Personalized vaccination against ovarian cancer: what are the possibilities?

KEYWORDS:

• Cancer vaccine
• immunotherapy
• neo-antigens
• personalized vaccine
• ovarian cancer

The basis of immunology is the discrimination between self and nonself. All pathogens have molecular signatures that can be recognized by the host’s immune system and activate immune responses. Vaccinations against viruses and bacteria, which have successfully exploited this vulnerability, have become an integral part of preventive healthcare. With regard to cancer therapy, prophylactic vaccines against human papilloma virus have evolved over the last decade for the prevention of cervical cancer and have become part of routine vaccination schedules [1]. Theoretically, vaccines for ovarian cancer patients directed against private tumor neo-antigens derived from nonsynonymous somatic mutations is a promising treatment strategy. Such an approach will activate endogenous immune cells to recognize specific tumor associated antigens (TAAs), thus killing cancer cells with minimal harm to the surrounding healthy tissue. Additionally, by increasing the pool of available tumor-specific T cells, therapeutic cancer vaccines could contribute to combination immunotherapy.

There is still an ongoing debate about the immunogenicity of ovarian cancer. Some evidence indicates that a proportion of these tumors express a large number of known TAAs [2], have immunoreactive gene signatures, and are infiltrated by intraepithelial tumor–infiltrating lymphocytes (TILs) [3]. On the other hand, others have demonstrated that ovarian tumors have a very heterogeneous and comparatively low mutational load, thus making immune recognition of neo-antigens dubious [4]. In spite of this comparatively low mutational load, however, one type of ovarian tumor, epithelial ovarian cancer (EOC), is susceptible to immune recognition [2,3]. Unfortunately, the projected potential of cancer vaccines based on these preclinical findings has not translated into the clinical setting thus far. The explanation for this can be the wrong choice of antigens or can be related to the inability of tumor-specific T cells to home in the tumor microenviroment. For example, barriers that prevent T cell homing in ovarian tumors include the peripheral tolerance mediated by regulatory T cells (T Reg) or the vascular endothelial growth factor A (VEGF-A) mediated vascular endothelial barrier [5]. Another major reason for clinical failure includes that most molecularly defined tumor vaccines, up till now, have used a single ‘self’ antigen. Therefore, the poor outcome of this approach in the clinical trials could be credited, at least in part, to immune-selection of antigen loss variants after vaccination.

There are two main classes of targetable TAAs in ovarian cancer: the shared common TAAs and the exclusive, individually mutated neo-antigens. The shared antigens can be separated into three main groups: the overexpressed antigens, which are normal surface proteins expressed in elevated levels on cancer cells but in lower levels in normal cells (i.e. mesothelin); tissue-specific TAAs, which are shared between tumors and the normal tissue of their origin; and TAAs whose expression is normally restricted to male germline cells (i.e. cancer testes (CT) antigens such as NY-ESO-1) [6]. In the past decades, the mutation-based cancer vaccines were restricted to using synthetic peptides featuring frequent driver mutations in many human cancers such as mutated p53 or Ras [7]. Also, the available pool of immunogenic mutations increased very slowly and most of the published shared immunogenic mutations were limited to small subsets of cancer patients [8]. Further, there was no feasible concept to use individual mutations. Instead, the therapeutic application of TAAs was focused mainly on shared TAAs. One example of a shared TAA tested in early phase clinical trials is the cancer testes antigen NY-ESO-1 [9]. In this small phase I trial decitabine, which has been shown to enhance NY-ESO-1 expression, was used in combination with a NY-ESO-1 vaccine and liposomal doxorubicin chemotherapy in recurrent ovarian cancer. Stable disease was noted in 50% (5/10 patients) that lasted a median of 6.3 months and a partial response was seen in 1 patient with duration of response of only 5.8 months [9]. Since then others have studied cancer testes antigens with conflicting results in clinical trials [10]. They have also found that some of the CT antigens are expressed at low levels in some normal tissues and that no single CT antigen is universally expressed and that the frequency and expression levels can be heterogeneous across tumors [10]. The high-avidity T cell clones recognizing these shared antigens may be deleted by central tolerance mechanisms, which may be one other explanation of the unsatisfactory outcome of clinical vaccination trials [11].

