Introduction

Ovarian cancer is the fifth most common cause of cancer-related deaths in women, surpassing that of any other gynecological cancer. In the USA, an estimated 22,240 new cases of ovarian cancer will be diagnosed in 2018, and an estimated 14,070 women will succumb to the disease in this year alone [1]. In addition, there are more than 200,000 new cases of ovarian cancer and more than 150,000 deaths per year worldwide [2]. Initial treatment options for epithelial ovarian cancer (EOC) include cytoreductive surgery followed by platinum and taxane chemotherapy [3, 4] or neoadjuvant chemotherapy followed by interval debulking surgery [5, 6]. Due to the late stage at time of diagnosis (80%) [7], a high rate of recurrence (70–80%), and few treatment options for patients who develop resistance to frontline therapies, prognosis for ovarian cancer is poor, and the overall 5-year survival rate is only 47.4%. Thus, there is a great need to improve therapies available for ovarian cancer patients, particularly for patients with platinum-resistant disease.

Immunotherapy has shown promise in other cancers such as melanoma, bladder, lung, leukemia, and breast [8,9,10]. The tumor immune environment, such as the presence of CD3+ tumor-infiltrating T cells and intraepithelial CD8+ tumor-infiltrating lymphocytes (TILs), correlates with survival and progression in ovarian cancer [8, 11,12,14], indicating a role for modulation of the immune system in this disease site is possible. Current immunotherapeutic strategies for ovarian cancer consist mainly of (1) monoclonal antibodies as receptor mediators, including immune checkpoint inhibitors (ICPI); (2) cancer vaccines; or (3) adoptive immunotherapies alone or in combination with other approaches.

Immune checkpoint inhibitors for ovarian cancer

ICPIs impede the ability of the tumor to activate checkpoint proteins on the surface of T cells, thereby preventing the cancer from evading immune response and allowing the immune system to generate an antitumor response [15, 16]. Immune regulatory checkpoints are essential for cultivating peripheral tolerance and prevention of autoimmunity. Among the most well understood are cytotoxic lymphocyte-associated antigen 4 (CTLA-4) and programmed death receptor (PD-1). CTLA-4 abrogates autoreactive T cells in lymph nodes early in T cell activation, while PD-1 regulates previously activated T cells in non-lymphatic tissues later in the immune response and controls apoptosis of regulatory T cells [17, 18]. PD-1 expression occurs in instances of high T cell stimulation, such as cancer. Binding of PD-1 to its antigens PD-L1 and PD-L2 inhibits T cell proliferation/survival, via changes in interferon (IFN)-gamma, tumor necrosis factor-alpha, and IL-2, as well as phosphorylation downstream of the TCR, and increases in regulatory T cells [19]. Blocking these immune checkpoints with monoclonal antibodies is an attractive strategy [20].

A variety of ICPCs have gained FDA approval, yet the study of ICPIs in gynecologic cancers lags behind other disease sites. Nivolumab (Bristol-Meyers-Squibb) and pembrolizumab (Merck) target PD1. Both are IgG4 complexes, but differ subtly in binding site (N-loop versus CD-loop) and affinity (Kd = 3.06 pM versus 29 pM) [21]. Nivolumab has indications in melanoma, lung, renal cell, Hodgkin’s lymphoma, hepatocellular carcinoma, head and neck, urothelial, and MSI-high colorectal carcinomas. Pembrolizumab has indications in melanoma, lung, head and neck, Hodgkin’s lymphoma, PD-L1-expressing cervical, or gastric cancers and is notable as the first drug to gain site agnostic approval for tumors that exhibit microsatellite instability. Ipilimumab (Bristol-Meyers-Squibb; targets CTLA-4, with indications in melanoma, urothelial, and lung cancers. Atezolizumab (Genentech-Roche), darvulamab (Astra-Zeneca), and avelumab (EMD-Serono) target PD-L1 and are approved for urothelial/lung cancers, bladder/lung, and for lung/Merckel cell cancers, respectively.

