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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
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Review
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Aptamers in immunological research
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Roald Nezlin ∗ Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel
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Article history: Received 23 September 2014 Accepted 5 October 2014 Available online xxx
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Keywords: Aptamers Complexes of aptamers with immunoglobulins Modulation of immune reactions Reactions of aptamers with cell receptors Tumor immunology Aptamers in immunoassays
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1. Introduction
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In recent years aptamers, synthetic DNA or RNA single-chain oligonucleotides, have been used in various immunological studies to bind specific ligands. Detailed data on the interactions of an RNA aptamer with a human Fc fragment were obtained by X-ray crystallography. The complex formation involves multiple weak interactions that resemble protein–protein interactions. Aptamers specific to cell surface receptors may serve as antagonists or agonists blocking or stimulating cell activities. As aptamers can modify T-cell reactions, they could be useful in the treatment of chronic diseases such as autoimmune and oncological pathologies. In chimeras constructed for the delivery of active substances to defined targets, aptamers specific to surface proteins may be used to transport constructs directed to targets such as tumor cells. Aptamers are also employed as highly specific reagents in immunological assays after being labeled with reporter groups such as fluorescent dyes or following immobilization on insoluble carriers such as membranes or microspheres. © 2014 Published by Elsevier B.V.
Aptamers (Latin aptus – fitting) are short single-stranded RNA or DNA oligonucleotides that bind specific ligands with high affinity and specificity. Small molecules such as organic dyes, nucleotides, amino acids, nucleotides, and drugs; and biopolymers such as peptides, polysaccharides, and proteins can all be bound by aptamers. The reactions of substrates with aptamers occur in solution as well as on solid surfaces (e.g. cell surfaces). In recent years such properties of aptamers have enabled their application to various biomedical, as well as pharmaceutical and clinical purposes. The formation of complexes with aptamers involves various types of interactions (hydrogen bonding, polar groups, stacking interactions, and shape complementarity). Aptamers in solution are unstructured; association with their ligands causes them to fold into arrangements in which the ligand becomes an intrinsic part of the nucleic acid structure [1]. Aptamers are obtained by a selection procedure known as SELEX (Systematic Evolution of Ligands by Exponential enrichment) [2,3]. The SELEX process is characterized by repeated cycles of selection and enzymatic amplification steps, yielding RNA or DNA oligonucleotides that react specifically and with high affinity with their corresponding ligands.
∗ Tel.: +972 8 9343562; fax: +972 8 9344141. E-mail address: [email protected]
Intensive studies of aptamers began in the 1990s. Threedimensional structures for complexes of aptamers with their ligands were determined, and the molecular basis of aptamer specificity was clarified [4]. It was shown that aptamers can be applied as potential therapeutic [5,6] and diagnostic agents and biosensors [7]. During the past decade studies of aptamer properties and activity intensified as reflected in numerous publications. Several effective modifications of the SELEX technique were incorporated into the process. Chemical modifications of aptamers were introduced, to prevent their degradation by nucleases. Other methods were developed to combine aptamers with nanoparticles, and to use such conjugates for detection of cancer cells and for drug delivery [8,9]. As stated above, aptamers bind many ligands with high affinity which differ in chemical structure and they are widely utilized in various analytical methods [10]. At the end of 2004 the US food and Drug Administration approved an RNA aptamer as therapy to treat of choroidal neovascularization, a form of age-related macular degeneration. As a therapeutic agent it targets vascular endothelial growth factor [6]. Several other aptamers are currently undergoing clinical trials as therapeutic agents for a variety of oncogenic and non-oncogenic diseases [11–13]. In recent years aptamers have more often been used in immunological research. Interactions of aptamers with immunoglobulin molecules in solution as well as on the cell surface have been examined. Today, aptamers tend to be more widely applied for the modulation of immune reactions as well as in immunological
