Abstract
Natural killer (NK) cells are lymphocytes of the innate immune system that have the ability to recognize malignant cells through balanced recognition of cell-surface indicators of stress and danger. Once activated through such recognition, NK cells release cytokines and induce target cell lysis through multiple mechanisms. NK cells are increasingly recognized for their role in controlling tumor progression and metastasis and as important mediators of immunotherapeutic modalities such as cytokines, antibodies, immunomodulating drugs, and stem cell transplantation. Recent advances in manipulating NK cell number, function, and genetic modification have caused renewed interest in their potential for adoptive immunotherapies, which are actively being tested in clinical trials. Here, we summarize the evidence for NK cell recognition of osteosarcoma, discuss immune therapies that are directly or indirectly dependent on NK cell function, and describe potential approaches for manipulating NK cell number and function to enhance therapy against osteosarcoma.
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Keywords
- Natural killer cell
- Adoptive immunotherapy
- Innate immunity
- Antibody-dependent cell cytotoxicity
- Chimeric antigen receptor
- Immunomodulating drugs
Brief Overview of NK Cell Biology
The number of natural killer (NK) cells in humans varies widely, comprising 1–32.6% (median 7.6%) of all peripheral blood lymphocytes [1]. They are identified by the lack of CD3 and the presence of CD56 and/or CD16, and make up 85% of the large granular lymphocyte population [2]. NK cells are a major component of the innate immune system whose primary function is to serve as “first responders” against virally infected and transformed cells [3]. They have direct antiviral and anticancer activity through multiple cytokine and cytotoxic effector functions, but also serve to establish a pro-inflammatory microenvironment that recruits and primes adaptive immune responses [4, 5]. Unlike adaptive T and B lymphocytes, NK cells are characterized by their ability to recognize such targets without prior sensitization. Instead, NK cells base their response to targets on a balance of activating and inhibitory receptors that recognize danger and self, respectively. Activating receptors typically recognize proteins that are upregulated by cell stress or are of non-self-origin, and inhibitory receptors primarily bind human leukocyte antigens (HLA) as a form of self-recognition. NK cell effector function, including target cytotoxicity, is triggered when the balance of activating and inhibiting signals is tipped toward activation.
Activating and Inhibitory Receptors
NK cells express several families of activating receptors, including CD16 (FcRγIIIa), natural cytotoxicity receptors (NCRs), NK Group 2 (NKG2) family lectin-like receptors, DNAM-1, and 2B4. In general, these activating receptors serve to recognize signs of stress or danger on target cells during immune surveillance. CD16 is the low-affinity Fc receptor which binds the Fc portion of human IgG1 and IgG3, mediating antibody-dependent cell cytotoxicity (ADCC) of antibody-labeled cells [6]. The NCRs (NKp30, NKp44, and NKp46) are activating receptors that bind virus- and stress-related proteins (such as B7-H6) [7]. The receptors of the NKG2 family are expressed as heterodimers with CD94, except for NKG2D which is expressed as homodimer [8]. NKG2D, the major activating receptor in this family, recognizes MHC class I-related chain A or B (MICA/B) and members of the UL-16 binding protein (ULBP) family, which are increased in response to cellular stress. 2B4 (a SLAM family member) recognizes other ligands of the SLAM family such as CD48, and DNAM-1 recognizes the viral receptors PVR and Nectin which are highly expressed on pediatric sarcomas [9].
The primary inhibitory receptors in NK cells are the long-tailed KIRs (which possess an immunoreceptor tyrosine-based inhibition motif (ITIM) [8]) and NKG2A, both of which bind to HLA class I molecules, preventing NK-mediated lysis of cells with normal HLA expression.
Inhibitory KIRs are specific for HLA isotypes on the basis of conserved amino acid residues at position 80. Approximately half of HLA-C alleles have the amino acid asparagine (N) at residue 80—referred to as Group C1—which confers binding to KIR2DL2 and KIR2DL3. The other half of the C alleles code for lysine (K) at residue 80 (Group C2), which confers binding to KIR2DL1. Similarly, about 40% of HLA-B alleles carry the supertypic serologic epitope HLA-Bw4 (defined primarily by threonine (T) at residue 80), which confers binding to KIR3DL1. The presence of the HLA ligand regulates the activity of these KIRs during NK cell development through a process called licensing. Thus, given both parental alleles, it is possible for the HLA type of an individual to restrict NK cell licensing to as few as one (e.g., C2/C2 homozygous and Bw4−) or as many as three (C1/C2 heterozygous and Bw4+) inhibitory KIRs.
