG2A. Tumor cells, cDCs, and tumor macrophages send a negative signal to NK cells by overexpressing HLA-E; (C) NK cells obtain PD-1 from tumor cells by trogocytosis. Glucocorticoids together with IL-12, IL-15, and IL-18 upregulate the transcription of PDCD1 in NK cells, thereby increasing PD-1 expression. PD-L1 expression is upregulated in response to multiple inflammatory stimuli in the TME, of which interferon-γ is the most potent. IFN-γ upregulates the expression of the ligand on tumor cells and increases the expression of the ligand on myeloid cells, thereby transmitting inhibitory signals to NK cells; (D) TIGIT ligand CD155 is highly expressed in tumor cells and inhibits NK cell functions by binding to TIGIT. MDSCs in the TME produce ROS, which upregulate the expression of CD155 on MDSCs. Interaction of the faap2 protein on the surface of Fusobacterium nucleatum with TIGIT might also inhibit the cytotoxicity of NK cells. NK, natural killer; MICA/B, major histocompatibility complex class I (MHC I) polypeptide-related sequence A and B; TGF, transforming growth factor; TME, tumor microenvironment; cDC, conventional dendritic cell; HLA-E, histocompatibility leukocyte antigen E; PD-1, programmed cell death 1; IL, interleukin; IFN, interferon; TIGIT, T-cell immunoglobulin and immunoreceptor tyrosine inhibitory motif; MDSC, myeloid-derived suppressor cell.

sMIC negatively reprograms the expression of genes relevant to the homeostatic survival and proliferation of NK cells (21). Ligands present in tumor-secreted exosomes (22) also downregulate NKG2D expression. The accumulation of vesicle-derived NKG2D ligands in the tumor microenvironment (TME) suppresses the activity of NK cells either by downregulating NKG2D expression or inducing the fratricide of NK cells dressed with EV-derived NKG2D ligands (Figure 1) (23). These processes impede tumor recognition and cytotoxicity of a large number of NK cells, leading to immune evasion. Moreover, some tumor stem cells are unable to express the NKG2D ligand (24). In acute myeloid leukemia (AML), the stemness of chemotherapy-resistant leukemic stem cells (LSCs) was believed to be related to immune evasion mediated by the NKG2D ligand because the absence of the NKG2D ligand defines LSCs, further indicating the importance of immune evasion mediated by the NKG2D ligand (25).

Downregulated expression of NKG2D

Downregulation of NKG2D receptor expression is a major mechanism of immune evasion by tumor cells. NKG2D expression decreases in NK cells in the TME due to several factors (26,27), such as transforming growth factor (TGF)-β (28,29) (Figure 2). Platelet occlusion also causes NKG2D ligand shedding (30). Mechanistically, platelet-derived TGF-β may be the main factor inhibiting the cytotoxicity of NK cells (31). IDO1 was also suggested to cause dysregulate NK cell activity via downregulating NKG2D expression in NK cells (32).

Polymorphisms of MICA/B

The effects of MICA/MICB ligand polymorphisms have been studied (33), and approximately 100 and 40 alleles have been reported for these ligands, respectively. MICA/B polymorphisms can also impair the recognition and binding of NKG2D, thereby reducing the cytotoxicity and recognition ability of NKG2D-expressing NK cells against tumor cells and causing NKG2D-based immune evasion.

High expression of NKG2A and HLA-E

NK cells express inhibitory receptors, including CD94/NKG2A heterodimer that recognize MHC class I molecules. These inhibitory receptors belong to the C-type lectin family (34). The cytoplasmic tail of NKG2A contains sequences of the immunoreceptor tyrosine-based inhibition motif (ITIM), which forms a heterodimer with the CD94 chain and identifies HLA-E as its ligand. After binding to the NKG2A/CD94 heterodimer, the function of T and NK cells is inhibited by recruiting SHP-1 tyrosine phosphatase to the ITIM of NKG2A (35). This axis is characterized by limited polymorphisms, and HLA-E has been proven to be highly expressed in a wide array of tumors (36,37), indicating that this axis may play an immunosuppressive role in the TME (36). Exploring the immunoregulatory mechanism of the NKG2A-HLA-E axis can help in developing improved and more efficient tumor targeting drug combinations.

