Cancer immunotherapy via nucleic acid aptamers
Introduction
There are established therapies employing a variety of manipulations to activate antitumor immunity. One type constitutes passive immunization with monoclonal antibodies, the introduction of adjuvants into the tumor microenvironment, and the systemic injection of cytokines. However, active transfer of antigen-loaded dendritic cells, tumor-activated T cell, tumor antigens formulated with either adjuvant or included in different delivery systems comprise second approach. Currently, immune therapy has become a standard treatment for a variety of cancers, and in some instances, could be considered as a replacement for nonresponsive cases to chemotherapy and other traditional strategies for cancer treatment [1].
Investigational immune therapies for cancer, involve devising more efficacious and less toxic cancer therapies upon established treatment regimens. A wide variety of novel strategies have been developed based on our understanding of the interactions between tumors and the immune system. Collectively, these strategies attempt to augment protective antitumor immunity and to disrupt the immune regulatory circuits that are critical for maintaining tumor tolerance [1]. The immune system has three basic roles in the prevention of tumors. First, it can protect the host from virus-induced tumors by eliminating or suppressing viral infections. Second is the timely and effective elimination of pathogens and prompt clearance of inflammation preventing the establishment of an inflammatory microenvironment conducive to tumorigenesis. Finally, some tumor cell express some antigens including tumor-associated antigens (TAS) and tumor specific antigens (TSA) which immune system can specifically identify them resulting in the elimination of tumor cells [2].
Aptamers are short nucleic acids (almost 12–80 nucleotides long) capable of specific and tight binding to their target molecules. The term aptamer is originated from the Latin word aptus (to fit) and the Greek word meros (part) [3]. Aptamer selection or isolation was accomplished by a process called SELEX (systematic evolution of ligands by exponential enrichment), which was first applied independently by Ellington and Szostak [4], Tuerk and Gold [5], and Robertson and Joyce [6] in 1990. A typical SELEX experiment starts with a library of up to 1015 random oligonucleotide sequences which can be DNA, RNA, or modified RNA (e.g., 2-OMe or 2-F modified RNA). Some members of this rich library have strong affinity for binding to a desired target. Numerous aptamers (also termed Oligonucleotide Aptamer Ligands), have been developed during the past 20 years which can bind and inhibit the activity of many proteins. The concept that nucleic acid ligands could modulate the activity of proteins emerged from basic studies of viruses and early works in the field of gene therapy [7]. Research on HIV and adenoviruses in the 1980s, discovered that these viruses encode a number of small-structured RNAs that bind to viral or cellular proteins with high affinity and specificity. For example, the human immunodeficiency virus has evolved a short-structured RNA ligand (TAR). The HIV TAR element binds to the viral proteins such as tat, as well as the cellular protein cyclin T1. HIV uses the TAR element to control viral gene expression and replication. Adenovirus has evolved a short-structured RNA aptamer, termed VA-RNA, which inhibits interferon-induced PKR activity and thus blocks one of the antiviral strategies employed by mammalian cell's [7]. In the late 1980s, the observation that viruses utilize RNA ligands in their biological activities particularly for escaping from immune-responses suggested that RNA ligands might also be useful for therapeutic and diagnostic purposes. The first study in 1990 indicated that CD4 + T cells containing the TAR aptamer were highly resistant to viral replication and cytotoxicity. Thus, for the first time in these studies, it was demonstrated that nucleic acid aptamers could be used as an agent to directly bind and inhibit the activity of proteins, suggesting possible clinical outcome [8]. This approach has been widely used during the past two decades to generate RNA ligands for many proteins. In 2003, Gilboa et al. demonstrated that aptamers can be employed as a potential therapeutic agent for cancer treatment by targeting immune-receptors described below. His work offered a new strategy for cancer immunotherapy [9].