The NK cell repertoire varies greatly between individuals. The KIR family also contains members with short cytoplasmic domains, which generally deliver an activating signal and are present or absent in many different haplotype combinations such that most individuals lack one or more KIR genes. In addition to their haplotype variability, KIR genes are highly polymorphic and are variably expressed between NK cells, and functional reactivity is educated by interaction with the host HLA haplotypes. The allelic variations in KIR have been grouped into A and B haplotypes [10], with B haplotypes having greater numbers of activating KIR genes. Individuals with the “B” haplotype are predicted to have superior NK cell-mediated antitumor effects [11].
This HLA-biased education without HLA-restricted antigen recognition (as for T cells) gave rise to the “missing-self hypothesis,” which postulates that NK cells recognize and destroy autologous cells with lost or altered self-HLA class I molecules [12]. However, classical HLA class I is not always required to protect from NK cell-mediated cytotoxicity, nor is it always sufficient to prevent NK cell cytotoxicity [13].
Mechanisms of NK Cell-Mediated Killing
Upon receiving a predominance of activating signals, NK cells release granules containing perforin and granzymes directed toward the target cell. The perforins form a pore in the cell membrane, allowing entry of the granzymes to the cytoplasm to induce apoptosis by direct activation of caspase-3 [14]. NK cell activation also results in increased expression of death receptor ligands on the NK cell, such as Fas ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) [15], which induce apoptosis via associated death receptors on target cells [16,17,18]. In addition to these pathways, NK cells also produce several cytokines such as IFN-γ, which are important in mediating the adaptive immune response against cancer [19].
Evidence for NK Cell Activity in Osteosarcoma
NK Cell Function in Patients with Osteosarcoma
The critical role of anticancer immune surveillance by NK cells is well established. NK cells also appear to play an important role in osteosarcoma (OS) prevention and treatment response. Whereas NK cells in patients with several types of cancer have been shown to have poor function, NK cells isolated from patients with OS were shown to be functionally and phenotypically unimpaired, have intact IFN signaling, and demonstrated cytolytic activity against autologous and allogeneic OS cells and other target cells [20, 21]. However, children and adolescents with osteosarcoma demonstrate a statistically significant reduction in peripheral blood NK cells at the time of diagnosis compared to healthy controls [22]. NK cells also confer a survival benefit during treatment of osteosarcoma, as the rapidity of absolute lymphocyte recovery while receiving standard frontline osteosarcoma chemotherapy regimens (MAP or MAPIE) correlates significantly with an improved event-free survival [23]. Further, IL-2 support during neoadjuvant and adjuvant chemotherapy for osteosarcoma demonstrated a significant correlation between the magnitude of NK cell expansion and enhanced survival [24].
In addition, low expression of PD-L1 (an important suppressor of immune effector function) in osteosarcoma correlates with significantly increased infiltration of NK cells into the tumor microenvironment and is associated with improved event-free survival [25]. Lastly, genomic data obtained from analysis of mRNA and miRNA from patients diagnosed with relapsed osteosarcoma show that the density of the patient’s activated NK cells calculated by CIBERSORT algorithm correlates positively with a good prognosis [26]. These findings all point to the critical role NK cells have in disease-free survival of patient with osteosarcoma.
Expression by Osteosarcoma of Ligands Recognized by NK Cells
The susceptibility of tumor cells to NK cell lysis is regulated by the proportion of inhibiting and activating signals perceived upon interaction of NK cells with the target cell. It correlates negatively with expression of HLA class I antigens and positively with intercellular adhesion molecules and activating ligands on the surface of tumor cells.
Downregulation of HLA class I antigens on the cell surface can be induced by stress conditions and is correlated with increased susceptibility to NK cell killing through decreased signaling by inhibitory KIRs, a phenomenon described as “missing-self.” In vitro experiments with OS cell lines of varying levels of HLA class I antigen expression show that OS cells with surface expression of HLA are less susceptible to killing by NK cells compared to cells lacking cell-surface HLA; moreover, downregulation of cell-surface HLA enhances the sensitivity of NK-resistant OS cells to NK killing. Similarly, OS target cell killing correlates with their degree of KIR-HLA incompatibility with the NK cells [27]. In vivo, OS primary and metastatic tumors have been shown to lose or downregulate HLA class I expression, thus becoming more susceptible to NK cell killing [28].