Cytokines such as TGF-β can upregulate the expression of CD94/NKG2A in the TME (Figure 2) (38). Tumor-derived interferon (IFN)-γ inhibits the activity of cytotoxic cells via NKG2A/CD94 induced by ICB (39). Moreover, the expression of NKG2A in NK cells was dominant in the TME, while its expression was higher in breast cancer-infiltrating NK cells than in circulating NK cells (26). Although the mechanism is still unknown, this finding suggests that NKG2A contributes to immune evasion not only in tumor tissues but also in lymph nodes. Thus, even though the potential of NKG2A as a therapeutic target for immune evasion has been explored, its contribution to tumor metastasis remains to be elucidated.

Several tumor cells highly express HLA-E to escape the cytotoxicity of NK cells (36,37). The accumulation of HLA-E in cancer tissues originates from epithelial-derived cancer cells, CD141+ conventional dendritic cells (cDCs), and tumor macrophages (Figure 2) (40). Further studies are warranted to shed light on this mechanism of immunosuppression.

Mechanism based on PD-1/programmed cell death 1 ligand (PD-L1) axis

PD-1 is encoded by PDCD1, which is a member of the immunoglobulin gene superfamily. PD-1 is an inhibitory receptor primarily expressed on T-cells and plays a vital physiological role in the maintenance of peripheral tolerance, and it is also expressed in NK cells (41). Although CD8⁺ T-cells may have difficulty detecting some common tumors with low neoantigen load and HLA loss (42,43), PD-1/PD-L1 blocking immunotherapy was still effective in such patients (44), and NK cells played a vital role. These findings demonstrate the notable contribution of NK cells to PD-1/PD-L1 blocking immunotherapy (44), thus highlighting the importance of PD-1 in NK cells as an inhibitory checkpoint.

Induced expression of PD-1 on NK cells

The expression of PD-1 in NK cells is upregulated in many patients with cancer (45,46); however, the effect of this upregulated expression remains controversial (47). Most studies exploring whether or not PD-1 is expressed by NK cells still focus on the phenotype. However, many patients develop resistance to immunotherapy. Therefore, confirming the molecular mechanisms that control the expression of PD-1 and PD-L1 is important, so that strategies can be developed to enhance the efficacy of blockers of the PD-1/PD-L1 axis. Here we summarize recent studies investigating these mechanisms.

Studies on viruses have shown that endogenous glucocorticoids can induce the selective and tissue-specific expression of the checkpoint receptor PD-1 in NK cells (48). Mechanistically, glucocorticoids, together with interleukin (IL)-12, IL-15, and IL-18, not only upregulate PDCD1 transcription but also promote a transcriptional program that leads to increased protein translation and enhances the surface expression of the protein (Figure 2) (49). Moreover, this regulatory mechanism of human NK cells is not transferable to mouse NK cells, indicating that species specificity should be considered in studies exploring the mechanisms underlying NK cells.

In a study of leukemia, NK cells were shown to acquire PD-1 entirely from leukemia cells via trogocytosis in a SLAM receptor-dependent manner (Figure 2) (50). This should also be considered in the development of future therapies.

Highly expressed PD-L1 in TME

The PD-L1 protein is highly expressed in the TME. Notably, tumor cells activate local immune tolerance by overexpressing PD-L1 to keep NK cells and T-cells under control. PD-L1 is expressed in various kinds of cancer and myeloid cells in the TME, and its expression is upregulated in response to various inflammatory stimuli (51-53), of which IFN-γ is the most potent (Figure 2) (54). These inflammatory mediators induce PD-L1 and PD-L2 expression not only in cancer cells but also in other cell types in the TME (including macrophages, dendritic, and stromal cells) (51,52). The expression of PD-L1 in antigen-presenting cells (APCs) (but not tumor cells) has been suggested to play an essential role in checkpoint blockade therapy (53). These results provided insights into the mechanistic details of this treatment (53). In another study, a previously unknown strategy mediated via a shift toward an exhausted PD-1-enriched CD3-CD56hiCD16-ve NK cell phenotype facilitated immune evasion (55). Through this mechanism, NK cells are indirectly suppressed by PD-L1/PD-L2-expressing tumor-associated macrophages (TAMs) as well as directly inhibited by malignant B cells (55).