In this review, we describe aptamers which are implicated in the treatment of cancer, particularly those which target proteins involved in tumor immunotherapy thereby preventing tumor immune-escaping (Table 1). Aptamers are not only a new and promising alternative to antibodies in tumor diagnostics, but also can be used in direct tumor immunotherapy and delivery systems. Aptamer can be used directly for either therapeutic applications or delivering drug molecules to disease-related cells or tissues of interest thereby minimizing the exposure of these possibly harmful agents to surrounding healthy tissues (decrease bystander and adverse effect of the main drug). Aptamer immunotherapy can replace monoclonal antibody therapy. There are some promising examples of cancer antibody therapy which potentially can be replaced by aptamer therapy. Ipilumimab is a FDA approved antibody that blocks the inhibitory action of CTLA-4 [10], [11], and clinical trials targeting 4-1BB and PD-1 or PD-L1, have demonstrated the therapeutic potential of using immunomodulatory antibodies to stimulate protective immunity in human patients [12]. Nonetheless, systemic administration of immunomodulatory antibodies has been associated with dose-limiting autoimmune pathologies, conceivably reflecting also the activation of resident auto-reactive T cells [8]. Aptamers have notable advantages over antibodies which include, a) as a cell-free chemical synthesis is used for aptamer selection, they are more simple and cost-effective in comparison to cell-based products like antibodies, b) conjugation of aptamers to other entities such as toxins or drug carriers is easier compared to antibodies, and c) antibody-based therapeutic agents have at least one foreign motif which may promote immune response in receiving hosts, whereas aptamers due to their nucleic acid nature and size, initiate less or no immune response. However, translating aptamers into clinic faces some major limitations such as fast renal clearance and digestion by serum nuclease [13] which are explained in the next section.
In traditional approach of cancer therapy by aptamers, they were mainly developed for targeting tumor markers. These include aptamers targeting MUC-1 (CD227) [14], prostate specific membrane antigen (PSMA) [15], human epidermal growth factor receptor-3 (HER3), tenascin-c [14], [16] and some other receptors [17]. Among these, only aptamers specific for HER3 as one of the tumor markers, exhibited antagonistic activity [2]. Nowadays, aptamers are not only considered as a new and promising alternative to antibodies in tumor diagnostics tests, but also they can be used in direct tumor immunotherapy and drug delivery system [18]. There are several advantages for aptamers which make them suitable alternative to mAbs in clinical applications. Aptamers are not recognized by the body's immune system as foreign, and do not evoke a negative immune response. Although aptamers are small molecules and they are subject to kidney filtration, resulting in shortened half-lives but further chemical modifications to overcome the susceptibility to kidney filtration can be easily introduced during synthesis. Tumor cells are different quantitatively and qualitatively from healthy cells in their protein repertoire and surface antigens making them suitable for targeting by antibodies and aptamers [19]. SELEX is performed using two approaches including ligand or specific molecule SELEX and cell SELEX in which surface molecules on whole cell are targeted. Cell SELEX can be employed for the generation of aptamers against known or unknown target proteins displayed on live cells. Cell SELEX is a particularly promising selection strategy for various applications such as cancer research, cancer diagnosis and therapy. Highly specific aptamers can be developed against tumor cells differentiated from normal cells. Different cancer cells have been used in this process, and as a result, a significant number of aptamers have been generated for most cancers studied. Cell SELEX and its clinical application were well discussed in other reviews [20], [21].
Extracellular targets are divided in two types including, a) secretory targets such as antibodies, cytokines, tumor secreted immunosuppressive proteins (Fig. 1) and b) cell surface targets such as co-stimulatory molecules, adhesive molecules, tumor immunosuppressive induction molecules, activation and inhibitory receptors (for agonist and antagonist targeting) on T cells, NK cells, macrophages and dendritic cells. In the case of extracellular targets, aptamer stability becomes a major issue. Therefore, stabilized RNA (e.g., by 2-O-methyl or 2-fluoro modifications) or DNA is preferable when extracellular proteins are targeted [20]. Even then, degradation and renal clearance is inevitable, and repeated administration will be required until treatment is complete. In this paper, some strategies have been suggested to overcome rapid aptamer clearance and degradation. On the other hand, immunotropism aptamers can be classified into three categories on the basis of their application including (i) agonistic or (ii) antagonistic activity on target protein and (iii) aptamer as a drug delivery agent [22]. Two excellent reports have explained use of oligonucleotide aptamer ligands to modulate the function of immune receptors [15], [23].
