Aptamers as potential therapeutic agents for ovarian cancer

Highlights

• Current ovarian cancer treatments do not convey acceptable survival advantages and is plagued by drug resistance.

• Ovarian oncology has put considerable effort into understanding tumour characteristics to develop targeted treatments.

• Antibodies have made significant progress towards targeted therapies but there is a need for novel targeting agents.

• EpCAM has been identified as a suitable ovarian cancer biomarker and its targeting has been investigated with varied success.

• Aptamers are attractive alternatives to antibodies as targeting ligands and use in ovarian cancer is rapidly emerging.

Abstract

Current therapy for ovarian cancer typically involves indiscriminate chemotherapies that can have severe off target effects on healthy tissue and are still plagued by aggressive recurrence. Recent shifts towards targeted therapies offer the possibility of circumventing the obstacles experienced by these traditional treatments. While antibodies are the pioneering agents in targeted therapies, clinical experience has demonstrated that their antitumor efficacy is limited due to their high immunogenicity, large molecular size, and costly and laborious production. In contrast, nucleic acid based chemical antibodies, also known as aptamers, are ideal for this application given their small size, lack of immunogenicity and in vitro production. As aptamers have begun to demonstrate their promise through targeting Epithelial Cell Adhesion Molecule (EpCAM), as well as a number of ovarian cancer biomarkers, in in vivo and in vitro models, their clinical applicability is slowly being realised. This review explores some of the current progress of aptamers targeting cancer biomarkers and their potential role as ovarian cancer therapeutics.

Introduction

Ovarian cancer can be considered the most lethal amongst any gynaecologic malignancy, causing more fatalities annually than any other female reproductive system cancer [1]. Of the estimated 240,000 new ovarian cancer diagnoses each year, close to two thirds are fatal, and the poor 5-year survival rates have remained stagnant since 2008 despite prolific drug development [1], [2], [3]. The reasons for the lower survival outcomes for ovarian cancer have been postulated to be due to the relatively high proportion of diagnoses made at an advanced stage, attributable to the non-specific nature of the symptoms of this cancer [4]. Nearly all ovarian tumours originate from one of three cell types: epithelial cells, sex cord stromal cells and germ cells, though most are epithelial [4]. It is important to note that ovarian cancers reflect a heterogeneous group of diseases, each with their own histologic subtypes that differ in cellular origin, the molecular alterations that mark their initiation and progression, and subsequently their reactions to common treatments. Moreover, the aetiology of this lethal disease is poorly understood and research to identify strategies for early detection and treatment are needed.

Current treatment options for ovarian cancer are relatively non-specific. Many studies stress the importance of debulking surgery, though this can cause menopause and infertility, especially in younger patients. Additionally, surgery places a life-threatening stress to the patient and has been found to have several associated risks such as the triggering of metastasis [5]. The current gold standard chemotherapy is intravenous platinum based carboplatin or cisplatin and paclitaxel combination therapies [6], [7], [8], [9]. The platinum analogues cisplatin, carboplatin and oxaliplatin (amongst others) have been investigated for therapeutic efficacy and toxicity [10], [11]. Carboplatin has now assumed widespread adoption as it was found to exhibit less toxicity with similar efficacy as cisplatin and therefore suitable for more aggressive high-dose chemotherapy [12]. Another example is the renewed interest of anthracycline class drugs such as doxorubicin (DOX), which was originally used in first line treatment in the 1970s [10], [13]. Today, a new formulation of DOX known as PEGylated liposomal DOX, is being preferentially used for its favourable pharmacokinetic properties when compared with the conventional form, resulting in different and more convenient toxicity and efficacy profiles [10], [13]. Unfortunately, even with these minor improvements to their formulations, these conventional forms of chemotherapy are still notorious for the poor quality of life associated with the non-specific cytotoxic side effects targeting the normal healthy cells as well as the intended cancer cells. Furthermore, despite the extensive research that has gone into the best combination/application of chemotherapies, only marginal progress has been made to improving their non-specific toxicity profiles and overall survival.

In addition to the problems associated with the non-specific side effects, drug resistance is a major problem that contributes to limited efficacy. This resistance can be defined as either pre-existent (intrinsic) or drug induced (acquired), depending on whether most the tumour cells already possess resistance-mediating factors, or whether the tumour cells develop resistance during treatment [14].