Upregulated expression of TIGIT and CD155

TIGIT, also known as WUCAM, Vstm3, and VSIG9, is a receptor of the Ig superfamily that is expressed mainly in NK cells and T-cells. Researchers have revealed that the CD155/TIGIT axis is necessary and sufficient to promote and maintain immune evasion in pancreatic adenocarcinoma (PDAC) (56). Recent studies emphasized the specificity and importance of TIGIT in NK cells, including high expression of TIGIT in NK cells present in the TME (57) and its specificity in NK cell exhaustion, but not that of the other checkpoint molecules CTLA-4 and PD-1 (57). Anti-TIGIT monotherapy or in combination with PD-1 blockade was shown to mainly affect NK cells, not only by promoting NK cell-dependent tumor immunity but also by enhancing NK cell-dependent tumor-specific T-cell immunity (57). These findings demonstrated the role of TIGIT in the NK cell-based mechanism of immune evasion of tumor cells.

The expression of TIGIT on mouse NK cells is upregulated during tumor progression. In patients with colorectal tumors, the constitutive expression of TIGIT in NK cells is reportedly further upregulated in the tumor compared with that in the peritumoral area (57). Likewise, the expression of TIGIT in NK cells from ascites of patients with ovarian cancer was higher than that in NK cells from healthy donors (58). TIGIT/CD155 binding suppresses IFN-γ production via the NF-κB pathway (59). Specifically, a novel adaptor β-arrestin 2, which associates with phosphorylated TIGIT to recruit SH2-containing inositol phosphatase 1 (SHIP1) via the ITT-like motif, impairs the autoubiquitination of TNF receptor-associated factor 6 (TRAF6), thus abrogating the activation of NF-κB, and in turn leading to suppressed production of IFN-γ in NK cells (59).

CD155 is highly expressed in many tumor tissues (60,61), although its mechanism of function in tumor immunity is relatively complex. CD155 can inhibit NK cell functions via PI3K and MAPK signaling after binding to TIGIT (62). Its intrinsic roles in tumor cells include promotion of tumor migration (63), shortening of the cell cycle of tumor cells, and promotion of tumor cell proliferation (64). In addition, CD155 affects the cytotoxicity of NK cells by downregulating the expression of CD226 on their surface (65).

Bacteria present in the TME may also inhibit NK cell functions through TIGIT. Mechanistically, TIGIT binds directly to the fap2 protein of Fusobacterium nucleatum (a bacterium commonly found in the TME), inhibiting the cytotoxicity of NK cells (Figure 2) (66). Similarly, myeloid-derived suppressor cells (MDSCs) in the TME inhibit NK cell functions through TIGIT. Interestingly, TIGIT signaling in NK cells lead to a decrease in the phosphorylation of ZAP70/Syk and ERK1/2 after coculture with MDSCs, which in turn produced reactive oxygen species (ROS) that upregulated the expression of TIGIT ligand CD155 on their surface (Figure 2) (67).

Therapeutic status and opportunities based on above mechanism

Therapeutic status and opportunities related to NKG2D

Antibody-mediated inhibition of MICA/B shedding

Numerous studies have explored methods of improving the cytotoxicity of NK cells by targeting NKG2D-NKG2DL axis-mediated immune evasion mechanisms. The clinical application of α3 domain-targeted MICA/B antibodies may offer a new strategy for cancer immunotherapy (Figure 1) (68). Antibodies targeting tumor-derived sMIC not only reprogrammed the homeostatic survival and function of NK cells but also enhanced the response to PD-L1 blockade therapy in melanoma (21). These findings highlight the importance of antibodies targeting sMIC to improve the effect of anti-PD1/PD-L1 antibody-based treatment in patients with MIC/sMIC+ metastatic melanoma (21).