Expression of cell adhesion molecules renders tumor cells more susceptibility to NK-mediated lysis; these molecules fortify cell-to-cell interactions and provide co-stimulatory signals that enhance the cytotoxic activity of NK cells [29, 30]. Expression of the adhesion molecules CD54 and CD58 increases the bond between target and effector cells and correlates positively with the susceptibility of OS cells to NK lysis [31,32,33]. In vivo, lack of CD54 expression allows the circulation of tumor cells, avoids establishing stable cytolytic conjugates, and provides means of evading NK spontaneous lysis [34]. In contrast, NK cells can enhance the inflammatory microenvironment in tumors through release of IFNγ, which upregulates these adhesion molecules and increases recognition by NK cells [9].
Several activating receptor-ligand interactions have been implicated in the interaction of NK cells with OS cells. Ligands for NKG2D and DNAM-1 activating receptors (MICA/B, ULBP, PVR, and nectin-2) are widely expressed on OS cell lines and OS tumor samples [20, 35], rendering them more sensitive to NK recognition and killing. Cytolysis of OS cells is dependent on NKG2D and DNAM-1 pathways, and blockade of both pathways is required for optimal inhibition of activated NK cells; activation through NKG2D and DNAM-1 pathways also overcomes inhibition of NK cells mediated by KIR-HLA interaction [20]. In vivo, the level of MICA expression on OS cells has been correlated with staging; expression of MICA is higher in patients with early stage disease compared to late stage, suggesting a role for MICA-NKG2D-mediated NK control of OS [35], and downregulation of MICA appears to be a common immune escape mechanism [36]. Unlike other tumor types, MICA expression on OS tumor cells is unaltered by exposure to chemotherapy [20]. NK cell recognition of OS tumor cell has also been described via the NCR receptors, although the ligands on OS cells for these receptors are unknown.
Mechanisms of Killing
True to their name, NK cells exhibit a wide range of robust direct and indirect antitumor activities. NK cells can kill tumor cells via the secretion of cytotoxic granules that contain perforin and granzyme and secretion of cytokines and other effector molecules that impact tumor survival and recruitment of adaptive immunity, ligation, and activation of death receptors (e.g., TRAIL, Fas) on tumor cells and ADCC through CD16 when combined with tumor-targeting antibodies. Moreover, their release of pro-inflammatory cytokines has a profound impact on recruitment and maturation of adaptive immune responses [19].
Several early studies demonstrated the in vitro cytotoxicity of NK cells against osteosarcoma cell lines [30, 32, 37]. The mechanism by which NK cells induce apoptosis in osteosarcoma cells may depend on both the activation status of the NK cells and the death receptor and apoptotic pathways that are intact in the osteosarcoma. The predominant pathway for activated NK-mediated lysis of some osteosarcoma cell lines is via granule-mediated release of granzyme B, such that blocking this pathway leads to complete abrogation of cytolysis [20]. However, NK cells may also induce apoptosis of osteosarcoma via granule-independent mechanisms, depending more on Fas-Fas ligand or TNF-TRAIL interactions (see Book 2, Chap. 12 “Fas Signaling as a Potential Target for the Treatment of Osteosarcoma Metastasis in the Lungs”). The importance of these pathways may be underappreciated, as they are kinetically slower and therefore less apparent in classic 4-hour in vitro cytotoxicity assays that measure loss of membrane integrity [38, 39].
As previously discussed, the cytolytic activity of NK cells is mediated by the balance of activating and inhibitory receptors. NK cells isolated, propagated, or activated by different approaches may differ as to which activating receptor(s) play the dominant role in recognition of osteosarcoma. IL-15-stimulated NK cells target osteosarcoma predominantly through DNAM1, though NKG2D remains important [20]. IL-2-stimulated NK cells target osteosarcoma predominantly through NKG2D-NKG2D ligand interactions [40]. In vitro study of IL-15-stimulated NK cells co-cultured with an osteosarcoma cell line demonstrated decreased expression of activating receptors (NKG2D, DNAM-1, and NKp30), inhibiting direct killing [41]. In contrast, IL-21-expanded NK cells increase both NKG2D and DNAM-1 [42, 43], and TGFβ-imprinted NK cells express much higher levels of TRAIL and FasL [44]. Thus, the type of NK cell applied to immunotherapy of osteosarcoma may be an important consideration in optimizing outcomes.