Section snippets
Methods used for developing immunotherapeutic aptamers and technical challenges involved
Methods used for the isolation of potential aptamers to specifically target the immune system are dependent on the type of target and the purpose for which aptamer is developed. Collectively, target cells or proteins are incubated with a complex library consisting of up to 1015 individual single stranded oligonucleotide molecules (ssDNA or RNA library).
SELEX is an iterative in vitro process which includes three main steps, a) binding step in which the target molecule is incubated with a random
Immune costimulatory-based cancer therapy with aptamers
Costimulatory (co-stimulatory) molecules expressed on the surface of APCs are responsible for the second signal, known as costimulatory signal. The interactions between costimulatory molecules and cognate receptors on the surface of T cells result in clonal T-cell expansion and differentiation, as well as carrying out their effector functions [31]. The presence of an efficient costimulation is crucial for improving antitumor immunity [32]. In fact, one of the mechanisms through which tumors are
Aptamers against immune-suppressive cytokines
Tumors and tumor-associated immune cells can produce a range of cytokines with the ability to suppress tumor-immunity (Fig. 1). The anti-inflammatory cytokines, IL-10 and transforming growth factor-β (TGF-β), are among the most important immunosuppressive cytokines produced or induced by tumor cells. Strategies to reverse this immune suppression represent an attractive target for immune therapy [60], [61], [62]. Targeting TGF-β can substantially augment antitumor immunity in animal models [63],
Bi-specific aptamers for cancer immunotherapy
The first bi-specific cancer immunotherapy was accomplished by bi-specific antibodies (bsAb). The earliest bsAbs were generated by two methods including, chemical cross-linking of whole or parts of antibodies and fusion of two hybridomas resulting in hybrid hybridoma (quadroma). This quadroma secreted bi-specific IgG molecules [74]. However these bsAbs have two major drawbacks. The first problem is associated with the production approach. It was difficult to generate large and homogeneous
Aptamer-targeted antigen delivery in cancer immunotherapy
Aptamers can also serve as modules that selectively recognize and bind to defined cell types or tissues. Conjugation of aptamers to drug, toxin or nanoparticulate systems enable them to deliver cargo molecules to or into specific cells or tissues of interest [82]. Utilizing aptamers for specific targeting and directional antigen transfer into antigen presenting cells has currently emerged as a replacement for antibodies. Targeting immune cells for acquiring potent immune response as well as
Ex vivo and in vivo use of aptamers in cancer immunotherapy
Although cancer vaccines have not yet been successful in patients, an alternative approach of cell-transfer cancer therapy which involves sensitization, ex vivo expansion of autologous lymphocytes with the ability to recognize, reach, and destroy cancer can overcome some barriers to the development of an effective cancer treatment [90]. There are a couple of advantages to this approach including the possibility of administering a very large number of highly selected cells with high affinity for
Conclusion
Rare and sporadic regressions of progression tumors were observed in both cell-based immunotherapy strategy and peptide vaccine emulsified in immune adjuvants. The lack of clinical responses points to the need of interfering with the escape mechanisms of tumors. These strategies have failed because of the inability of immune lymphocytes for homing and infiltrating into tumor tissues, loss of antigen expression by the tumor, the local presence of immunosuppressive factors or cells, and the
Acknowledgments
The authors are very grateful to Eli Gilboa for his excellent critical comments during the preparation of this review. Financial supports from Mashhad University of Medical Sciences are appreciated.
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