Anti-MICA/B and tumor vaccine

Anti-MICA/B antibodies have been used to enhance the response of immunotherapy applied to human colorectal tumor spheroids by increasing the activation and infiltration of NK cells (69). Interestingly, tumor cells responded to immunotherapy by upregulating the expression of HLA-E, which is the ligand of NKG2A expressed by NK and CD8⁺ T-cells (69). This demonstrated the antitumor potential of combination immunotherapy targeting MICA/B and NKG2A (69); however, the specific mechanism remains unclear.

In a preclinical study of osteosarcoma, acetylation of the NKG2D ligand protected RAE-1 from shedding, thereby revealing potential new targets for immunotherapy based on NKG2D (70). Recently, a new tumor vaccine targeting the MICA/B protein was reported. This vaccine induced the generation of antibodies that increased the levels of MICA/B by restraining protein hydrolysis and shedding, thereby facilitating a stronger cytotoxic function of NK cells (Figure 1) (71). Moreover, the design of this vaccine enabled protective immunity even target tumors with common escape mutations (71). Researchers have also attempted to remove these soluble ligands from the plasma of patients to recover NK cell functions (Figure 1). They proposed the adsorption apheresis of these soluble ligands as a future preprocessing method to increase the efficacy of autologous and adoptively transferred immune cells in cancer immunotherapy (72). The mechanism underlying therapeutic potential of chemotherapy-resistant LSCs has also been investigated, and poly-ADP-ribose polymerase 1 (PARP1) was found to repress NKG2D ligand expression. The genetic or pharmacological inhibition of PARP1 can thus induce NKG2DL expression on the LSC surface, thereby providing strong evidence for combining PARP1 inhibition and NK cell functions (25).

NKG2D-CAR-NK cells

Researchers have identified a CAR cell subpopulation that contains the transmembrane domain of NKG2D and can mediate potent antigen-specific NK cell signaling (Figure 1) (73). It remarkably prolonged survival and reduced tumor burden in xenograft models of ovarian cancer, showed similar activity in vivo to that of CAR-T cells, and was less toxic (73). Autologous NKG2D-CAR-NK cells showed enhanced antimyeloma activity in both in-vivo and in-vitro experiments (14), supporting the potential of NKG2D-CAR-NK cells in the treatment of multiplemyeloma (MM). By fusing the extracellular domain of NKG2D to DAP12, a NKG2D RNA CAR was engineered to improve antitumor response of NK cells. Its expression remarkably enhanced the cytotoxicity of NK cells to several kinds of tumor cell lines in preclinical models (13) and led to decrease in ascites and tumor cells in patients with metastatic colorectal cancer (13). The combination of immune metabolism and NKG2D-CAR-NK cells can thus result in remarkable antitumor responses in vivo within the hypoxic TME of solid tumors (74).

However, the therapeutic effect of NKG2D-CAR-NK cells might be limited if the immune evasion mechanism associated with the ligand is not resolved. Therefore, researchers developed a homologous recombinant antibody called NKAB-ErbB2, which enables tumor-specific NKG2D-expressing effector cells to be independent of membrane-anchored NKG2D ligands (75). This molecule carries an NKG2D-based CAR, which enables its relocalization to ERBB2⁺ tumor cells and increases tumor antigen specificity and NKG2D-CAR and NKG2D-mediated therapeutic efficacy (75). This was shown to be an alternative method of exploiting the molecular properties of NKG2D to avoid immune evasion. Therefore, NKG2D-CAR-NK therapy that simultaneously targets sMICA might be a new strategy to enhance NK cell functions.