On the other hand, the current technological breakthroughs and extensive deep sequencing analyses have discovered that solid tumors contain as few as tens to several thousand private non-synonymous somatic mutations, and these mutations are different even among tumors from the same histologic subtype [12]. These neo-antigens result from the large number of somatic mutations that are fully tumor specific and consequently give us the opportunity to use them to design immunotherapeutic vaccinations. These neo-antigens, as compared to non-mutated tumor antigens in ovarian cancer, are unlikely to induce tolerance or to induce normal tissue toxicity. Thus, vaccination strategies directed against private tumor neo-antigens derived from non-synonymous somatic mutations is the future approach of personalized cancer immunotherapy.

As further evidence was provided by checkpoint inhibitor and adoptive T cell therapies, novel treatment approaches unfolded. Tumors with high mutation load and high number of neo-epitopes responded better to immunotherapy. Thus, each patient’s mutanome can be mapped with next-generation sequencing technologies, and these cancer mutations can be used as target antigens for vaccines. The use of multiple tumor-restricted antigens, such as neo-epitopes, can be done simply with the application of autologous whole-tumor or whole tumor lysate vaccination which makes the recognition of each single defined neo-antigen unnecessary [13]. Advantages of vaccination with autologous tumors and lysates include targeting the full repertoire of multiple, patient specific TAAs, including the private neo-antigens, independent from the HLA haplotype of the patients and priming of both CD4+ and CD8+ T cells, thus make it a truly personalized approach of active immunotherapy. One main restraint of these cell or lysate vaccines is the lack of ability of the vaccines to reach the lymph nodes to directly prime T cells. One solution of this problem can be the use of biodegradable particles such as liposomes, synthetic polymers, and acid degradable hydrogels that can deliver TAAs to dendritic cells (DCs) in vivo [14]. Further major benefits of using nanoparticles is that they decrease antigen degradation thus making antigen release longer, augmenting antigen uptake by DCs and co-delivering adjuvants with the vaccine simultaneously to stimulate an effective and specific immune response [14]. Another approach, instead of activating DC in vivo, is the pulsing of tumor material ex vivo with DCs. DCs can be generated ex vivo from the patient’s peripheral blood monocytes, using different forms of antigens and then the activated DCs then can be injected back to patients’ lymph nodes under ultrasound guidance [15]. Another problem can be with vaccine strategies that the stimulated endogenous antitumor T cells’ effectiveness is decreased by the T-cell receptor repertoire, which can be identified within the thymus as central tolerance and made nonfunctional by the postthymic peripheral tolerance. Several studies provided evidence that DC-based vaccines are clinically safe and can elicit even stronger CD4+ and CD8+ T cell responses than the vaccination with irradiated autologous whole tumor cells only [16]. This study showed that vaccination with tumor-pulsed DCs resulted in longer survival (72 % and 31% 2-year survival, respectively), stronger expansion in CD8+ antigen-specific T-cells against known TAAs and probably also undefined TAAs compared to vaccination with the autologous whole tumor cells only [16]. Unfortunately, however, there is often times an insufficient amount of tumor tissue available which makes creation of the autologous tumor cell, -lysate or DC vaccines difficult. In these situations, the DCs can be pulsed with whole tumor RNA that can be amplified and isolated from even microscopic amounts of tumor tissues or the tumor RNA can be introduced into DCs, typically with the use of electroporation [17]. Another novel vaccination strategy that showed promise in the preclinical setting involved DC-tumor fusion cells (DC/tumor) [18]. Using electroporation or polyethylene glycol (PEG) as a membrane-destabilizing agent the DC/tumor cell fusion can be created. During this fusion, the tumor cells and the DCs become hybrid cells sharing some of their cytoplasm, but keeping separate nuclei. This makes it possible that all TAAs are delivered to the DCs, processed and presented by MHC-I and MHC-II molecules stimulating both CD4+ and CD8+ tumor-specific T cells responses [18]. Unfortunately, the clinical responses the DC/tumor cell fusion vaccines were far inferior than expected. Patients enrolled in these studies tended to have advanced, heavily pretreated disease, which can create an immunosuppressive tumor microenvironment thus limiting the efficacy of vaccination strategies [19]. Further, the DC/tumor cells fusion themselves secreted immunosuppressive mediators such as TGF-β1, which could also contribute to poor treatment response [18].

