Ovarian cancer is the most lethal gynecologic malignancy, with an estimated 14,080 deaths expected in the United States for 2017 (95). Despite optimal treatment with surgery and adjuvant chemotherapy, the recurrence rate approaches 70–80% (96). Although the disease tends to remain confined to the abdominal cavity, women with recurrent ovarian cancer progress and ultimately die. Therefore, there is an urgency to develop new and effective therapies.

Important to effective therapeutic development is the understanding of the immunologic interactions within the tumor and its physiologic impacts, which often includes the development of profuse ascites. It is well documented that the ascites fluid from patients with advanced stage ovarian cancer suppress the function of otherwise normal immune effectors, including NK cells (97–101). The ascites contains large numbers of growth factors and cytokines that promote the proliferation of tumor cells (102–104). While fresh NK cells (CD56⁺CD3⁻CD16⁺) isolated from the ascites fluid are found in relatively high concentrations compared to peripheral blood, they are functionally deficient (105, 106). This population of NK cells demonstrates decreased CD16 expression and have reduced proliferative, cytolytic, and cytokine production compared to peripheral blood NK cells (107). Signaling proteins vital to interpreting the activating and inhibitory signals become defective and alter the expression of cytokine transcripts and proteins (100). As detailed above, tumor-associated ligands MICA/B and B7-H6 are often found within the peritoneal fluid of serous ovarian cancer patients and impair NK cell effector function (46, 64). These deficiencies act in combination and likely influence the ability to control the spread and proliferation of tumor cells within the peritoneal cavity of patients with advanced ovarian cancer.

For ovarian cancer, the role of the immune response has been well documented with immunohistochemistry. Multiple studies document a positive correlation between the number of tumor-infiltrating lymphocytes within the tumor and overall survival (OS) (108–110). The absence of CTL infiltration (CTL) also predicts platinum resistance (111). More recently, genomic profiling studies also support using immunophenotype as a method to predict response to therapy and clinical outcomes (112–114). However, most of the published studies document limited infiltration of NK cells within the primary ovarian tumor and cells that suppress immune response and support tumor growth dominate (108, 115–120). The presence of infiltrating NK cells impact on OS is also controversial. Infiltrating NK cells have largely not been associated with better outcomes, and in one case, predicted worse OS (121). However, it was recently shown that CD103⁺ tumor-infiltrating NK cells were almost always found with CD8⁺ T cells and were the second best predictor of positive outcomes in primary ovarian cancer (122). In light of this evidence, further study regarding the role of infiltrating NK cells is warranted. Regardless of the uncertainty of the role of NK cells and OS, freshly isolated NK cells from the tumor are drastically impaired through a variety of mechanisms (123, 124).

Within the tumor tissue, NK cells have complex interactions with other immune cells with suppressive functions, including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (T_reg) (125). The MDSCs typically suppress effector function of T cells, thereby promoting tumor growth. In mice, a subset of MDSCs expresses the NKG2D ligand Rae-1 and is capable of elimination through NK cell-mediated cytolysis (126). If present, NK cells are also potent producers of IFN-γ and prevent the macrophage polarization toward the M2 phenotype that support tumor progression (127).

Another mechanism interfering with NK effector function within the microenvironment is the secretion of immunosuppressive cytokines. The hypoxic environment induces transcription of interleukin (IL)-8, a chemokine important for tumor growth, angiogenesis, and metastasis (128–130). Women with ovarian cancer have significantly elevated concentrations of IL-8 compared to benign controls (131). In addition to promoting angiogenesis and tumor growth, in vitro studies demonstrate an immunosuppressive effect of IL-8 by inhibiting TNF-induced apoptosis (132). In addition, the cytokine transforming growth factor-β (TGF-β) can contribute to the immunosuppressive microenvironment (133). Overproduction of TGF-β by tumor cells suppresses CD16-mediated NK cell IFN-γ production and ADCC (134). These immunosuppressive cytokines attenuate NK cell effector function and limit the antitumor response.

Cervical cancer is a human papillomavirus (HPV)-induced cancer. Mediated by an adaptive immune response against viral proteins, the greatest success involving immunotherapy in gynecologic malignancies is the development of vaccines against HPV and prevention of cancer (135). The two early viral proteins E6 and E7 are defined tumor-associated antigens and are processed and expressed on MHC class I molecules. However, HPV-induced cervical cancers often show altered expression of MHC class I molecules resulting in the inability of CTLs to recognize the peptide epitopes (88, 136–138). The HPV16 E6 and E7 oncoproteins also inhibit NK cell IL-18-induced IFN-γ production likely contributing to viral pathogenesis (139). In addition, HPV infection is non-lytic and produces only a modest inflammatory infiltrate of macrophages and lymphocytes (140). These changes likely affect the efficacy of the innate immune response and provide opportunities to escape immune surveillance. However, the “loss of self” may render these HPV-related cancers susceptible to NK cell attack.