NKG2A-related monotherapy and combination therapy

Anti-NKG2A monotherapy

Monalizumab has high affinity for CD94/NKG2A and blocks inhibitory HLA-E signaling (76). Preclinical studies have suggested that blocking NKG2A on NK cells in chronic lymphocytic leukemia (CLL) is sufficient to directly restore the cytotoxicity of NK cells against HLA-E⁺ targets, without affecting NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) (37). The low efficacy of monotherapy might be attributed to the fact that only 50% of NK cells in peripheral blood express NKG2A, which may limit the potential of NKG2A therapy (77,78). Nevertheless, the importance of anti-NKG2A antibodies in combination therapy has been well recognized by researchers.

Importance of anti-NKG2A in combination therapy

The expression of NKG2A and PD-1 in NK cells in tumor-draining lymph nodes (TD-LNs) supports their potential for antitumor immunotherapies using anti-NKG2A, anti-PD-1, or both antibodies, and it also suggests that anti-NKG2A and anti-PD-1 combination therapy might be a preferred adjuvant therapy for breast cancer (79). Monalizumab in combination with PD-1/PD-L1 axis blockade enhanced NK cell functions against a variety of tumor cells, improved survival, and rescued the cytotoxic T-cell-dependent response (76). In preclinical mouse tumor models, the combination of NKG2A and PD-L1 blocking therapy (monalizumab and durvalumab) has shown better therapeutic efficacy and synergistic effects (76). In a phase II clinical trial called COAST, the therapeutic efficacy of durvalumab alone or in combination with monalizumab or oleclumab (the anti-CD73 antibody) as consolidation therapy was assessed in patients with unresectable stage III non-small cell lung cancer (80). The confirmed objective response rate (ORR) was reported to be higher and progression-free survival (PFS) was prolonged in patients treated with durvalumab combined with monalizumab as compared to those treated with durvalumab. In addition, adverse events of grades ≥3 occurred at the lowest frequency (27.9%) in patients treated with durvalumab combined with monalizumab but at a higher frequency (39.4%) in patients treated with durvalumab alone (80). This highlights the role of anti-NKG2A therapy, demonstrates the potential of combination therapy with anti-NKG2A and anti-PD-1, and raises expectations for future phase III clinical trials. Table 1 summarizes ongoing clinical trials of NKG2A monotherapy and combination therapy.

Table 1. Ongoing clinical trials involving anti-NKG2A alone or in combination with other therapies.

Trial identifier	Phase	Participants	Drugs	Condition or diseases	Status
PD-(L)1, programmed cell death 1/programmed cell death 1 ligand.
NCT04307329	Phase 2	38	Monalizumab, trastuzumab	Breast cancer	Active, not recruiting
NCT04590963	Phase 3	369	Monalizumab, cetuximab	Squamous cell carcinoma of the head and neck	Active, not recruiting
NCT05221840	Phase 3	999	Durvalumab with monalizumab or oleclumab	Non-small cell lung cancer	Recruiting
NCT05414032	Phase 2	200	Monalizumab, cetuximab	Locoregionally advanced head and neck squamous cell carcinoma	Not yet recruiting
NCT02643550	Phase 1, Phase 2	143	Monalizumab and cetuximab with or without anti-PD-(L)1	Head and neck neoplasms	Active, not recruiting
NCT03822351	Phase 2	188	Durvalumab alone or with monalizumab or oleclumab	Stage III non-small cell lung cancer, unresectable	Active, not recruiting
NCT05061550	Phase 2	210	Durvalumab with oleclumab or monalizumab	Non-small cell lung cancer	Recruiting
NCT03088059	Phase 2	340	Monalizumab alone or with durvalumab and other interventions	Carcinoma, squamous cell of head and neck	Recruiting
NCT03833440	Phase 2	120	Durvalumab with monalizumab or oleclumab or other interventions	Non-small cell lung cancer	Recruiting

Loss of the function of Qa-1b (HLA-E in humans) in mice improved the efficacy of PD-1 immunotherapy (81) in preclinical studies. In addition, disruption of the NKG2A/Qa-1b axis significantly enhanced the effect of therapeutic vaccines, even in PD-1 refractory mouse models (82). Of note, in bladder tumors with limited PD-1 efficacy, NKG2A was associated with improved survival and PD-L1 blocking of immunotherapy reactivity (83). These data support the strategy of combining monoclonal antibodies that block PD-1/PD-L1 with NKG2A/HLA-E in clinical applications.