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methods. In the present review, the results obtained in these studies will be briefly discussed. 2. Interaction of aptamers with immunoglobulins Japanese scientists studied in detail the interaction of an RNA aptamer with human IgG [14,15]. The aptamer Apt8-2, a 23 nucleotide-long molecule, containing 2 -fluoropyrimidines reacted with high affinity to the Fc fragment of human IgG of all four subclasses but was not able to bind the IgG of any animal species. Apt8-2 competed with bacterial protein A for Fc binding but not with cellular Fc-receptors. The reaction of Apt8-2 with Fc was observed only in the presence of the divalent ions Mg2+ and Ca2+ . Removing these ions by means of EDTA-buffer solution released the aptamer bound to IgG molecules. For the release of IgG from protein A, acid buffers are usually used. However, acidic conditions could be harmful to IgG antibodies. Therefore, the use of the Apt8-2 aptamer for the purification of IgG is preferable, as the separation of IgG from Apt8-2 can be performed by neutral buffers containing EDTA. In this study the complex containing Apt8-2 aptamer and the Fc fragment of human IgG1 was crystallized and its structure determined at 2.15 A˚ by X-ray crystallography. Formation of the complex did not change the structure of the protein. The interaction of the aptamer with Fc involves multiple weak interactions as hydrogen bonds and van der Waals contacts. Electrostatic forces did not play significant role in the Apt8-2-Fc complex formation. The complex resembles protein–protein complexes rather than nucleic acid–protein interactions. It was found that divalent ions support a specific aptamer structure, which is important for its specificity. Two amino-acid residues – Gln342 and Phe404 – which are present in all subtypes of human Fc-IgG, played an important role in the contacts with Apt8-2. One of them, Gln342, is located in a loop between the C␥2 and C␥3 domains. The protein A binding site is also located at the C␥2–C␥3 interface (for a review see [16]) which explains the fact that both polymers compete for Fc-IgG binding. Aptamers specific to other immunoglobulins have also been obtained. DNA and RNA aptamers with specificity to the Fc region of mouse IgG subclasses and rabbit IgG were isolated using SELEX [17–19]. A DNA aptamer with specificity to human IgE has also been identified [20]. 3. Reaction of aptamers with cell receptors During the past decade aptamers have been applied to modify the activity of cells participating in immune reactions. Aptamers can block cell receptors and inhibit their reactions with corresponding ligands; other aptamers in contrast are active as agonists promoting cell reactions. Gilboa and colleagues synthesized several aptamers that can modulate immune responses. In a series of experiments they used aptamers specific to CD28, a co-stimulatory receptor, which is responsible for activation of T-lymphocytes. The CD28 ligand (B7) is located on activated antigen-presenting cells. One of these aptamers blocked binding of B7 to CD28. In contrast, dimers of anti-CD28 aptamers introduced a co-stimulatory signal that acted as an agonist and enhanced the cellular antitumor immune response. Anti-CD28 aptamers were also able to enhance immune responses induced by idiotype vaccines used to treat murine lymphoma [21]. In other experiments aptamers specific to T-cell receptors – an inhibitory CTLA-4 and co-stimulatory 4-1BB were applied [22–24]. The aptamer to the first receptor blocked the inhibitory activity of T cells, and a tetrameric derivative of the same aptamer promoted protective, T-cell dependent anti-tumor immunity in murine tumor Please cite this article in press as: http://dx.doi.org/10.1016/j.imlet.2014.10.001
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models. Dimers of aptamers to the 4-1BB receptor which contribute to the expansion of activated T cells induced rejection of tumors in mice similarly to agonistic anti-4-1BB monoclonal antibodies. In control experiments non-specific aptamers had no inhibitory effect. An RNA aptamer specific to the OX40 receptor was used to stimulate proliferation and cytokine production by T cells [25]. OX40 is a member of the tumor necrosis factor (TNF) receptor superfamily. Aptamer monomers were found to be inactive because the receptor has to be dimerized on the cell surface in order to act as a receptor agonist. To prepare a receptor-activating form of the aptamer, a DNA oligonucleotide scaffold was used to connect two aptamer monomers in close proximity. This construct was able to bind OX40 on murine cells and to crosslink receptor monomers. The resulting reaction induced cytokine production and proliferation of antigenstimulated lymph-node cells. The agonistic aptamer was shown to be effective in the treatment of aggressive metastatic melanoma in mice. Interleukin 10 (IL-10) is a multifunctional cytokine that functions as an important immune inhibitor of inflammation in chronic infections and cancer. Suppression of IL-10 activity by blocking the corresponding surface cell receptor IL-10R by means of an RNA aptamer inhibits the development of chronic infections and tumor growth. The aptamer to IL-10R was as effective as anti-IL-10R antibodies in eliminating viral infection and cancer development in mice [26]. Dimers and tetramers of the anti-IL-10 