Multiple early-phase clinical trials are ongoing to examine ICPIs in ovarian cancer (Table 1). Preliminary results from the JAVELIN Solid Tumor [22••] clinical trial of avelumab (anti-PD-L1) in 124 patients with refractory/recurrent ovarian cancer found an overall response rate (ORR) of 9.7%; stable disease (SD) was observed in 55 patients (44.4%), yielding a DCR of 54.0%. Only 6.5% of patients experienced a grade 3/4 treatment-related adverse event (AE). PD-L1 expression was present in 77% of patients; ORR was 12.3% in PDL-1+ versus 5.9% in PD-L1- patients. Preliminary results from a trial of pembrolizumab in 16 patients with PD-L1+ advanced ovarian cancer reported an ORR of 11.5%, with 1 patient with a complete response (CR), 2 with partial responses (PR), and 6 with SD. Progression-free survival (PFS) was reported as 1.9 months and overall survival (OS) as 13.1 months, with 73.1% of patients experiencing an AE but only 1 patient with a grade 3 treatment-related AE. A phase II study of ipilimumab in 40 patients with recurrent platinum-sensitive ovarian cancer reported a best overall response rate (BORR) of 10.3% by RECIST 1.1 (n = 39); however, 50% (20/40) of the patients experienced a grade 3 or higher treatment-related AE [23•]. Toxicities related to ICPIs most commonly include skin (rash, pruritus), gastrointestinal tract (diarrhea), infusion-related reactions, and fatigue, which may limit the clinical effectiveness of this class of drugs [24, 25].

Table 1 Selected ongoing clinical trials of ICPIs in ovarian cancer
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There is immense interest in ICPIs in combination with poly-ADP-ribose polymerase inhibitors (PARPi) or vascular endothelial growth factor receptor (VEGF) inhibitors, including both monoclonal antibodies (e.g., bevacizumab) and small molecule inhibitors (e.g., cedirinib). There are currently numerous ongoing early-phase clinical trials focused on ICPIs in combination with these agents [26,26,27,28,30] (Table 1). A phase I study of durvalumab in combination with olaparib or cediranib in 26 patients with women’s cancers found an 83% disease control rate (DCR) in patients who received durvalumab plus olaparib and a 75% DCR and 50% response rate in patients who received durvalumab plus cediranib [31]. IMagyn050 is an ongoing phase III clinical trial of atezolizumab (anti-PD-L1) versus placebo with standard frontline chemotherapeutics and bevacizumab on yet untreated patient with advanced EOC and fallopian or primary peritoneal cancers (NCT03038100) [32].

Biomarkers to predict response to ICPI in ovarian cancer must be validated. The definition of PD-L1 “positivity” varies greatly. Microsatellite instability (MSI) leading to high mutational burden was first shown to be predictive of response to ICPIs in colorectal carcinomas [33]. Rates of MSI may vary by histologic subtype of ovarian cancer. One group identified a subset of ovarian clear cell carcinomas, typically characterized by worse prognosis compared to other histological subtypes, associated with increased MSI and increased tumor immunogenicity [34]. BRCA1/2 mutations may also be associated with increased mutation load, immunogenicity, and greater expression of PD-1/PD-L1, indicating that these patients may respond well to ICPIs [35]. Others have suggested that whole exome sequencing may also identify disruptions of the PD-L1 gene, signifying that genetic characteristics may, one day, be used to identify ICPI responsiveness [36]. Such observations underscore the need for further study of the effects of genomic instability, homologous recombination status, or BRCA 1/2 mutations on ICPI outcome [29, 37, 38].

Vaccination strategies for ovarian cancer

The first attempt to use vaccination to stimulate immune function occurred in 1891 when William Coley injected Streptococcus pyogenes and Serratia marcescens intra-tumorally, after observing regression of sarcoma in a patient with erysipelas [39]. Modern cancer vaccines consist of autologous whole cells, dendritic cells loaded with autologous tumor-specific antigens, or plasmids that result in antigen expression and immune activation after repeated exposure. Each vaccination strategy may be further augmented by a variety of immune-modulatory agents.