Preliminary results in patients with squamous cell carcinoma of the head and neck (SCCHN) treated with monalizumab in combination with cetuximab (an anti-EGFR antibody) revealed improved overall response rates, even in refractory patients. Importantly, blockade of NKG2A showed little toxicity and no evidence of autoimmunity (76). In-vitro experiments revealed that monalizumab promoted NK cell-mediated ADCC against SCCHN tumor cells coated with cetuximab. This action was likely mediated by NK cells expressing NKG2A rather than activated cytotoxic T-cells (76). These clinical data suggested that NKG2A is a promising checkpoint inhibitor for combination therapy.

Highly functional NK and ML NK cells

To bypass HLA-E-mediated inhibition, researchers have attempted to develop a method to generate highly functional NK cells that lack NKG2A expression on the surface (84). These NKG2A protein expression blockers (PEBLs) were generated by linking a single-chain variable fragment derived from an anti-NKG2A antibody to endoplasmic reticulum retention domains. Interestingly, PEBLs eliminated the expression of NKG2A, bypassed HLA-E cancer immunity checkpoints, and enhanced the function of NK cell infusions (84).

A group studied the phenotypic characteristics of cytokine-induced memory-like (ML) NK cells and demonstrated that NKG2A is a dominant checkpoint (85). The potent cytotoxic therapeutic effect of ML NK cells relies on the activating receptors NKG2D and NKp46 (86). When anti-MICA/B was used to treat colon tumors, tumor cells responded to the increased cytotoxicity by upregulating the expression of HLA-E (69). Therefore, combined therapy based on targeting the mechanisms of immune evasion through NKG2A and NKG2D may overcome the obstacles of NK cell therapy in the future.

Targeting of PD-1/PD-L1 pathways for improving NK cell functions

CAR-NK cells targeting PD-L1 have an enhanced effect against solid tumors in vitro and in vivo. Combined treatment with CAR-NK cells and nivolumab resulted in a synergistic response against solid tumors (87). Fabian et al. developed a new NK cell line, PD-L1-targeting high-affinity natural killers (t-haNKs), from NK-92 cells that express high-affinity CD16, endoplasmic reticulum-retained interleukin-2, and a PD-L1 specific CAR (88). They demonstrated that PD-L1 t-haNK cells had good antitumor activity and preferentially lysed the myeloid-derived suppressor cell population but not other immune cell types when cocultured with human peripheral blood mononuclear cells (PBMCs) (88). In another study, NK cells were chemically equipped with a TLS11a aptamer targeting HepG2 cells and PD-L1-specific aptamer without genetic alteration. This improved the specificity of NK cells, remarkably upregulated the expression of PD-L1 in HepG2 cells, and enhanced checkpoint blockade. This holds significant potential for improved adoptive immunotherapy against solid tumors (89).

In some tumors deficient in the expression of MHC class I proteins, the efficacy of PD-1 and PD-L1 blockers depends on the activity of NK cells (44). In the model of PD-1-resistant B16-F10 lung metastasis, anti-PD-1 monotherapy failed to achieve an antitumor effect, whereas a lipid nanoparticle containing a stimulator of an interferon gene (STING) and an agonist (STING-LNP) achieved synergistic antitumor effects when combined with anti-PD-1 (90). Mechanistically, NK cells are activated by stimulating the STING pathway to produce IFN-γ, leading to an increase in the expression of PD-L1 on tumor cells, resulting in a synergistic antitumor effect when anti-PD-1 was administered (90). NKTR214 (bempegaldesin) is a novel agonist of the IL2 pathway that provides sustained signaling via heterodimeric IL2 receptor βγ to drive the increased proliferation and activation of CD8+ T-cells and NK cells (91). A phase I trial of the combination of NKTR214 and nivolumab (anti-PD-1 mAb) was conducted in patients with advanced solid tumors (NCT02983045) (92), in which the combination was shown to be safe, with an ORR of 59.5% (92). Based on the assumption of activating NK cells while targeting PD-1/PD-L1, researchers also developed N-809, which is fused with the IL-15 superagonist N-803 in 2 αPD-L1 domains and stimulates the expression of IL-15 and blocks PD-L1 (93). N-809 had a stronger antitumor effect and immunomodulatory effects in the TME compared with the N-803+αPD-L1 combination (94).