aptamer have enhanced binding affinity and biological activity comparable to that of anti-IL-10 monoclonal antibodies. Aptamers specific to cell membrane proteins were used to construct chimeras that could target cells and stimulate their inhibitory or stimulatory activity. Such an approach was applied to inhibit the mTOR1 complex, a regulator of cell growth, in circulating CD8+ T cells [27]. The construct was composed of an aptamer to the 4-1BB T-cell receptor, coupled with a small interfering RNA (siRNA), which specifically downregulated the mTOR1 complex. As a result CD8+ T-cell memory was enhanced and protective antitumor immunity in mice was increased. Administration of the construct was more effective than use of rapamycin, an agent that specifically inhibits mTOR1 activity. Successful inhibition of tumor growth and rejection in mice was achieved by a bispecific aptamer composed of the aptamer to the 4-1BB receptor and an aptamer specific to prostate specific membrane antigen (PSMA) [28]. As T-cell activation causing the stimulation of 4-1BB occurred near the tumor site, the dose of the therapeutic bi-specific aptamer could be reduced. In this way, two barriers of successful cancer therapy could be overcome, by diminishing drug toxicity and avoiding autoimmune reactions that could develop during immunotherapy. The anti-tumor immune response is weak, as tumors do not produce strong rejection antigens. To stimulate anti-tumor immunity by inducing new potent tumor antigens, inhibiting the activity of the nonsense-mediated decay (NMD) complex was suggested [29,30]. This complex comprised of several factors, prevents the formation of mRNA with a premature termination codon. The small interfering RNA (siRNA) specific to the NMD factors inhibits their activity which in turn stimulates the expression of peptide products with new antigenic properties. As NMD is active in all body cells, siRNA must be delivered directly to cancer cells. For this purpose, RNA heterodimers composed of siRNA and an RNA aptamer targeted to tumor antigen PSMA were prepared. The construct inhibited tumors displaying PSMA antigen on the cell surface in vivo in a mouse model. Tumor rejection was immune-mediated as it was dependent on both CD4+ and CD8+ T cells and not observed in immune deficient mice. A multimeric DNA aptamer specific for membrane-bound IgM (mIgM) was constructed from a 37 nucleotide long monomer [31]. Monomer units were connected with flexible polyethylene glycol (PEG) linkers. Trimeric and tetrameric DNA aptamers bound
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effectively to mIgM but not to soluble serum IgM. Membrane IgM molecules have a membrane portion of mu-chains, absent in soluble IgM. Fifteen amino-acids of this portion are located extracellularly; this sequence could therefore serve as a target for the DNA aptamer. B cells from healthy people and CD3 T cells rarely bound the DNA aptamer. B-cells from several lymphoma cell lines and B cells, obtained from leukemia patients, however, did react with the DNA aptamer. Polyvalent DNA aptamers with cell receptor specificities may be useful as drug-carriers via endocytosis and may also have wider clinical applications as blockers of cell receptors, preventing reactions with ligands that trigger cell division. The stimulation of immune cells by ligands, agonists of Toll-like receptor 9 (TLR9) has been described [32,33]. Localized primarily on B cells and plasmacytoid dendritic cells (pDC) this receptor is one of several involved in the innate immune system. It recognizes unmethylated cytosine-phosphate-guanine (CpG) dinucleotides that are commonly seen in bacterial and viral DNA but are inactive in vertebrate DNA, due to methylation. Interactions of ligands with the CpG motif with TLR9 induces conformational changes in the receptor, which in turn, stimulates both innate and adaptive immune responses, resulting in the activation of specific B cells and pDC as well as promoting of the development of T cells. Therefore, CpG oligonucleotides are potentially effective drugs for the treatment of various pathologies including cancer, allergies and infections and may also enhance effectiveness of vaccines. CpG oligonucleotides used in combination with other therapies are likely to be even more effective. 4. Complexes of aptamers with soluble proteins The C5 complement component, a part of the innate immune system, plays an important role attacking various infectious agents. C5a and C5b, proteolytic fragments of C5, are highly active inflammatory proteins. C5a is responsible for systemic inflammation in several pathologies, particularly in sepsis and autoimmune diseases. Several aptamers specific to complement component C5 and its proteolytic fragment C5a were synthesized. RNA aptamers to human and rat C5 effectively inhibited activity of this component. The binding of these aptamers to C5 also prevented proteolytic cleavage of C5 on C5a and C5b [34]. Aptamers resistant to nucleases were synthesized with unnatural oligonucleotides. An RNA/DNA aptamer NOX-D20 was prepared from l-oligonucleotides (l-ribose/l-deoxyribose nucleotides). NOX-D20 inhibited the activity of human and mouse C5a [35]. In an experimental mouse model of sepsis NOX-D20 effectively prevented pathology progression and improved survival of the mice. Non-natural RNA l-oligonucleotides with specificity to murine CCL2 (pro-inflammatory chemokine monocyte-chemoattractant protein-1) improved kidney status in a mouse model of diabetic nephritis. An aptamer specific to the homeostatic chemokine CXCL12 and the aptamer to CCL2 had additive protective effects in preventing glomerulosclerosis in diabetic kidney disease in mice [36]. 