Very little is known in regard to NK cell function and phenotype in women with cervical cancer. Freshly isolated peripheral blood NK cells in women with cervical cancer and benign healthy controls demonstrated no significant functional differences until the patient had distant metastatic disease (stage IVb) (141). Another study demonstrated infiltrating NK cells of patients with cervical cancer were present and of the CD56^brightCD16⁻ phenotype. They also observed the upregulation of the DNAM-1 ligand CD155 and the NKG2D ligand MICA in cervical cancer but not in cervical intraepithelial neoplasia or normal controls (142). A follow-up study demonstrated keratinocyte expression of HPV16 E6 and E7 produced rapid induction of intracellular adhesion molecule-1 protein levels (143). NK cells recognize expression of these ligands and adhesion molecules and may be a promising strategy to target for the treatment of cervical cancer.

Interestingly, certain combinations of KIRs and HLA loci associated with NK cell activation increase the risk of developing cervical cancer (144). Specifically, the presence of the activating KIR3DS1 on NK cells in the absence of ligands for inhibitory KIRs results in an increased risk of cervical neoplasia. In constrast, NK cell effector inhibition mediated by KIR2DL1 and KIR3DL1 in the absence of KIR3DS1 results in protection from cervical neoplasia. KIR receptors and HLA ligands interact through an epistatic relationship in which HLA ligands activate a genetic molecular cascade through the KIR receptor that influences NK functionality. This KIR/HLA interaction suggests that an inappropriate inflammatory response, mediated by NK cell KIR-ligand interactions, may lead to tumor progression. The precise role of NK cells in the context of cervical cancer is far from being defined.

Endometrial carcinoma arises from the lining of the uterine cavity and is the fourth most common malignancy in women (95). Most women are diagnosed with low-grade, early stage disease and are cured following surgery. There is very little information on the microenvironment of uterine NK (uNK) cells and cancer. There is much more known about NK cell biology and pregnancy.

Uterine NK cells are a tissue-specific, specialized population of cells that make up large percentage of both endometrial and decidual lymphocytes (145). uNK cells are almost exclusively CD56^brightCD16⁻, although they contain cytotoxic granules (146). uNK cells are thought to contribute to immunosuppressive mechanisms during pregnancy when immune tolerance is vital. As a result, these uNK cells also display less cytotoxicity against tumor targets compared to peripheral NK cells (147). uNK cells are not only immunosuppressive during pregnancy in order to protect the fetus but also may play a key role in modulating fetal growth, with activated uNK cells at the maternal–fetal interface producing factors that play a role in the regulation of trophoblast invasion and uterine vascular remodeling. These roles are critical to placental formation and healthy gestation (148, 149). A number of interesting questions remained unanswered with respect to NK cells and uterine cancer: (i) does the immunosuppressive nature of the maternal–fetal interface contribute to developing uterine carcinoma? (ii) Do peripheral NK cells migrate into the uterine cavity with tumorigenesis? (iii) Are uterine tumor cell lines susceptible to NK cell killing? More complete knowledge of the biology and function of uNK cells in endometrial cancer is required prior to developing strategies of NK cell immunotherapy for this malignancy.

Early clinical trials in ovarian cancer patients aimed to improve the antitumor activity of immune cells through intraperitoneal injection of biological response modifiers, including Corynebacterium parvum, bacillus Calmette-Guerin, leukocyte interferons (IFN), and irradiated autologous and allogeneic tumor cells (150). Overall, these agents had limited success in treatment response with relative toxic side effects. Another novel strategy using an attenuated strain of influenza virus to infect ovarian cancer tumor cell lines was later developed. The nonviable extracts from the tumor cells, termed viral oncolysate, were isolated and then injected intraperitoneally (IP) into patients with ovarian cancer with both clinical and pathological responses noted (151, 152). Follow-up studies noted the viral oncolysate enhanced the NK cell response (153). Although better tolerated, the treatment had limited clinical responses.

Advancement in recombinant DNA technologies led to the purified production of cytokines and their use to treat a variety of malignancies. Today, cytokines are easy to manufacture and administer. While the central goal of cytokine therapy is to potentiate the autologous antitumor response in vivo, they lack specific immunomodulatory effects (154). Table 1 lists selected clinical trials in ovarian cancer evaluating cytokine therapy and NK cell response if reported. The first generation of cytokines were recombinant (r) IFN-α, rIFN-γ, and rIL-2. The published results of IP therapy with recombinant IFN with or without chemotherapy have been comprehensively reviewed by Freedman and colleagues (155). They also reported on eight clinical trials evaluating IP therapy with IL-2 alone or in combination with cellular therapy (discussed below). The results demonstrated IP immunotherapy with cytokines was tolerated but had varying levels of success. Two important features were identified: (i) response was dependent on remaining tumor burden prior to initiation of therapy and (ii) efficacy of first-line therapy is critical in these patients. Most of these early trials did not assess NK cell response to therapy.