Acquired resistance is the result of mutations arising during treatment in combination with adaptive evolutionary responses. Inadequate amounts of therapeutic agents reaching the target site places a high risk of selection of drug resistant cell clones. A diverse range of molecular resistance mechanisms have been identified, some of which include drug efflux, and enhanced DNA damage repair. These factors, as well as other factors such as abnormal blood and lymphatic vessels, elevated interstitial fluid pressure, high tumour cell density, and acidic environment, affect the delivery of chemotherapeutic agents to solid tumours and present numerous obstacles to overcome with conventional chemotherapeutic agents [15]. Successful treatment very much depends on the ability to deliver sufficient doses of therapeutic agents to the tumour cells while overcoming these obstacles, as inadequate concentration at the tumour site can contribute to acquired drug resistance [15].

To compound this issue of drug resistance, should a sufficient amount of therapeutic agent reach the tumour, the presence of transporter proteins on cancer cell membranes which promotes drug efflux across the plasma membrane can pump drugs out of the cell (Fig. 1). This process is largely regulated by members of the ATP-binding cassette (ABC) transporter family of proteins [16]. Consisting of 49 members in total, each transporter is characterised by a highly-conserved nucleotide binding domain and a variable transmembrane domain, though each family member's structure varies from protein to protein [16], [17]. Upon substrate binding to the transmembrane domain, a change in conformation at the nucleotide binding site results in the substrate being fluxed out of the cell [16]. The overexpression of these transporters in cancer cell lines and tumours results in multidrug resistance and is one of the major contributors to the failure of chemotherapy [18]. Much of the research into these transporters and their role in chemo-resistance has been focused on multi-drug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). With each transporter having broad overlapping specificities, these transporters eliminate a number of chemotherapeutics employed in the treatment of ovarian cancer (Table 1) [14]. Recent progress in the understanding of the characteristics of the disease, tumour biomarkers and microenvironment however, has facilitated the development of various targeted agents with mechanisms of action that can circumvent these efflux pumps [13].

The field of cancer research and the knowledge of the disease has been advancing in recent years, revealing the complex and dynamic nature of the disease progression and development. These advancements include the discoveries of the various capabilities of cancerous cells and the knowledge of the various cell types that form a tumour. Beginning in the year 2000, there have been a number of capabilities of cancerous cells discovered that are acquired during the multistep development of human tumours. These have evolved with our understanding of tumorigenesis to include sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis and activating invasion and metastasis, deregulation cellular energetics and avoiding immune destruction, as well as genome instability and inflammation [22], [23]. In addition, the other area of cancer research that has advanced greatly is the formation of tumours and the cells that take part in forming a “tumour microenvironment”. By conceptualising the mechanisms that enable tumour survival, new research provides targets for treatments specific and essential to cancer pathogenesis, and translating to more effective and robust therapies with fewer off target effects and less non-specific toxicity.

Considerable effort in the past two decades has focussed on combining these hallmarks with biological indicators associated with ovarian cancer to not only understand the aetiology of the disease, but also to develop a ‘personalised’ treatment for this cancer. While biomarkers can take many forms, such as metabolites, karyotypes, methylation status, DNA, mRNA and protein expression, it is the cell surface proteins that have become synonymous with biomarkers and have been the main choice for targeting agents [24], [25]. Membrane proteins have been found to function in many biological process including; cell to cell interaction, adhesion, migration and signal transduction, and their expression has been associated to tumorigenesis. In 2006, Yildirim et al. demonstrated that membrane bound proteins were the target of 69% of US Food and Drug Administration (FDA) approved drugs, and while that number dropped to 36% after 2014, it remains a major site of drug targeting [26], [27], [28]. With these proteins being the target of choice, modalities with high specificity and sensitivity to these targets have been highly sought after.

The production and use of monoclonal antibodies (mAbs) to selectively target tumours became a reality with the advent of Hybridoma technology in 1975 [29]. These mAbs were typically immunogenic in humans due to their origins in mice and had poor abilities to induce human immune effector responses, which limited their clinical applicability. However, with the advances in antibody engineering, solutions to address these problems were devised and the development of chimeric, humanized and fully human mAbs emerged [30], [31], [32].

