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Journal: Expert Opinion on Therapeutic Patents (EOTP) Title: Aptamers: from bench side research towards patented molecules with therapeutic applications ID: 431548 Corresponding author: Henning Ulrich AUTHOR: Please complete the colour figure order form that accompanies your proof if you require figures to appear in colour in the print version of the journal; once you have completed your article and credit card details, email or fax the form back to the production editor. The following queries have arisen during the editing of your manuscript. Please answer the queries by making the necessary corrections on the CATS online corrections form. Once you have added all your corrections, please press the SUBMIT button.

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Aptamers: from bench side research towards patented molecules with therapeutic applications

1. Introduction 2. Aptamer development for in vivo applications

Paromita Majumder, Katia Gomes Neves & Henning Ulrich† †Universidade

de São Paulo, Instituto de Quimica, Departamento de Bioquímica, São Paulo, SP, CEP 05508-900, Brazil

3. Aptamers: towards clinical applications 4. Therapy of age-related macular eye disease 5. The anti-von Willebrand factor aptamer ARC1779 6. REG-1: an aptamer–antidote system for anticoagulation 7. Other promising aptamers in clinical trials: the AS1411 or AGRO100 aptamers 8. The AX102 aptamer 9. Aptamers in diagnosis: the tenascin and prostate specific membrane antigen-binding aptamers 10. Aptamers and patents 11. Expert opinion

Background: RNA and DNA aptamers recognize their targets with high specificity and affinity. These aptamers can be developed against almost any target protein through iterative cycles of in vitro screening of a combinatorial oligonucleotide library for target binding. Aptamer sequences from the final pool of in vitro selection are screened for pharmacological activity and possible medical applications. Methods: Chemical modifications and improvements of the identification of aptamer selection procedures made aptamers rival antibodies in diagnostic and therapeutic applications. This article reviews recent literature and patents and discusses the properties of aptamers as high-affinity and specificity target binders as well as their stability in biological fluids that turns them into therapeutic agents. Conclusion: The development of aptamers into compounds with therapeutic and diagnostic compounds has resulted in patents protecting the sequences and the use of these oligonucleotides. Several of these patented aptamers are currently being tested in Phase I or II clinical trials. Moreover, an anti-VEGF aptamer has already been approved by the FDA for treatment of age-related macular degeneration in humans. Keywords: aptamers, cancer, cardiovascular system, clinical applications, patents Expert Opin. Ther. Patents (2009) 19(11):1-11

1. Introduction

Synthetic and natural compounds, monoclonal antibodies and combinatorial synthesized aptamers are being developed as site-specific drugs to bind to proteins that are overexpressed or reveal altered activities in disease states. Antibodies have been considered for a long time as promising therapeutic candidates for the treatment of cancer and other diseases by blocking growth factor receptor interaction involved in proliferation induction [1]. For instance, the monoclonal antibody Trastuzumab has been employed for treatment of patients with human epidermal growth factor receptor 2 (HER2) tyrosine kinase positive metastatic breast cancer [2,3]. The anti- VEGF human monoclonal antibody bevacizumab (Avastin®, Genentech) was effective in combination therapy of recurrent malignant gliomas [4]. As alternative to monoclonal antibodies, high-affinity and specificity capture agents can be identified by screening of combinatorial oligonucleotide libraries (RNA and DNA) for binding affinity against a given target protein. The identified target binders, denominated aptamers, have similar properties to those of monoclonal antibodies such as high specificity of recognition and high-affinity binding. There are some obvious advantages of aptamers, also called ‘chemical antibodies’, compared to use of monoclonal antibodies in therapeutic applications. Aptamers are developed using in vitro selection procedures and can be reproduced by

10.1517/13543770903313746 © 2009 Informa UK Ltd ISSN 1354-3776 All rights reserved: reproduction in whole or in part not permitted

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enzymatic reactions. They can be developed against almost every target including toxins and molecules that do not elicit immune responses. Moreover, due to their low molecular mass and structural flexibility, aptamers may bind to epitopes against those antibodies cannot be raised. Their low immunogenic potential and production by chemical or enzymatic reactions without the presence of cell contaminants are further advantages for the use of aptamers as therapeutics when compared to antibodies. Unlike antibodies, aptamers do not require humanization procedures during their development. Furthermore, since aptamers can be truncated to small biological-active sequences and modified for optimization of pharmacokinetics and target accessibility, they combine the advantages of therapeutic antibodies and small molecule-based drugs [5-8]. As will be discussed later in this review, aptamers with therapeutic applications in pathologic neovascularization, cardiovascular disease and cancer have entered preclinical and clinical evaluation. Therefore, the use of these aptamers has been protected by patents. 2. Aptamer