Alternative approaches to target neoantigens include mRNA and peptide-based vaccines [20]. Peptide-based vaccines have evolved from ‘minimal epitope’ peptides of 9–10 amino acids in length to long peptides of 25–30 amino acids, which have started to show some promise possibly due to their different requirements for uptake and processing than the early generation peptide vaccines [20]. Personalized mRNA-based cancer vaccine approaches use high-throughput sequencing to identify unique mutations in an individual’s tumor (the mutanome). These can then be ranked according to predicted affinity to autologous HLA class II and high expression of the mutation-encoding RNA and predicted HLA class I binding. Selected mutations can then be engineered into synthetic RNAs. In the first-in-human application of this in melanoma, all patients developed T cell responses again the multiple vaccine neo-epitopes and there was T cell infiltration and net-epitope specific killing of tumor cells. There was vaccine-related objective responses in two of the five patients who were enrolled with metastatic disease [13].

While personalized vaccine immunotherapy is promising, alone it is not able to generate appropriate clinical responses. Thus, combinatorial immunotherapy strategies are vital to successful vaccination efforts. For this reason, combining next generation personalized vaccination with other methods and adjuvant treatments might be the answer to reach better therapeutic effect. For example, the immunogenicity of tumor-loaded DC vaccines can be enhanced by oxidative modification of the tumor lysate, by hypochlorous acid (HOCL), prior to the DC pulsing [21]. The proteins oxidized by HOCL have increased immunogenicity, as HOCL connects them with aldehydes and unfolding them makes them better engulfed and presented by DCs. Further, tumors escape immune surveillance through the immune checkpoint system, which can decrease the effectiveness of the vaccine induced antitumor T cells in the tumor microenvironment. Therefore, to overcome immune barriers generated by the tumor, monoclonal antibodies neutralizing immune checkpoint inhibitors can be used. So far, the most promising clinical outcomes were seen with anti-PD-1/PDL-1, anti-CTLA-4 and IDO checkpoint inhibitors [22]. These antibodies were synergist in causing tumor rejection in preclinical models when used in combination with cancer vaccines. In fact, dual blockade with vaccination augments tumor rejection and increases effector T cell activity [23]. Another area of interest includes the CD25 expressing regulatory T cells (T reg), which is a major factor contributing to decrease effectiveness of the vaccine primed T cell responses. Multiple strategies were introduced to decrease T Reg activity including the use of immuno-depleting chemotherapeutic agents such as low-dose fludarabine or cyclophosphamide and the application of monoclonal antibodies (anti-CD25) [24]. Additionally there is the tumor endothelial barrier, which can decrease T cell infiltration into the tumor microenvironment. This is another way that tumors can impede active vaccine immunotherapy with the stimulation of VEGF-based angiogenic signaling [5]. Many groups demonstrated that with the use of antiangiogenic therapies through the blockage of VEGF, this barrier can be lifted resulting in improved T cell homing into the tumor [5]. Finally, timing of vaccination may be critical to efficacy of vaccine strategies in ovarian cancer. Early use of vaccines in adjuvant setting may avoid the compromised immune system of heavily pretreated patients, the sheer number of tumor cells to mount an immune response to in the case of large tumor bulk, and the immunosuppressive microenvironment fostered by the growth and spread of disease.

In the future, combinations of various immunomodulatory treatments will be necessary to effectively stimulate the immune response and take advantage of the immunogenicity of ovarian cancer. The arrival of novel immunomodulatory molecules even further raised the possibility that cancer vaccines could reach their full therapeutic potential as a significant partner in combinatorial immunotherapy. Personalized vaccines targeting tumor neo-epitopes, whether they are whole tumor lysates or created peptides, could be particularly promising because they could expand neo-epitope-specific T cell clones [25]. It is highly expected, although not fully proven, that these vaccines induced neo-epitope-specific T cells could exhibit high avidity because they likely escape thymic selection, which depletes self-recognizing clones bearing high-affinity T cell receptors (TCRs) [25]. Recent discoveries provided evidence that DCs pulsed with whole-tumor lysate can expand preexisting as well as novel high-avidity T cell clones with markedly high-affinity TCRs against tumor neo-epitopes. These findings suggest that while peripheral tolerance mechanisms may suppress at the steady state, with the vaccination of exogenous DCs loaded with immunogenic lysate, the expansion of these high-affinity TCR clones can be stimulated. The recent clinical and biological observations provide promising data that DC vaccines pulsed with autologous whole-tumor antigen as personalized combinatorial immunotherapy will be an important future strategy. This approach is able to mobilize broad antitumor immunity and neo-epitope-specific T cells causing clinical benefit for the patients with ovarian cancer.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Funding

The paper was not funded.