The success of mAbs is exemplified by Rituximab in the treatment of non- Hodgkin's lymphoma, which was the first mAb to get FDA approval in 1997. Rituximab is a chimeric mAb which targets the CD20 cell surface marker, expressed on 90% of B-cell lymphomas. Its function is to mediate complement and antibody-dependent cell-mediated cytotoxicity (ADCC) and has direct antiproliferative effects against malignant B-cell lines in vitro. Its success has been demonstrated in several clinical trials when used in combination with CHOP (cyclophosphamide, DOX, vincristine, and prednisone) chemotherapy. The addition of rituximab to the CHOP regimen increases the complete-response rate and prolongs event-free and overall survival with diffuse large-B-cell lymphoma, without a clinically significant increase in toxicity [33], [34], [35].

Increasingly, mAbs have been used for the effective treatment of cancer over the past ten years, with tumour expressed antigens being the specific target of these mAbs. Monoclonal antibodies conjugated to radioactive isotopes or chemotherapeutic drugs have shown therapeutic efficacy mainly in haematological malignancies, and mAbs targeting growth factor receptors, such as the epidermal growth factor receptor and human epidermal growth factor receptor 2, are regularly used for the treatment of non-leukaemic cancers.

To be an effective therapeutic for ovarian cancer, mAbs need to penetrate tissues and the extracellular matrix to reach their target cells. Typically, mAbs have limited tissue penetration due to their large size and molecular weight and is considered a major limiting factor [15], [36], [37], [38]. As tumours are characterised with heterogeneous and tortuous vasculature, high interstitial fluid pressure and high viscosity of the tumour blood supply, a major determinant of speed of diffusion through tumours is molecular size. The rate of diffusion is approximately inversely proportional to the cube root of molecular weight. Consequently, large macromolecules such as mAbs diffuse poorly explaining why larger tumour masses may be more difficult to treat by mAb therapy [39]. It has been demonstrated in murine xenograft models, that mAbs directed against tumour-specific antigens largely remain in the blood and no more than 20% of the administered dose typically interacts with the tumour [39]. In addition, achieving optimal affinity is critical for tolerability and cytotoxicity, which is not always easy to engineer as these properties depend on several factors, including antigen density, internalisation, association and dissociation rates.

In addition to mAbs that target tumour antigens, mAbs that target the tumour microenvironment slow tumour growth by enhancing host immune responses to tumour-associated antigens or by curtailing pro-tumorigenic factors produced in the tumour stroma [30]. This ‘enhancing host immune responses’ relies on a functional immune response for treatment to be effective [40]. As described in 2011 by Hanahan and Weinberg, an emerging hallmark is the ability of tumours to evade immune destruction and that a competent immune system will routinely eliminate highly immunogenic cancer cell clones [22]. This process has been referred to as “immunoediting” which will select for weakly immunogenic variants to develop into solid tumours [22]. Tumours may also supress immune response by disabling components of the immune system. For example, cancer cells may paralyse infiltrating cytotoxic T lymphocytes and natural killer cells, by secreting TGF-β or other immunosuppressive factors or through the recruitment of inflammatory cells that are actively immunosuppressive, including regulatory T cells and myeloid-derived suppressor cells [41], [42], [43], [44]. This suggests that this type of therapy is limited due to the known hallmarks of cancer.

However, when considering the hallmarks of cancer, inducing angiogenesis is one target that has shown promising effects in ovarian cancer. Targeting vascular endothelial growth factor (VEGF) has provided the most success for the treatment of ovarian cancer, and bevacizumab has been adopted in combination therapy. Bevacizumab is an anti-angiogenesis antibody targeting VEGF and has demonstrated abilities to improve progression-free survival in women with ovarian cancer [45], [46], [47], [48]. While some believed that effective inhibition of angiogenesis could result in tumour dormancy and even remission, anti-angiogenesis therapies have demonstrated only transitory clinical responses and can lead to other off target effects [49], [50], [51], [52]. While mAbs have demonstrated efficacy in several cancers and for many patients, and we have gained invaluable insight into the mechanism of action of these therapeutic agents, it is evident that further investigation into other targeted therapies are required to improved long term survival in patients.