5. Aptamer-based immunological methods Aptamers have been utilized in many analytical methods as highly specific reagents [10,37]. A good example is the usage of DNA aptamers in biomarker discovery which enables the measurement thousands of proteins in small serum volumes [38]. Aptamers were also applied successfully in various immunological assays. A sensitive Enzyme Linked Aptamer Assay (ELAA) was developed for detection of thrombin using an aptamer specific to this protein [39]. A competition variant of the method is characterized by high sensitivity (1.8 nM thrombin) and short assay time (1.5 h). Please cite this article in press as: http://dx.doi.org/10.1016/j.imlet.2014.10.001
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RNA aptamers have also been used to detect rabbit IgG [19]. The aptamers were specific to the constant part of native IgG but did not bind the SDS-denatured form of the protein. One of the aptamers, R18, required magnesium ions for binding to rabbit IgG. After Mg ions are removed by EDTA, bound IgG molecules were released from immobilized R18. R18 columns could be used for mild isolation of rabbit IgG, similar to that described for the purification of human IgG by RNA aptamer Apt8-2 (see Section 1). A fluorescence-labeled DNA aptamer specific to IgE was used to detect this immunoglobulin by means of fluorescence polarization [20]. Two fluorescent dyes – fluorescein and Texas red – were applied and the latter fluorophore was found to be more sensitive. The labeling did not significantly influence the affinity of the aptamer. The detection of IgE was rapid and sensitive. A new, involving DNA aptamers method was developed for detection of antibodies to Toxoplasma gondu [39]. A sandwich complex was formed from two different aptamers specific to this infectious agent and anti-T. gondu antibodies. One aptamer immobilized on microplates was used for antibody capture as the other one labeled by biotin was a detection biosensor.
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Aptamers are often compared with antibodies as both display high specificity and affinity [11]. However, they are characterized by fundamental differences: antibodies are large protein molecules produced by immune cells whereas aptamers are products of chemical synthesis. Aptamers are much cheaper, can easily be produced and are not contaminated by viruses, are non-immunogenic and due to their small size, can penetrate more easily into cells as well as into solid tumors. Nevertheless, aptamers, small-sized molecules have shorter half-life due to renal filtration and are susceptible to nuclease degradation if not well protected. Application of aptamers in immunological research has revealed information of general interest. Detailed X-ray structural studies of an RNA aptamer in complex with the Fc fragment of human IgG [14,15] demonstrates the complex is formed from many weak interactions like hydrogen bonds and van der Waals contacts. Multiple such contacts could explain the high specificity and affinity of aptamers in formation of complexes with proteins. It was also shown that divalent ions are important for supporting aptamer structure and maintaining its specificity. Removing these ions disrupts formation of the protein–aptamer complex, implying that the relatively mild procedure for protein isolation could be applied for protein isolation. In recent years intensive studies of the interactions of aptamers with cell surface receptors have been undertaken. Initially, aptamers were used as modulators of cellular activity, acting as antagonists blocked the reaction of ligands with cell receptors, or on the contrary, stimulated cell reactions as agonists. Such investigations were related to the activation of T cells and the stimulation of the anti-tumor immunity. Polyvalent aptamer derivatives (dimers, trimers and tetramers) were more effective than monomers. The effect could be attributed to the crosslinking cell receptors. Recently it was also shown that a trimeric DNA aptamer to human epidermal growth factor receptor 2 reduced tumor growth more effectively than monomers [41]. In addition, aptamers were used in chimeras, constructed for delivery of inhibitors or stimulators of cellular activities targeting specific cells like a “guided bullet” [42,43]. In these constructs, aptamers to cell surface proteins transported active components to defined cellular targets such as tumor cells. A good example of the effectiveness of this technique involves the inhibition of prostate tumor growth by heterodimers composed of siRNA and the aptamer to an antigen located on the tumor cell surface [30].