Mechanisms of Immune Escape
Tumor cells may acquire diverse mechanisms to evade NK cell recognition [45]. No or low expression of adhesion molecules or ligands for activating receptors and/or increased expression of ligands for inhibitory receptors are described mechanisms adopted by tumor cells to evade NK cell surveillance. In addition, shedding of NKG2D ligands (soluble sMICA) from the membrane of tumor cells can impair NKG2D-mediated cytotoxicity by blocking the NKG2D receptors on NK cells. Furthermore, secretion of immunosuppressive cytokines and transforming growth factor-β has been associated with defective NK cell function, restricting tumor cell recognition and killing.
Both classical and nonclassical HLA class I molecules, which are ligands for inhibitory KIR and CD94/NKG2A receptors, are expressed on some OS naïve tumors and may be increased in OS cells when exposed to chemotherapy [20].
OS cell lines and tumor sample show higher expression of surface MICA compared to normal bone tissue and benign bone tumors making them theoretically more susceptible to NK cells killing. However, soluble MICA was detected in the serum of some patients with OS resulting in diminished NKG2D expression on NK cells and decreased tumor cell killing. Clinical correlation showed that in patients with OS, elevated MICA expression combined with increased soluble MICA was associated with decreased NKG2D expression on PBMC, and this combination correlated significantly with advanced and metastatic disease [35, 46]. With progression of OS, expression of MICA decreases, soluble MICA increases, and expression of NKG2D on NK cells decreases [35].
Indirect Activation of NK Cell Function
As described above, patients with osteosarcoma have important defects in NK cell function—including lower circulating peripheral NK cell numbers and decreased expression of activating receptors—and NK cell numbers are further impacted during treatment, as they are extremely sensitive to chemotherapy and radiation. As these functional and numeric NK cell deficits have been linked to poorer outcomes for patients, approaches to improve the antitumor activity of NK cells can improve clinical outcomes. These include monoclonal antibodies, cytokines, immunomodulators, and attention to chemotherapeutic regimens that enhance NK cell-mediated tumor lysis.
Monoclonal Antibodies
ADCC by NK cells requires interaction between the Fc receptor (CD16) on NK cells and the Fc region of an antibody binding to an antigen on the tumor cell surface, resulting in NK cell activation and degranulation toward the target cell.
EGFR is expressed on 90% of OS tumor samples [47]. Cetuximab, a MoAb-targeting EGFR, increases NK-dependent lysis of EGFR-expressing sarcomas. Importantly, the sensitivity to cetuximab-enhanced lysis by resting NK cells is comparable among most EGFR-expressing cell lines, including chemotherapy-resistant OS cells [48]. Although prolonged OS/NK cell co-cultures and excess of tumor cells in culture result in diminished NK cell cytotoxicity secondary to downregulation of activating receptors on NK cell surface, ADCC killing of OS by NK cells is unaltered by this suppressive mechanism [41].
NK cytotoxicity to OS cells is enhanced by Fc-FcγR interaction; epidermal growth factor receptor (EGFR)-expressing OS cells are more susceptible to NK killing in the presence anti-EGFR monoclonal antibody (MoAb) compared to EGFR-negative OS cells [48].
GD2 and GD3 are tumor-associated glycolipid antigens that are highly prevalent in osteosarcoma and are potential targets for antibody-based therapies. GD2 and GD3 are highly expressed in sarcomas of children, adolescents, and young adults [49]. Tumors that express ganglioside GD2 tend to have persistence of GD2 expression at the time of recurrence [50], including patients with osteosarcoma [49]. It has been shown that ganglioside GD2-specific antibodies can inhibit tumor cell viability without involving the immune system. Combination of GD2 with cisplatin induces apoptosis in osteosarcoma cell lines [51]. Chimeric antigen receptors (CARs) against GD2 have been used to enhance the activity of NK cells against Ewing sarcoma. The expression of CARs directed against the GD2 in activated NK cells increased the responses to GD2+ allogenic Ewing sarcoma cells and also overcame resistance of individual cell lines to NK cell lysis [52].
Cytokines
Cytokines may act directly on tumor cells as antiproliferative agents and indirectly via activation of cellular immune agents such as NK cells leading to increased lysis of tumor cells.