Engineered autologous tumor cell immunotherapy represents an elegant approach to restoration of T cell effector function. This may involve transfection of autologous irradiated tumor cells with a plasmid encoding for the upregulation of immunostimulants and downregulation of immunosuppressants. One notable example is Vigil® (Gradalis, Carrollton, TX), containing a proprietary plasmid leading to up regulation of granulocyte-monocyte colony stimulating factor (GM-CSF) and silencing of furin, an enzyme that activates transforming growth factor (TGF)-ß1/2. Expression of GM-CSF stimulates antigen presentation; inhibition of TGF-ß1/2 allows antigen-presenting cells to recognize tumor antigens. Early studies showed that response to vaccination (as manifested by positivity in gamma-interferon) using ≥ 107 cells with the construct for up to 12 monthly injections in solid tumors correlated with survival (25.7 versus 11.6 months) [40]. In 2017, similar immune results were found using a 1-log lower-dose formulation in a study of 15 patients, including 40% with heavily pre-treated ovarian cancer [41], which suggests smaller tumor samples are required for initial engineering. Additional studies of this technology as maintenance therapy remain in progress (NCT02346747, NCT01309230) [42, 43].

An antigen of recent interest in gynecologic malignancies is folate receptor (FR)-α (also known as folate-binding protein, FBP), as it is highly overexpressed in ovarian and type 2 endometrial cancers, coinciding with increased DNA production and turnover. Jackson and colleagues (2017) recently published results of a phase I/IIa trial employing E39 (GALE 301), an HLA-A2 restricted, FRα peptide in women without evidence of disease following treatment of initial disease (n = 24 with 16 controls) or recurrence (n = 5 with 6 controls). A total of 6 monthly injections (100, 500, or 1000 μg) with granulocyte macrophage-colony stimulating factor (GM-CSF) were provided. Delayed-type hypersensitivity increased in a dose-dependent fashion (13.3% vs 55% CG, p = 0.01). Estimated 2-year disease-free survival (DFS) was 85.7% in the 1000 μg group vs 33.6% in controls (p = 0.021). A subsequent phase Ib study of breast and ovarian cancer patients evaluated six serial immunizations using E39 and an attenuated variant (E39’) (E39 × 6, versus E39 × 3 then E39’ × 3, E39’ × 3 then E39 × 3). This study suggested benefit to incorporation of attenuated vaccine with no new safety signals [44]. Immune response was successfully generated without significant AEs in 90% of patients in a related phase I trial of 22 patients with a history of ovarian cancer or breast cancer and no evidence of disease who were treated with metronomic cyclophosphamide then vaccinated 6 times monthly [45••]. While this provides biologic plausibility, there is presently no estimate on clinical efficacy of this approach.

Of note, FRα is also the target for mirvetuximab soravtansine (IMGN853), a humanized monoclonal antibody attached via a disulfide-containing linker to the cytotoxic maytansinoid, DM4. Once released within the target cell, DM4 acts as an anti-mitotic agent that inhibits tubulin polymerization and microtubule assembly, resulting in cell cycle arrest and apoptosis. A phase III study of mirvetuximab soravtansine versus investigator’s choice chemotherapy (paclitaxel, pegylated liposomal doxorubicin, or topotecan) in platinum-resistant ovarian cancer is currently ongoing (FORWARD1, NCT02631876) [46]. Mirvetuximab soravtansine is not independently cytotoxic to FRα-negative endometrial cells in vitro; however, bystander killing on FRα-negative cells in co-culture with FRα-positive cells does occur [47]. This effect is presumably secondary to diffusion of metabolites to adjacent cells following lysosomal processing post-internalization [48]. Thus, antibody-drug conjugation may offer prominent advantages relative to other FRα-targeting strategies including farletuzumab (MORAb-003; Morphotek, Inc., Exton, PA), which relies only upon antibody-dependent cellular and complement-dependent cytotoxicity.