development for in vivo applications The SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique developed in the early 1990s simultaneously by Tuerk and Gold and Ellington and Szostak [9,10] is a combinatorial library approach based on iterative enzymatic amplifications of a partially randomized nucleic acid library where each amplification round is followed by an in vitro selection cycle against a target molecule. The original DNA template synthesized by organic phosphoramidite chemistry consists of an inner random sequence varying from 16 to 100 positions in which all four deoxyribonucleotides are incorporated with equal probabilities. This inner region is flanked by two constant regions for primer annealing and PCR amplification as well as by restriction enzyme sequences for cloning purposes. The amplified DNA template can now be denatured into its single strands for DNA aptamer selection or is in vitro transcribed into RNA, for RNA aptamer selection. Single-stranded DNAs or RNAs are induced to fold into secondary and tertiary structures and are then exposed to their target molecules, that is, peptides, purified proteins, proteins embedded in their membrane environment, whole plasma membranes or even entire cells or organisms [11-13]. Following removal of unbound DNA/RNA molecules, target binders are recovered by adding excess concentration of a competitor that binds to the desired target site of a protein. Following elution, the recovered DNA/RNA molecules are amplified by PCR or RT-PCR to originate the pool used for the next selection round against their target (Figure 1). The heterogeneity of DNA/RNA pools decreases with the progress of SELEX cycles, as selected species are being enriched in the SELEX pools. Consequently, binding affinities of the 2

selected pools to their target protein increase along the SELEX process. In vitro selection is complete when no further increase of binding affinity to the target is detected, indicating that the previously random pool has been purified to homogeneity. This final selection pool is cloned and sequenced, and the obtained sequences are searched for homologies in their previously random regions. Functional sequences are individually characterized by their target recognition and binding affinity. Instead of elution of target-bound DNA/RNA molecules by specific competitors, other strategies can be used for recovery of bound molecules. Alternating the exposure of the SELEX library to the surface of cells (i.e., cancer cells) containing the target molecule and to cell surfaces (healthy somatic cells) that do not express the target protein allows the selection of aptamers, which specifically recognize the molecular signature of a cancer cell, but do not bind to a healthy cell. This novel approach, also named Cell SELEX [7], has led to the development of aptamers as promising agents for diagnosis of cancer cells and as vehicles for targeting cancer cells by toxins or RNA interference [14,15]. Cell SELEX has also been used for the development of aptamers specifically binding to mesenchymal stem cells [16]. Moreover, aptamers binding to endothelial progenitor cells were coupled to polytetrafluoroethylene and polydimethylsiloxan surfaces. This coating promoted an anti-thrombogenic matrix and complete endothelialization by binding a sufficient number of endothelial progenitor cells and could serve as intelligent biomimetic aptamer implant for the enrichment of endothelial progenitor cells at the localization of vessel lesions [17]. However, as discussed by Li et al. [18], the selection of the aptamers against cell surface biomarkers is not a trivial procedure. The complexity and diversity of the cell surface proteome, the dependence on the activity of the aptamers in relation to the original selection conditions and the control of nonspecific binding to other sites than the target protein are critical points that need to be carefully studied. For in vivo use, DNA and RNA aptamers can be conveniently protected against degradation by nucleases present in biological fluids. For instance, modified SELEX procedures with chemically modified oligonucleotides revealed increased half times in body fluids from 8 sec [19] in case of unmodified RNA molecules to ≤ 48 – 72 h in case of 2′-fluoro or 2′-amino pyrimidine-modified oligonucleotides [5,20]. Stability of RNA molecules can be even further increased by introducing 2′-O-methyl (2′-OMe) modifications at the 2′-hydroxyl position of purines or by utilization of Spiegelmers (l- instead of d-enantiomers) [21]. For diagnostic purposes, aptamers can be either internally labeled with fluorescent bases [22] or a fluorescence reporter can be attached to the 5′ end of the oligonucleotide [5]. Aptamers as promising agents for diagnostics and therapeutics have now been identified against soluble proteins, intracellular proteins and cell surface antigens. Their

Expert Opin. Ther. Patents (2009) 19(11)

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Majumder, Neves & Ulrich

Random library synthetized by phosphoramidite chemistry

ssDNA or RNA library

dsDNA library Constant Random Constant

RT-PCR/PCR Membrane proteins ssDNA or RNA library enriched for binders to target binding

Cells

Target presentation

Cytosolic proteins

Selected aptamer

Validation

Antigens

Clinical applications

Figure 1. In vitro selection and development of aptamers into therapeutics. The chemically synthesized combinatorial DNA library is extended to double-stranded (ds) DNA. The PCR-amplified dsDNA is then either in vitro transcribed to RNA (in this case a T7-promoter sequence is incorporated in one of the constant primer binding sites) or denatured into its single strands. The single strand DNAs or RNAs folded into unique structural motifs are exposed to their selection targets (proteins or nonprotein antigens or intact cancer cells). Targetside bound DNA/RNA molecules are eluted and used for RT-PCR/PCR amplifications in order to restore the next generation DNA pool. Following various iterative SELEX (Systematic Evolution of Ligands by EXponential Enrichment) cycles, the final DNA pool is sequenced, and obtained aptamers are screened for their pharmacological properties. Aptamers are validated in animal models, and aptamer sequences and their applications are patented before submitting them to clinical tests.