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Aptamers are specific binding molecules that can influence cellular activity following their reactions with cell receptors. We can appreciate that next years they would be useful more widely in clinical practice as specific drugs for treatment of various diseases such as autoimmune disorders and cancer. Aptamers serve as recognition elements that are applied in diagnostic assays due to their high specificity and affinity to various targets. Aptamers are also effectively used in immunological methods especially after labeling with reporter groups such as fluophores or biotin. As powerful tools they can replace antibodies in many applications for example as detection elements in blotting experiments. It is anticipated that in the coming years, aptamers will also see wider use in clinical practice as drugs of choice in the treatment of various diseases such as autoimmune disorders and cancer. Uncited reference [40]. References
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
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Review
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Aptamers in immunological research
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Roald Nezlin ∗ Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel
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Article history: Received 23 September 2014 Accepted 5 October 2014 Available online xxx
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Keywords: Aptamers Complexes of aptamers with immunoglobulins Modulation of immune reactions Reactions of aptamers with cell receptors Tumor immunology Aptamers in immunoassays
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1. Introduction
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In recent years aptamers, synthetic DNA or RNA single-chain oligonucleotides, have been used in various immunological studies to bind specific ligands. Detailed data on the interactions of an RNA aptamer with a human Fc fragment were obtained by X-ray crystallography. The complex formation involves multiple weak interactions that resemble protein–protein interactions. Aptamers specific to cell surface receptors may serve as antagonists or agonists blocking or stimulating cell activities. As aptamers can modify T-cell reactions, they could be useful in the treatment of chronic diseases such as autoimmune and oncological pathologies. In chimeras constructed for the delivery of active substances to defined targets, aptamers specific to surface proteins may be used to transport constructs directed to targets such as tumor cells. Aptamers are also employed as highly specific reagents in immunological assays after being labeled with reporter groups such as fluorescent dyes or following immobilization on insoluble carriers such as membranes or microspheres. © 2014 Published by Elsevier B.V.
Aptamers (Latin aptus – fitting) are short single-stranded RNA or DNA oligonucleotides that bind specific ligands with high affinity and specificity. Small molecules such as organic dyes, nucleotides, amino acids, nucleotides, and drugs; and biopolymers such as peptides, polysaccharides, and proteins can all be bound by aptamers. The reactions of substrates with aptamers occur in solution as well as on solid surfaces (e.g. cell surfaces). In recent years such properties of aptamers have enabled their application to various biomedical, as well as pharmaceutical and clinical purposes. The formation of complexes with aptamers involves various types of interactions (hydrogen bonding, polar groups, stacking interactions, and shape complementarity). Aptamers in solution are unstructured; association with their ligands causes them to fold into arrangements in which the ligand becomes an intrinsic part of the nucleic acid structure [1]. Aptamers are obtained by a selection procedure known as SELEX (Systematic Evolution of Ligands by Exponential enrichment) [2,3]. The SELEX process is characterized by repeated cycles of selection and enzymatic amplification steps, yielding RNA or DNA oligonucleotides that react specifically and with high affinity with their corresponding ligands.
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Intensive studies of aptamers began in the 1990s. Threedimensional structures for complexes of aptamers with their ligands were determined, and the molecular basis of aptamer specificity was clarified [4]. It was shown that aptamers can be applied as potential therapeutic [5,6] and diagnostic agents and biosensors [7]. During the past decade studies of aptamer properties and activity intensified as reflected in numerous publications. Several effective modifications of the SELEX technique were incorporated into the process. Chemical modifications of aptamers were introduced, to prevent their degradation by nucleases. Other methods were developed to combine aptamers with nanoparticles, and to use such conjugates for detection of cancer cells and for drug delivery [8,9]. As stated above, aptamers bind many ligands with high affinity which differ in chemical structure and they are widely utilized in various analytical methods [10]. At the end of 2004 the US food and Drug Administration approved an RNA aptamer as therapy to treat of choroidal neovascularization, a form of age-related macular degeneration. As a therapeutic agent it targets vascular endothelial growth factor [6]. Several other aptamers are currently undergoing clinical trials as therapeutic agents for a variety of oncogenic and non-oncogenic diseases [11–13]. In recent years aptamers have more often been used in immunological research. Interactions of aptamers with immunoglobulin molecules in solution as well as on the cell surface have been examined. Today, aptamers tend to be more widely applied for the modulation of immune reactions as well as in immunological