Other tumor-associated antigens (TAA) of interest in ovarian cancer belong to cancer-testis family and p53, the latter of which is nearly universally mutated in ovarian cancers [49]. Vaccination strategies targeting cancer-testis antigen (e.g., MAGE, NY-ESO) are reviewed extensively elsewhere [50]. Despite much early excitement surrounding various vaccination strategies targeting various TAAs, phase III trials have historically been disappointing. One notable example is vaccination with anti-idiotypic antibodies against glycoprotein CA-125, an FDA-approved serum biomarker to monitor clinical response to therapy in ovarian cancer, which failed at the phase III level for reasons still not fully understood, but which remain under active investigation [51]. Recently, a phase I trial of a modified vaccinia ankara vaccine delivering wild-type human p53 (p53MVA) in combination with gemcitabine chemotherapy in patients with platinum-resistant ovarian cancer showed longer PFS in patients with p53 reactive CD4+/CD8+ T cells but unacceptable toxicity with chemotherapy administration [52•]. These studies serve as a humbling reminder of the complexity of the immune response in disease.

Adoptive immunotherapy in ovarian cancer

Adoptive immunotherapy is based on the infusion of autologous or allogeneic neoplastic targeting immune cells that have been expanded and/or activated ex vivo [53, 54]. Adoptive immunotherapy can be based on antigen-independent (innate immunity, e.g., natural killer (NK) and cytokine-induced killer (CIK) cells) or antigen-dependent (adaptive immunity, e.g., TILs, chimeric antigen receptor (CAR) T cells) strategies [53].

NK cells can kill tumor cells without prior sensitization or need for MHC expression and have an important role in tumor immunosurveillance. Individuals with impaired NK cell function display an increased risk of cancer development [55, 56]. Initial clinical trials involving NK cells focused on response modifiers and cytokine therapy to potentiate autologous antitumor response in vivo and demonstrated conflicting results [55].

Most of the clinical trials using NK cells remain in early-phase development; however, data thus far suggest that administration of large numbers of donor NK cells is safe, feasible, and efficacious in patients with leukemia and solid tumors (NCT01212341) [57, 58]. Administration of lymphodepleting chemotherapy and T cell suppression with allogenic NK cells has been shown to increase TTP from 52 days with fludarabine, cyclophosphamide, cyclosporine, NK cells, IL-2 (Arm 1) to 98 and 100 days using this regimen plus 10 mg methylprednisolone (Arm 2) or 1 mg methylprednisolone (Arm 3), respectively (NCT01105650). Arm 2 experienced a decrease in disease progression (33%, 1/3) compared to Arm 1 (67%, 2/3) and Arm 3 (71%, 5/7) suggesting benefit from higher doses of methylprednisolone. Both Arm 2 (1/3) and Arm 3 (3/7) treatments resulted in improved OS at 1 year compared to Arm 1 (0/3). CAR-NK cells, chimeric antigen receptor-engineered NK cells, have also been explored in vitro and in hematologic and MUC1-positive solid tumors, but have yet to be tested clinically in ovarian cancer [59, 60]. While data from many NK cell clinical trials demonstrates that this treatment is safe, one trial (NCT00652899) was terminated early due to toxicity.

While clinical trials involving cytokine-induced killer (CIK) cells are numerous for other cancers, data are much more limited for ovarian cancer. In one study, stage II ovarian cancer patients receive radiofrequency ablation (RFA) with and without autologous transfer of CIKs (Table 2). Several studies have been published analyzing the efficacy of CIK cells in the treatment of solid tumors including ovarian cancer [61,61,63]. Liu et al. in a phase II study tested the role of CIK cells in maintenance therapy after first-line treatment in advanced epithelial ovarian cancer [61]. Ninety-two patients underwent cytoreductive surgery followed by 6–8 courses of carboplatin and paclitaxel chemotherapy and 1 month after the last course half of the patients received monthly infusions of autologous CIK cells with the remaining patients receiving no treatment [53, 61]. In patients treated with CIK therapy, an increase in median PFS was observed (37.7 vs 22.2 months, p = 0.004) while OS differences did not reach statistical significance. Zhang et al. demonstrated that pre-treating CIK cells with an immunological adjuvant PA-MSHA (Pseudomonas aeruginosa-mannose-sensitive hemagglutinin) led to improved function of CIK cells; patients treated with PA-MSHA-stimulated CIK cells plus chemotherapy achieved PR or SD (with only one patient exhibiting PD), whereas no patients in the CIK plus chemotherapy group achieved partial remission [63].