Preclinical studies have suggested that blocking PD-L1 enhances the cytotoxicity of NK cells against tumor cells (95). Mechanistically, NK cells secrete a large amount of IFN-γ, which stimulates further expression of PD -L1 on tumor cells (95); conversely, anti-PD-L1 mAbs act directly on PD-L1⁺ NK cells against PD-L1^- tumors via the p38 signaling pathway (96). The endogenous glucocorticoids can induce the selective and tissue-specific expression of the checkpoint receptor PD-1 in NK cells (48). In a study of leukemia, NK cells were shown to acquire PD-1 entirely from leukemia cells via trogocytosis in a SLAM receptor-dependent manner (50). These studies support the combination of NK cells and PD-1 axis therapy.

The combination therapy of anti-PD-1/PD-L1 and NK cells is considered to have good clinical prospect. In non-small cell lung cancer (NSCLC) clinical trials, the anti-PD-1 antibody pembrolizumab in combination with allogeneic NK cells resulted in a higher ORR and significant improvement in survival in patients with advanced NSCLC and treated with PD-L1⁺ (97). In addition, the combination of highly activated allogeneic NK cells (“SMT-NK”) with pembrolizumab did not result in any serious adverse events directly related to the drug combination in patients with advanced biliary tract cancer (98). This combination therapy also exerted enhanced efficacy compared with monotherapy with pembrolizumab (98). Table 2 summarizes ongoing clinical trials of PD-1/PD-L1 combination therapy with NK-based treatment.

Table 2. Ongoing clinical trials involving anti-PD-1/PD-L1 in combination with NK cell-based therapy.

Trial identifier	Phase	Participants	Intervention/treatment	Condition or diseases	Status
PD-1, programmed cell death 1; PD-L1, programmed cell death 1 ligand; IL, interleukin; DC, dendritic cell; NK, natural killer; CAR, chimeric antigen receptor.
NCT05334329	Phase 1	21	Umbilical cord blood natural killer cells (CB-NK) expressing soluble IL-15 (sIL-15) and PD-L1 +/− atezolizumab	Advanced/metastatic/recurrent/refractory non-small cell lung carcinoma	Recruiting
NCT05461235	Phase 2	/	Anti-PD-1 antibody combined with autologous DC or NK cells	Digestive carcinoma	Not yet recruiting
NCT03383978	Phase 1	42	Intracranial injection of NK-92/5.28.z cells in combination with the anti-PD-1 antibody ezabenlimab (BI 754091)	Glioblastoma	Recruiting
NCT04847466	Phase 2	55	Irradiated PD-L1 CAR-NK cells plus pembrolizumab plus N-803	Recurrent/metastatic gastric or head and neck cancer	Recruiting
NCT04050709	Phase 1	16	PD-L1 t-haNK	Locally advanced or metastatic solid cancers	Active, not recruiting
NCT04927884	Phase 1, Phase 2	79	Sacituzumab plus cyclophosphamide, N-803, and PD-L1 t-haNK	Advanced triple negative breast cancer	Active, not recruiting
NCT04390399	Phase 2	328	Standard-of-care chemotherapy versus standard-of-care chemotherapy in combination with aldoxorubicin HCl, N-803, and PD-L1 t-haNK	Pancreatic cancer	Recruiting
NCT03228667	Phase 2	145	PD-1/PD-L1 checkpoint inhibitor, N-803, and PD-L1 t-haNK cellular therapy	Patients who have previously received treatment with PD-1/PD-L1 immune checkpoint inhibitors	Active, not recruiting