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methods. In the present review, the results obtained in these studies will be briefly discussed. 2. Interaction of aptamers with immunoglobulins Japanese scientists studied in detail the interaction of an RNA aptamer with human IgG [14,15]. The aptamer Apt8-2, a 23 nucleotide-long molecule, containing 2 -fluoropyrimidines reacted with high affinity to the Fc fragment of human IgG of all four subclasses but was not able to bind the IgG of any animal species. Apt8-2 competed with bacterial protein A for Fc binding but not with cellular Fc-receptors. The reaction of Apt8-2 with Fc was observed only in the presence of the divalent ions Mg2+ and Ca2+ . Removing these ions by means of EDTA-buffer solution released the aptamer bound to IgG molecules. For the release of IgG from protein A, acid buffers are usually used. However, acidic conditions could be harmful to IgG antibodies. Therefore, the use of the Apt8-2 aptamer for the purification of IgG is preferable, as the separation of IgG from Apt8-2 can be performed by neutral buffers containing EDTA. In this study the complex containing Apt8-2 aptamer and the Fc fragment of human IgG1 was crystallized and its structure determined at 2.15 A˚ by X-ray crystallography. Formation of the complex did not change the structure of the protein. The interaction of the aptamer with Fc involves multiple weak interactions as hydrogen bonds and van der Waals contacts. Electrostatic forces did not play significant role in the Apt8-2-Fc complex formation. The complex resembles protein–protein complexes rather than nucleic acid–protein interactions. It was found that divalent ions support a specific aptamer structure, which is important for its specificity. Two amino-acid residues – Gln342 and Phe404 – which are present in all subtypes of human Fc-IgG, played an important role in the contacts with Apt8-2. One of them, Gln342, is located in a loop between the C␥2 and C␥3 domains. The protein A binding site is also located at the C␥2–C␥3 interface (for a review see [16]) which explains the fact that both polymers compete for Fc-IgG binding. Aptamers specific to other immunoglobulins have also been obtained. DNA and RNA aptamers with specificity to the Fc region of mouse IgG subclasses and rabbit IgG were isolated using SELEX [17–19]. A DNA aptamer with specificity to human IgE has also been identified [20]. 3. Reaction of aptamers with cell receptors During the past decade aptamers have been applied to modify the activity of cells participating in immune reactions. Aptamers can block cell receptors and inhibit their reactions with corresponding ligands; other aptamers in contrast are active as agonists promoting cell reactions. Gilboa and colleagues synthesized several aptamers that can modulate immune responses. In a series of experiments they used aptamers specific to CD28, a co-stimulatory receptor, which is responsible for activation of T-lymphocytes. The CD28 ligand (B7) is located on activated antigen-presenting cells. One of these aptamers blocked binding of B7 to CD28. In contrast, dimers of anti-CD28 aptamers introduced a co-stimulatory signal that acted as an agonist and enhanced the cellular antitumor immune response. Anti-CD28 aptamers were also able to enhance immune responses induced by idiotype vaccines used to treat murine lymphoma [21]. In other experiments aptamers specific to T-cell receptors – an inhibitory CTLA-4 and co-stimulatory 4-1BB were applied [22–24]. The aptamer to the first receptor blocked the inhibitory activity of T cells, and a tetrameric derivative of the same aptamer promoted protective, T-cell dependent anti-tumor immunity in murine tumor Please cite this article in press as: http://dx.doi.org/10.1016/j.imlet.2014.10.001
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models. Dimers of aptamers to the 4-1BB receptor which contribute to the expansion of activated T cells induced rejection of tumors in mice similarly to agonistic anti-4-1BB monoclonal antibodies. In control experiments non-specific aptamers had no inhibitory effect. An RNA aptamer specific to the OX40 receptor was used to stimulate proliferation and cytokine production by T cells [25]. OX40 is a member of the tumor necrosis factor (TNF) receptor superfamily. Aptamer monomers were found to be inactive because the receptor has to be dimerized on the cell surface in order to act as a receptor agonist. To prepare a receptor-activating form of the aptamer, a DNA oligonucleotide scaffold was used to connect two aptamer monomers in close proximity. This construct was able to bind OX40 on murine cells and to crosslink receptor monomers. The resulting reaction induced cytokine production and proliferation of antigenstimulated lymph-node cells. The agonistic aptamer was shown to be effective in the treatment of aggressive metastatic melanoma in mice. Interleukin 10 (IL-10) is a multifunctional cytokine that functions as an important immune inhibitor of inflammation in chronic infections and cancer. Suppression of IL-10 activity by blocking the corresponding surface cell receptor IL-10R by means of an RNA aptamer inhibits the development of chronic infections and tumor growth. The aptamer to IL-10R was as effective as anti-IL-10R antibodies in eliminating viral infection and cancer development in mice [26]. Dimers and tetramers of the anti-IL-10 aptamer have enhanced binding affinity and biological activity