Recently, a randomized phase III trial of 847 women with stage III or IV ovarian cancer evaluating front-line combination carboplatin/paclitaxel plus subcutaneous Escherichia coli-derived recombinant IFNγ-1b was stopped early following a second interim analysis (156). At the time of analysis, patients receiving IFNγ-1b plus chemotherapy compared to chemotherapy alone demonstrated significantly shorter OS (60 vs 70%). It should be noted IFNγ-1b has biological activity identical to natural human IFN-γ (157). The authors speculate IFNγ-1b may have resulted in activation of T_regs and immunosuppression. They also suggest treatment with IFNγ-1b is more toxic and leads to decreased treatment adherence or dose reductions of chemotherapy. The ability to complete all six cycles of chemotherapy was compromised in patients receiving IFNγ-1b (77 vs 83%). Regardless, it was concluded that IFNγ-1b does not have a role in first-line treatment of advanced ovarian cancer.

Finally, a phase II trial of advanced ovarian cancer patients treated with second-line therapy followed by maintenance low-dose subcutaneous IL-2 with oral 13-cis-retinoic acid reported an overall response rate of 57% (158, 159). Treatment was associated with an improvement of peripheral NK cell counts and a decrease in VEGF compared to baseline. In this cohort of 65 patients, the progression-free survival (PFS) and OS was 29 and 38%, respectively.

Similar to IL-2, the cytokine IL-15 can potently increase NK cell numbers. While IL-2 and IL-15 share common signaling mechanisms, they differentially control the development, activation, and proliferation of NK cells (160). IL-2 activates a broad range of T cells, including T_regs. In contrast, IL-15 preferentially stimulates CD8⁺ T cells and non-terminally differentiated NK cells and has been shown to enhance NK cell function in the ovarian cancer setting (101, 161). Several clinical trials evaluating IL-15 are underway (162).

In summary, biologic response modifiers and cytokine therapy have demonstrated conflicting results in the limited clinical trials. In addition, the small number and heterogeneity of study participants limit interpretations. Additional investigations examining the role of cytokines in ovarian cancer and reporting standard immune and clinical responses is warranted.

Initial efforts in adoptive transfer of immune cells aimed to improve the autologous antitumor responses through cytokine stimulation (155, 163–165). Immune cells removed from the peripheral blood of patients were activated with various cytokines and then infused back into the same patient. Table 2 lists selected clinical trials in ovarian cancer evaluating adoptive transfer of NK cell-related therapy and response if reported.

The early phase I clinical trials evaluating the adoptive transfer of autologous lymphokine-activated killer (LAK) cells with high-dose IL-2 therapy demonstrated limited clinical responses with high rates of peritoneal fibrosis (165–167). Similar to LAK immunotherapy, cytokine-induced killer (CIK) cells arise from peripheral blood mononuclear cell cultures with stimulation of anti-CD3 mAb, IFN-γ, and IL-2 (168). CIK cells are characterized by a mixed T-NK phenotype (CD3⁺CD56⁺) and demonstrate enhanced cytotoxic activity compared to LAK cells against ovarian and cervical cancer (169, 170). A recent phase III clinical trial investigated adoptive transfer of autologous CIK cells following primary debulking surgery and adjuvant carboplatin/paclitaxel chemotherapy (171). Advanced epithelial ovarian cancer patients (n = 92) were paired to receive maintenance monthly CIK transfusions (n = 46) vs standard of care observation (n = 46). Median PFS was 37.7 months in the treatment group and 22.2 months in the control group (p = 0.004). Median OS in the treatment group was 61.5 months, compared to 55.9 months in the control group (p = 0.289). The therapy was well tolerated with no grade III or IV adverse reactions. Interestingly, the proportion of T_regs in peripheral blood decreased following two courses of immunotherapy (p = 0.006). While only a small, non-randomized phase III study, these results are promising and follow-up studies are warranted.

Recently, insights into the molecular mechanisms regulating NK cell function shifted the focus toward allogeneic NK cell immunotherapy. The mismatch between donor KIR repertoire and recipient MHC class I molecules can improve the antitumor activity of NK cells (172). In a phase II clinical trial, we studied haplo-identical related IV infused NK cells in patients with recurrent ovarian (n = 14) and breast cancer (n = 6) (173). Following a lymphodepleting chemotherapy regimen ± radiation, women received adoptive transfer of a CD3/CD19-depleted NK cell product and were treated with subcutaneous IL-2 injections. No successful NK cell persistence or expansion was noted, likely as a result from recipient T_reg expansion and reconstitution following therapy. Only two other small reports using allogeneic NK cell therapy in ovarian cancer have been published (174, 175). Together, these studies suggest allogeneic NK cell therapy is feasible. However, further investigation into strategies to augment in vivo NK cell persistence and expansion are needed.