Interleukin (IL)-15 potentiates the cytolytic activity of NK cells by increasing NKG2D expression on cell surface and enhancing GrB release upon activation. IL-15 activation reverses impaired expression of NKG2D and DNAM-1 and impaired NK cell cytotoxicity induced by prolonged co-cultures of NK cells with OS cells, and NK cells activated with IL-15 prior to co-culture with OS cells do not downregulate activating receptors and preserve functional activity despite prolonged exposure to target cells [41]. IL-2 and IL-12 increase cytotoxicity of NK cells to NK-sensitive and NK-resistant OS cell lines by increasing the density of CD18 and CD2 receptors on the NK cell surface, enhancing the conjugate-forming capacity of NK cells to OS targets [53]. Importantly, targeted application of IL-2 to the lung by aerosolized delivery markedly improves the migration of adoptively transferred NK cells into lung metastasis, resulting in enhanced control of metastatic disease [54].
IL-12 increases expression of ICAM-1 (a ligand for CD18) on OS cell lines co-cultured with PBMCs in cell-to-cell contact [55]. In a mouse model of metastatic osteosarcoma, mice bearing pulmonary metastasis treated with IL-12 showed decreased number and size of pulmonary metastasis mediated by NK cells [56]. IFN potentiates NK-mediated lysis of OS cell lines; IFN-conjugated antibodies specifically localize tumor cells in a mouse xenograft tumor model and further increase NK cell activation and tumor cell lysis [57, 58]. IL-17 augments expression of fibronectin on OS cell lines that express the IL-17 receptor, mediating increased adhesion of NK cells to OS cells and thus enhancing NK cytotoxicity. IL-17 has no direct effect on NK cells function [37].
The common γ chain cytokines IL-15 and IL-2 have both been used successfully to activate and expand NK cells ex vivo for adoptive transfer [59]. These cytokines both activate trimeric receptors on NK cells that share two subunits in common—IL-2Rβ and IL-2Rγc—with the third subunit conferring cytokine specificity [60]. Despite this similarity, they have disparate effects on NK cell expansion that can be leveraged against osteosarcoma. K562 have been induced to express membrane-bound IL-15 to serve as a platform for NK cell expansion [61]. Use of this IL-15 platform leads to a multifold expansion of activated NK cells with increased NKG2D expression on cell surface, enhanced granzyme B release, and thus increased cytolytic activity against tumor targets. However, NK expansion to a clinically usable product is limited by senescence caused by telomere shortening. More recently, recombinant IL-15 has been used for NK cell activation and expansion through mTOR-dependent activation of STAT-5 signaling leading to improved NK cell metabolic function and antitumor cytotoxicity [62]. Allogeneic and autologous NK cells expanded with this recombinant IL-15 have proven cytotoxicity against even chemotherapy-resistant osteosarcoma cell lines in vitro [20]. Expansion and activation of NK cells for adoptive transfer as a cancer immunotherapy has been accomplished with IL-2 stimulation as well with demonstrably increased expression of NKG2D, CD16, CD94, and NKp46 and cytolytic activity [63]. NK cells cultured as briefly as 18 hours in IL-2 have shown markedly improved cytotoxicity against both NK cell-sensitive and NK cell-resistant osteosarcoma cells in vitro with similar results seen with the use of IL-12 as the activating cytokine [53]. These cytokines were seen to increase the density of CD18 and CD2 receptors on the NK cell surface, enhancing the conjugate-forming capacity of NK cells to osteosarcoma targets. Aerosolized IL-2 has been used successfully to expand adoptively transferred NK cells in vivo in a canine model of metastatic osteosarcoma [64, 65]. This aerosolized delivery of cytokine led to better specificity in terms of expansion and activation only of the pulmonary NK cells without systemic IL-2 toxicities and was associated with improved therapeutic efficacy against pulmonary metastasis.
IL-21 is another common γ chain cytokine known to play a pivotal role in NK cell activation and maturation. Ex vivo expansion utilizing feeder cells expressing membrane-bound IL-21 can yield 30,000-fold expansion of NK cells in 21 days, with retained KIR repertoires, increased expression of CD16 and NKG2D, and superior cytokine secretion [42]. In a canine patient-derived xenograft model of osteosarcoma, adoptive transfer of membrane-bound IL-21 expanded canine NK cells led to tumor regression and suppression of metastasis. Notably, NK cell homing and antitumoral cytolytic activity against osteosarcoma were enhanced by radiotherapy [66] (see Book 2, Chap. 14 “Comparative Immunology and Immunotherapy of Canine Osteosarcoma”).