comparable to that of anti-IL-10 monoclonal antibodies. Aptamers specific to cell membrane proteins were used to construct chimeras that could target cells and stimulate their inhibitory or stimulatory activity. Such an approach was applied to inhibit the mTOR1 complex, a regulator of cell growth, in circulating CD8+ T cells [27]. The construct was composed of an aptamer to the 4-1BB T-cell receptor, coupled with a small interfering RNA (siRNA), which specifically downregulated the mTOR1 complex. As a result CD8+ T-cell memory was enhanced and protective antitumor immunity in mice was increased. Administration of the construct was more effective than use of rapamycin, an agent that specifically inhibits mTOR1 activity. Successful inhibition of tumor growth and rejection in mice was achieved by a bispecific aptamer composed of the aptamer to the 4-1BB receptor and an aptamer specific to prostate specific membrane antigen (PSMA) [28]. As T-cell activation causing the stimulation of 4-1BB occurred near the tumor site, the dose of the therapeutic bi-specific aptamer could be reduced. In this way, two barriers of successful cancer therapy could be overcome, by diminishing drug toxicity and avoiding autoimmune reactions that could develop during immunotherapy. The anti-tumor immune response is weak, as tumors do not produce strong rejection antigens. To stimulate anti-tumor immunity by inducing new potent tumor antigens, inhibiting the activity of the nonsense-mediated decay (NMD) complex was suggested [29,30]. This complex comprised of several factors, prevents the formation of mRNA with a premature termination codon. The small interfering RNA (siRNA) specific to the NMD factors inhibits their activity which in turn stimulates the expression of peptide products with new antigenic properties. As NMD is active in all body cells, siRNA must be delivered directly to cancer cells. For this purpose, RNA heterodimers composed of siRNA and an RNA aptamer targeted to tumor antigen PSMA were prepared. The construct inhibited tumors displaying PSMA antigen on the cell surface in vivo in a mouse model. Tumor rejection was immune-mediated as it was dependent on both CD4+ and CD8+ T cells and not observed in immune deficient mice. A multimeric DNA aptamer specific for membrane-bound IgM (mIgM) was constructed from a 37 nucleotide long monomer [31]. Monomer units were connected with flexible polyethylene glycol (PEG) linkers. Trimeric and tetrameric DNA aptamers bound
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effectively to mIgM but not to soluble serum IgM. Membrane IgM molecules have a membrane portion of mu-chains, absent in soluble IgM. Fifteen amino-acids of this portion are located extracellularly; this sequence could therefore serve as a target for the DNA aptamer. B cells from healthy people and CD3 T cells rarely bound the DNA aptamer. B-cells from several lymphoma cell lines and B cells, obtained from leukemia patients, however, did react with the DNA aptamer. Polyvalent DNA aptamers with cell receptor specificities may be useful as drug-carriers via endocytosis and may also have wider clinical applications as blockers of cell receptors, preventing reactions with ligands that trigger cell division. The stimulation of immune cells by ligands, agonists of Toll-like receptor 9 (TLR9) has been described [32,33]. Localized primarily on B cells and plasmacytoid dendritic cells (pDC) this receptor is one of several involved in the innate immune system. It recognizes unmethylated cytosine-phosphate-guanine (CpG) dinucleotides that are commonly seen in bacterial and viral DNA but are inactive in vertebrate DNA, due to methylation. Interactions of ligands with the CpG motif with TLR9 induces conformational changes in the receptor, which in turn, stimulates both innate and adaptive immune responses, resulting in the activation of specific B cells and pDC as well as promoting of the development of T cells. Therefore, CpG oligonucleotides are potentially effective drugs for the treatment of various pathologies including cancer, allergies and infections and may also enhance effectiveness of vaccines. CpG oligonucleotides used in combination with other therapies are likely to be even more effective. 4. Complexes of aptamers with soluble proteins The C5 complement component, a part of the innate immune system, plays an important role attacking various infectious agents. C5a and C5b, proteolytic fragments of C5, are highly active inflammatory proteins. C5a is responsible for systemic inflammation in several pathologies, particularly in sepsis and autoimmune diseases. Several aptamers specific to complement component C5 and its proteolytic fragment C5a were synthesized. RNA aptamers to human and rat C5 effectively inhibited activity of this component. The binding of these aptamers to C5 also prevented proteolytic cleavage of C5 on C5a and C5b [34]. Aptamers resistant to nucleases were synthesized with unnatural oligonucleotides. An RNA/DNA aptamer NOX-D20 was prepared from l-oligonucleotides (l-ribose/l-deoxyribose nucleotides). NOX-D20 inhibited the activity of human and mouse C5a [35]. In an experimental mouse model of sepsis NOX-D20 effectively prevented pathology progression and improved survival of the mice. Non-natural RNA l-oligonucleotides with specificity to murine CCL2 (pro-inflammatory chemokine monocyte-chemoattractant protein-1) improved kidney status in a mouse model of diabetic nephritis. An aptamer specific to the homeostatic chemokine CXCL12 and the aptamer to CCL2 had additive protective effects in preventing glomerulosclerosis in diabetic kidney disease in mice [36]. 5. Aptamer-based immunological methods Aptamers have been utilized in many analytical methods as highly specific reagents [10,37]. A good example is the usage of DNA aptamers in biomarker discovery which enables the measurement thousands of proteins in small serum volumes [38]. Aptamers were also applied successfully in various immunological assays. A sensitive Enzyme Linked Aptamer Assay (ELAA) was developed for detection of thrombin using an aptamer specific to this protein [39]. A competition variant of the method is characterized by high sensitivity (1.8 nM thrombin) and short assay time (1.5 h). Please cite this article in press as: http://dx.doi.org/10.1016/j.imlet.2014.10.001