Future efforts generating novel NK cell products for adoptive transfer are likely to be investigated in the ovarian cancer setting. Ex vivo inhibition of GSK3 kinase in peripheral blood enhances CD57 expression and late-stage maturation of NK cells (176). These NK cells demonstrated significantly higher production of cytokines (TNF-α and IFN-γ) and ADCC when exposed to cancer cells. Recruitment for an ovarian cancer clinical trial using the product generated from this method has opened at the University of Minnesota. Another recent study evaluated the potency of NK cells derived from human CD34⁺ hematopoietic stem and progenitor cells (HSPC) against a mouse xenograft model for ovarian cancer (177). Mice that received IP HSPC-NK cell infusions had significantly reduced tumor progression compared to controls. Finally, efforts to generate ovarian cancer specific NK chimeric antigen receptors are underway (178). These engineered proteins consist of a fused single-chain variable fragment (scFv) to an intracellular signaling domain to enhance NK effector function. A combination of these different techniques of generating NK cell products hold great promise and may make IP adoptive transfer effective against ovarian cancer following primary cytoreductive surgery and adjuvant chemotherapy.

Other immunotherapeutic strategies are currently being characterized for antitumor activity (162). The development of drugs known to influence NK cell presence and function include mAb therapy, immunomodulatory drugs, vaccines (peptide, viral-based, tumor antigens, dendritic cells), and the adoptive transfer of T cells, dendritic cells, and macrophages. Even commonly used cytotoxic agents increase expression of NK cell-activating ligands and enhance NK cell recognition and killing more than others (179). A thorough discussion of each method is outside the scope of this review, but we will comment on several key issues.

Antibody-based immunotherapy has transformed the treatment of many malignancies, but is not yet standard of care for ovarian cancer. The mAbs function through two separate mechanisms. First, treatment is aimed at antigens present on tumor cells to facilitate an antitumor response through opsonization and activation of ADCC. Several tumor-associated antigens targeted with mAb for ovarian cancer have been identified, including NY-ESO-1, CA 125 (MUC16), MUC1, and epithelial cell adhesion molecule (EpCAM) (154). mAbs can also function in a non-immune-mediated manner to block vital growth and survival pathways, such as Her2/Neu, membrane folate receptor, and VEGF. Clinical trials evaluating the efficacy of mAbs should include investigations into both mechanisms.

A newer approach involves engineered bispecific antibodies and bispecific/trispecific killer engagers (BiKEs or TriKEs), which are molecules that cross-link antigens on tumor cells with CD16 on NK cells, activating and enhancing ADCC (180). One example utilized anti-CD16 scFv spliced to anti-EpCAM scFv (181). This BiKE promoted immune synapse and ADCC between NK cells and EpCAM-expressing tumor cells. More recently, a fully humanized TriKE utilized a modified IL-15 to cross-link the anti-CD16 scFv and EpCAM scFv (182). The 1615EpCAM TriKE was specific and active against EpCAM bearing ovarian cancer cells and mediated NK proliferation, sustained ADCC activity, improved lytic degranulation, and cytokine production. A TetraKE construct incorporating the cancer stem cell marker anti-CD133 scFv was recently engineered to simultaneously target EpCAM and CD133 bearing cells (183). These engineered small molecules combine the specificity of mAbs with the NK cell expansion and survival benefits of cytokine therapies via IL-15 into a single product. This novel strategy to target NK cells for antigen-specific immunotherapy has recently been reviewed and will hopefully prove effective in supplementing traditional therapies against gynecologic malignancies (184, 185).

Immune checkpoints are inhibitory pathways that serve to prevent self-tissue damage. During tumorigenesis, cancer cells often express ligands to bind and induce immune suppression. A number of antibodies have been developed to block checkpoint pathways expressed on certain T cells, B cells, monocytes, and NK cells, including CTL-associated protein 4, programmed death protein 1 (PD-1), TIM-3, NKG2A/CD94 complex, and CD96/CD226/TIGIT receptors (186, 187). A recent publication identified a population of NK cells within the ascites of women with ovarian cancer where PD-1 is highly expressed suggesting therapies targeting PD-1/PD-L may be effective (188). In fact, in vitro studies have shown that PD-1 and CD96/TIGIT blockade augments NK cell-mediated tumor lysis (189, 190). Future research is needed to clarify the effects checkpoint inhibitors have on the NK cell response and the potential to enhance adoptive NK cell immunotherapy.