IFNγ-conjugated antibodies specifically localized to tumor cells in a mouse xenograft tumor model and increased NK cell activation and tumor cell lysis [57, 58].
Although it is typically associated with NK cell suppression within the osteosarcoma tumor microenvironment, the inclusion of transforming growth factor-beta (TGFβ) during NK cell expansion and activation results in NK cells with enhanced functionality [44]. This process of TGFβ imprinting results in activated NK cells with increased cytokine secretion in response to osteosarcoma cells, improved cytolytic activity against an osteosarcoma, and resistance to the suppressive effects of TGFβ.
Cytokines can also act directly on osteosarcoma cells to make them more susceptible to lysis by NK cells. IL-12 increases expression of ICAM-1 (a ligand for CD18) on osteosarcoma cell lines, making them more susceptible to NK cell-mediated lysis [55] and improving NK cell-mediated metastatic control with decreased number and size of pulmonary metastases mediated by NK cells [67]. IL-12 may also increase expression of Fas on osteosarcoma [68], making it more susceptible to Fas-mediated lysis. IL-17 can increase NK cell-mediated lysis of osteosarcoma cells through augmented expression of fibronectin on osteosarcoma cells and subsequent increased NK cell adhesion [37].
Chemotherapy
As mentioned above, chemotherapy appears to increase expression of inhibitory ligands, but does not increase MICA [20]. Chemotherapy does increase sensitivity to ADCC by NK cells [41], and both gemcitabine [69] and cisplatin [70] may increase sensitivity of OS to direct NK cell lysis by upregulation of Fas or downregulation of anti-apoptotic proteins.
Several chemotherapeutic agents commonly used for osteosarcoma have direct effects on the osteosarcoma cells that enhance NK cell-mediated lysis. Doxorubicin, cisplatin, and etoposide have all been shown to downmodulate expression of the inhibitor of apoptosis X-IAP, sensitizing osteosarcoma cell lines to NK cell-mediated lysis via TRAIL [71]. This sensitization to TRAIL was specific to osteosarcoma cells and was not seen in normal human osteoblasts. Cisplatin has also been shown to sensitize osteosarcoma cells to Fas/Fas ligand-mediated apoptosis via downregulation of FLICE inhibitory protein long form (FLIP-L) [70].
The taxane docetaxel and the nucleoside analog gemcitabine are commonly used in relapsed or refractory pediatric sarcoma patients, including those with osteosarcoma [72, 73]. Gemcitabine has been shown to upregulate NKG2D ligand in several other solid tumor cancer types [74,75,76]. While docetaxel has been shown to upregulate NKG2D expression on NK cells in vivo [77], it also inhibits NK cell cytotoxicity [78]. Irinotecan and temozolomide have similarly been used in relapsed and refractory osteosarcoma patients. Temozolomide has been shown to cause minimal reduction in NK cell cytotoxicity, but may suppress proliferation of NK cells in response to activation with IL-2.
Immunomodulators
In addition to monoclonal antibodies (mAb) and cytokines, a variety of immunomodulatory drugs have been successfully combined with NK cells to potentiate their antitumor activity and treat human malignancies [79,80,81].
In the setting of OS, the activity of NK cells may be weakened or enhanced by immune-modulating agents. Sodium valproate (an HDAC inhibitor) and hydralazine (a DNA methylation inhibitor) increase the expression of MICA and MICB on OS cells, but not sMICA in serum, and therefore increase the susceptibility of tumor cells to NK cell lysis [82, 83]. Moreover, hydralazine increases cell-surface expression of Fas and augments Fas-induced OS cell death, whereas valproic acid sensitizes OS cells to Fas-mediated cell death and decreases production of soluble Fas [82, 83], thus further potentiating OS sensitivity to NK cell killing. However, both HDAC inhibition [84] and DNA hypomethylation [85] can have an adverse direct effect on NK cell function, necessitating approaches that sequence drug therapy and cell therapy. A narrow-spectrum HDAC inhibitor, SNDX-275, has been shown to increase osteosarcoma killing through upregulation of Fas [86], c-FLIP [87], and MICA [85], and also augments NK cell function through upregulation of NKG2D [36] (see Book 2, Chap. 4 “Targeting the Cancer Epigenome with Histone Deacetylase Inhibitors in Osteosarcoma”).