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RNA aptamers have also been used to detect rabbit IgG [19]. The aptamers were specific to the constant part of native IgG but did not bind the SDS-denatured form of the protein. One of the aptamers, R18, required magnesium ions for binding to rabbit IgG. After Mg ions are removed by EDTA, bound IgG molecules were released from immobilized R18. R18 columns could be used for mild isolation of rabbit IgG, similar to that described for the purification of human IgG by RNA aptamer Apt8-2 (see Section 1). A fluorescence-labeled DNA aptamer specific to IgE was used to detect this immunoglobulin by means of fluorescence polarization [20]. Two fluorescent dyes – fluorescein and Texas red – were applied and the latter fluorophore was found to be more sensitive. The labeling did not significantly influence the affinity of the aptamer. The detection of IgE was rapid and sensitive. A new, involving DNA aptamers method was developed for detection of antibodies to Toxoplasma gondu [39]. A sandwich complex was formed from two different aptamers specific to this infectious agent and anti-T. gondu antibodies. One aptamer immobilized on microplates was used for antibody capture as the other one labeled by biotin was a detection biosensor.
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Aptamers are often compared with antibodies as both display high specificity and affinity [11]. However, they are characterized by fundamental differences: antibodies are large protein molecules produced by immune cells whereas aptamers are products of chemical synthesis. Aptamers are much cheaper, can easily be produced and are not contaminated by viruses, are non-immunogenic and due to their small size, can penetrate more easily into cells as well as into solid tumors. Nevertheless, aptamers, small-sized molecules have shorter half-life due to renal filtration and are susceptible to nuclease degradation if not well protected. Application of aptamers in immunological research has revealed information of general interest. Detailed X-ray structural studies of an RNA aptamer in complex with the Fc fragment of human IgG [14,15] demonstrates the complex is formed from many weak interactions like hydrogen bonds and van der Waals contacts. Multiple such contacts could explain the high specificity and affinity of aptamers in formation of complexes with proteins. It was also shown that divalent ions are important for supporting aptamer structure and maintaining its specificity. Removing these ions disrupts formation of the protein–aptamer complex, implying that the relatively mild procedure for protein isolation could be applied for protein isolation. In recent years intensive studies of the interactions of aptamers with cell surface receptors have been undertaken. Initially, aptamers were used as modulators of cellular activity, acting as antagonists blocked the reaction of ligands with cell receptors, or on the contrary, stimulated cell reactions as agonists. Such investigations were related to the activation of T cells and the stimulation of the anti-tumor immunity. Polyvalent aptamer derivatives (dimers, trimers and tetramers) were more effective than monomers. The effect could be attributed to the crosslinking cell receptors. Recently it was also shown that a trimeric DNA aptamer to human epidermal growth factor receptor 2 reduced tumor growth more effectively than monomers [41]. In addition, aptamers were used in chimeras, constructed for delivery of inhibitors or stimulators of cellular activities targeting specific cells like a “guided bullet” [42,43]. In these constructs, aptamers to cell surface proteins transported active components to defined cellular targets such as tumor cells. A good example of the effectiveness of this technique involves the inhibition of prostate tumor growth by heterodimers composed of siRNA and the aptamer to an antigen located on the tumor cell surface [30].
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Aptamers are specific binding molecules that can influence cellular activity following their reactions with cell receptors. We can appreciate that next years they would be useful more widely in clinical practice as specific drugs for treatment of various diseases such as autoimmune disorders and cancer. Aptamers serve as recognition elements that are applied in diagnostic assays due to their high specificity and affinity to various targets. Aptamers are also effectively used in immunological methods especially after labeling with reporter groups such as fluophores or biotin. As powerful tools they can replace antibodies in many applications for example as detection elements in blotting experiments. It is anticipated that in the coming years, aptamers will also see wider use in clinical practice as drugs of choice in the treatment of various diseases such as autoimmune disorders and cancer. Uncited reference [40]. References
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