Table 2 Selected ongoing clinical trials of biologicals in ovarian cancer
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Ovarian cancer is not generally considered an immunogenic cancer, however, evidence of immune evasion, clinical response to immunotherapy, and the correlation of CD8+ CD3+ T cells with improved survival suggest otherwise [64]. TILs (tumor-infiltrating lymphocytes) are endogenous autologous T cells derived from resected tumors or the peripheral blood of naïve or vaccinated patients and expanded ex vivo [64]. Non-reactive autologous T cells can be stimulated by tumor antigen-specific stimulation in vitro or by genetic engineering to express either an exogenous tumor antigen-specific T cell receptor (TCR) or chimeric antigen receptor (CAR).

TILs have been explored in clinical trials since the 1990s with early human trials showing mixed results [65]. Prospective studies demonstrate spontaneous recognition of tumor neo-epitopes in immunotherapy naïve ovarian cancer patients validating the resurgence of TIL clinical trials [66•, 67]. Results for a pilot study determined that TIL treatment for platinum-resistant metastatic ovarian cancer with a decrescendo IL-2 was feasible and tolerable. All patients had SD for a minimum of 3 months (NCT02482090) [68]. TIL therapy in combination with checkpoint inhibitors or new methods for TIL expansion could results in improved clinical responses in ovarian cancer.

Numerous clinical trials using engineered T cells, TCR, and CAR-T cells to target antigens such as HER2, NY-ESO-1, FR-alpha, MSLN, MUC16, and p53 are currently recruiting. These TAAs are also targets in cancer vaccine development [64]. ALT-801 is a biologic compound composed of IL-2 genetically fused to a humanized soluble T cell receptor directed against p53-derived antigen which recognized an epitope displayed on cancer cells in the context of HLA-A*0201 [69]. In patients with progressive metastatic malignancies, this regimen was well tolerated and elicited a clinical antitumor response at the three doses analyzed (0.015 mg/kg, 0.040 mg/kg, and 0.080 mg/kg, the 0.040 mg/kg demonstrated the highest production of IFN-gamma and immunogenicity titer (NCT00496860).

A variant of adoptive T cell immunotherapy, CAR-T cell immunotherapy allows for the combination of antigen specificity through conjugation of a specific antibody with T cell activating properties in a single fusion molecule [70]. CARs bypass the immune escape mechanism of cancer cells because they endow T lymphocytes with cytotoxic effector features in an MHC-unrestricted manner. The current CAR-T cell trials mainly target antigens mesothelin and HER2. In a trial of CAR-T cells directed against mesothelin (CART-meso) (NCT02159716) [71], six recurrent serous ovarian cancer patients were treated with either a single dose (3 × 10^7 cells/m2) of CART-meso or CART-meso (3 × 10^8 cells/m2) with or without lymphodepletion. This therapy was found to be safe and feasible with patients showing SD after 1 month of treatment. Preclinical data demonstrated that established and primary ovarian cancer cells were recognized and reacted against by anti-HER2-CAR-T cells with little to no reactivity to normal ovarian surface epithelium, thus showing feasibility for the current HER2 clinical trials [72].

Cytokine release syndrome is a potentially severe systemic toxicity seen after adoptive T cell therapy caused by T cell activation and proliferation. It is associated with elevated levels of circulating cytokines and has been previously described as a systemic response in hematologic malignancies. Tanyi et al. (2017) described a new phenomenon, compartmental cytokine release syndrome, in a patient with advanced recurrent serous ovarian cancer treated with autologous mesothelin-redirected CAR-T cells [73]. This was thought to arise from antigen-specific T cell activation and innate immune activation targeted to the site of tumor in the pleural cavity rather than other compartments that harbored tumor or compartments in which CART-meso cells were detected. The unique nature of ovarian cancer growth and spread could therefore lead to unexpected responses to CAR-T cell therapy compared to other solid tumors.

Conclusions

Immunotherapeutic strategies for the treatment of ovarian cancer must be explored and refined to improve morbidity and mortality of this disease and render therapies available to these patients which are on par with those available to other tumor sites.