PD-1 and its ligands play a role in evasion of malignant tumor cells from the immune system. Recently, immunotherapy with anti-PD-1 inhibitors has been approved for treatment of non-small cell lung carcinoma, urothelial cell carcinoma, and Hodgkin lymphoma. In vitro studies have shown increased cytoplasmic expression of PD-1 in bone sarcomas [98]. Pembrolizumab, an anti-PD-1 antibody, has been studied as a treatment option for patients with advanced soft tissue sarcoma or bone sarcoma. Even though the primary endpoint of overall response was not met for either cohort, promising activity was seen in certain histologies and further study is underway [99].
Lenalidomide is an immunomodulatory thalidomide derivative with activity against a wide variety of cancers. Lenalidomide may enhance NK cell number and maturation through increased IL-15 levels [88]. Lenalidomide augments the activity of NK cells by enhancing ADCC of mAb against solid tumors [89], including trastuzumab and cetuximab activity against bone sarcomas [90]. Mifamurtide (MTP-PE), discussed extensively in Book 1, Chap. 11, may exert some of its anticancer effects by enhancing NK cell activity [91].
Heat treatment of OS cell lines increases their susceptibility to NK cell-mediated lysis through upregulation of heat shock protein 72 (HSP72) expression [92]. Hypoxia decreases the expression of MICA on OS cell lines in a hypoxia-inducible factor 1α (HIF-1α)-dependent manner and consequently decreases the susceptibility of tumor cells to NK cell lysis [93]. However, hypoxia does not interfere with MoAb-mediated target cell killing by ADCC [94].
NK Cell Adoptive Immunotherapy
Clinical NK Cell Sources and Trials
NK cells may be obtained in numbers sufficient for clinical use in adoptive immunotherapy by apheresis and CD3 depletion or by ex vivo expansion. NK cells have been successfully expanded from peripheral blood, cord blood, and pluripotent or embryonic stem cells. Expansion methods have included various combinations of cytokines, cytokine fusion proteins, cytokines and OKT3, cytokines and stromal support, antibody-coated beads, and feeder cells obtained from peripheral blood or derived from EBV-lymphoblastoid cell lines or K562 (reviewed in [42]).
NK cells have been delivered by adoptive transfer to very few patients with osteosarcoma. Expanded NK cells were given as adjuvant immunotherapy after matched allogeneic transplant (C. Mackall, personal communication, ClinicalTrials.gov Identifier NCT01287104). As mentioned above, KIR-ligand incompatibility is associated with increased NK cell activity against osteosarcoma cell lines [27]. Thus, similar to the observed benefit in AML, it is likely that approaches using mismatched allogeneic donors for NK cell therapy of osteosarcoma will have a greater antitumor effect than matched or autologous NK cells. NK92 is a cell line derived from a patient with NK cell leukemia and has NK cell-like activity against tumor cell lines. Clinical grade irradiated NK92 cells have been infused in a patient with advanced osteosarcoma, though no response to treatment was observed [95]. Newer studies evaluating the use of expanded natural killer cells following cytotoxic chemotherapy are being utilized in neuroblastoma and CNS tumors, as well as other sarcomas such as Ewing sarcoma and rhabdomyosarcoma.
Future Approaches
The recent availability of clinically viable approaches for obtaining large number of NK cells now enables the clinical testing of combination therapies to enhance NK cell function and osteosarcoma sensitivity. The antigen-binding domains of all of the mAb mentioned above have been identified and genetically manipulated to generate chimeric antigen receptors (CARs) that mediate enhanced killing by T cells (see Book 1, Chap. 10). As an alternative to T cells, genetic modification to express CAR may also be applied to NK cells to further enhance their activity against osteosarcoma [96]. These CARs also have potential application for clinical development in NK cells, and CAR with NKG2D-like specificity can further improve the NK cell immunotherapy of osteosarcoma in murine models [97]. The ability to deliver large cell doses, combination with sensitizing chemotherapy, radiation, or immunomodulatory drugs, and genetic modifications will be the subjects of cutting-edge trials in the decade to come.
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Tullius, B.P., Setty, B.A., Lee, D.A. (2020). Natural Killer Cell Immunotherapy for Osteosarcoma. In: Kleinerman, E.S., Gorlick, R. (eds) Current Advances in Osteosarcoma . Advances in Experimental Medicine and Biology, vol 1257. Springer, Cham. https://doi.org/10.1007/978-3-030-43032-0_12
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