TIGIT in NK-cell-based therapy

TIGIT is considered a previously neglected checkpoint in NK cells, and studies have recently highlighted its crucial role in NK cells. For instance, blockade of TIGIT reversed the exhaustion of tumor-infiltrated NK cells and promoted NK cell-dependent tumor immunity (57). Blockade of TIGIT led to NK cell-dependent tumor-specific T-cell immunity and enhanced the efficacy of anti-PD-L1 therapy, which in combination with other checkpoint receptors is a promising therapeutic strategy against cancer (57). Administration of IL-15 in combination with TIGIT blockers increased NK cell-mediated melanoma cytotoxicity in vitro and reduced tumor metastasis in mouse melanoma models. These results support the novel combinatorial immunotherapy with IL15 and TIGIT blockade for promoting NK cell-mediated destruction of MHC class I-deficient melanoma that was refractory to CD8+ T-cell-mediated immunity (99). Current research on the role of TIGIT in NK cells is still in the preclinical stage. However, based on the reported findings of the effects of TIGIT on NK cells, clinical trials on combined treatment with NK cells are expected in the future.

TIGIT is highly expressed in tumor-infiltrating T-cells of humans and mice; hence, most studies were initially focused on the function of TIGIT in T-cells. Combined blockade of TIGIT and administration of PD-L1 antibodies synergistically and specifically improved the effector function of CD8⁺ T-cells (100). Further, the expression of TIGIT was upregulated in T-cells and in dysfunctional PD-1⁺TIM-3⁺ tumor antigen-specific (TA-specific) CD8⁺ T-cells upon PD-1 blockade (101).

Collectively, these findings suggest that TIGIT might play a complementary role in PD-1/PD-L1-based therapies and support the possibility of combination therapy of anti-PD-1 and anti-TIGIT. In a phase I clinical trial, the anti-TIGIT (T-cell immunoglobulin and ITIM domain) antibody vibostolimab in combination with pembrolizumab was well tolerated and demonstrated antitumor activity in patients with advanced solid tumors, including advanced NSCLC (102). Phase II studies of anti-TIGIT in combination with anti-PD-L1 have also shown a clinically significant improvement in ORR and PFS compared with a placebo plus atezolizumab in patients with NSCLC (103). In addition, a combination of the two drugs was reported to be well tolerated, and its safety profile was similar to that of atezolizumab alone (103). These results suggest the importance of TIGIT in future immunotherapy.

Conclusions and perspectives

NK cells belong to a family of innate immune cells, and they kill virus-infected cells and malignantly transformed cells without recognizing specific antigens, thereby exhibiting immune surveillance activity and cytotoxic functions in tumor immunotherapy (5). Moreover, NK cells exhibit effective cytotoxicity, do not require the involvement of specific antigens, do not rely on MHC molecules to perform allogeneic adoptive transfer, do not cause cytokine release syndrome, and do not cause graft-versus-host disease (6). Because of these properties, NK cells are attractive targets for tumor immunotherapy and have received increasing attention from clinicians and researchers. The effector function of NK cells is based on the integration of activating and inhibitory signals. Here, we elaborated on the mechanism of immune evasion associated with the activating receptor NKG2D and the current status of related treatments, and proposed that activation of NKG2D and simultaneous inhibition of MICA shedding might enhance the activity of NK cells. The inhibitory molecule NKG2A on the surface of NK cells exerts low toxic effects when used in combination therapy, making it a better partner for future combination therapies. Although anti-PD-1/PD-L1 therapies have played a prominent role in the immunotherapy phase, they provide benefits to only a small number of patients. When combined with NK cells with enhanced functions, such as increased activity or specificity, these combination therapies might not only overcome immune evasion but also achieve a significant therapeutic outcome through NK cell-mediated ADCC. Future studies should aim at sustaining and enhancing the immune infiltration of NK cells. The combination of these functions of NK cells with PD-1/PD-L1 targets is expected to result in optimal therapeutic effects. The function and therapeutic benefits of TIGIT in NK cells require data from preclinical studies and clinical trials. Future studies should focus on the combination of anti-TIGIT and anti-PD-1 treatments as well as treatment combinations that enhance NK cell functions, such as CAR-NK cells.