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properties have been described in several patents (Table 1). The application for a patent follows the identification of the aptamer, the validation of its biological activity in vitro and in vivo prior to evaluation of its therapeutic performance in clinical trials (Figure 1). Pharmacologically useful aptamers, including oligonucleotides specifically binding to VEGF [23], platelet-derived growth factor (PDGF) [24], prostate-specific membrane antigen (PSMA) [25], C5 complement [26], von Willebrand factor, factor IXa [27] and anthrax spores [28] are protected by patents [29-42]. 3. Aptamers:

towards clinical applications

The idea of using nucleic acids as drug therapy agents is not new. In November 1998, Vitravene (Novartis Ophthalmics) was approved by FDA, thereby becoming the first nucleic acid drug available. This drug is an oligonucleotide discovered by Isis Pharmaceuticals using the antisense technique for the treatment of cytomegalovirus retinitis in persons suffering from AIDS [43]. In addition to the development of aptamers to study target-protein function in basic research, aptamers have been developed for different diagnostic purposes. They have been tested in protein arrays, ELISA and biosensor assays [8]. However, since its discovery, the SELEX technique aims to

identify aptamers for therapeutic purposes. One aptamer has already been approved for therapeutic applications, and several aptamers are being evaluated in ongoing clinical trials [44]. We will summarize some of the breakthroughs of these promising aptamers in blood therapy, pathological neovascularization and cancer therapy. 4. Therapy

of age-related macular eye disease

An RNA molecule binding to vascular VEGF-165, initially developed at the laboratory bench [23], turned into the first aptamer approved by the US FDA in December 2004 to reach the drug market for therapeutic purposes. This aptamer effectively antagonizes the action of VEGF-164/165, but does not bind to VEGF-120/121 [45-47]. Following various chemical modifications for improvement of stability and pharmacokinetics, the pegaptanib sodium aptamer (also known as Macugen, Pfizer-Eyetech Pharmaceuticals) has been shown to be effective for treatment of neovascular age-related macular degeneration (AMD), the leading cause of blindness in people > 50 years of age in developed nations [48]. Abnormal ocular vascularization results from metabolic stress that promotes the production of a high range of growth factors including VEGF. This growth factor can induce ocular neovascularization by increasing endothelial


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Table 1. Patented aptamers for therapy and diagnosis. Patent number

Assignee(s)

Inventor(s)

Subject

Drug name

US Pat. App 10/862084 (filed 4 June 2004)

Gilead Sciences, Inc.

Janjic N, Gold L

High-affinity VEGF receptor binding nucleic acid ligands and inhibitors of VEGF function

Macugen [29]

WO07103549

Archemix Corp.

Epstein D, Kurz JC

Complement binding aptamers and anti-c5 agents useful in the treatment of ocular disorders

ARC1905 or ARC187 [30]

US Pat. App 11/318227 (filed 22 December 2005)

Archemix Corp.

Benedict C, Diener J, Epstein D, Grate D, Keene SC, Kurz J, Kurz M, McCauley TG, Rottman J, Thompson K, Wilson C, Zoltoski AJ

Aptamer therapeutics for the treatment of complement-related disorders

ARC1905 or ARC187 [31]

US Pat. App 11/985827 (16 November 2007)

Antisoma Research Ltd

Green C, Jones D

Methods, compositions and kits for modulating tumor cell proliferation

Agro100/ AS1411 [32]

US Pat. App 12/322078 (29 January 2009)

Archemix Corp.

Diener JL, Lagasse HAD, Benedict C

Aptamers binding to von Willebrand factor and their use as thrombotic disease therapeutics

ARC1779 [33]

WO08150495

Archemix Corp.

Gilbert J, Hutabarat R, Schaub RG

von Willebrand factor aptamer formulations and methods for use

ARC1779 [34]

US Pat. App 11/805950 (filed 25 May 2007)

Regado Biosciences, Inc.

Rusconi CP, Tonkens RM

Administration of the REG1 anticoagulation system

Reg1 [35]

US Pat. App 11/789992 (filed 26 April 2007)

Duke University

Sullenger BA, Rusconi CP

Modulators of pharmacological agents

Reg1 [36]

US Pat. App 12/252994 (filed 16 October 2008)


Rusconi CP

Steady-state subcutaneous administration of aptamers

Reg1 [37]

US Pat. App 11/821183 (filed 22 June 2007)


Rusconi CP

Modulators of coagulation factors

Reg1 [38]

US Pat. App 11/666954 (filed 2 November 2005)

Archemix Corp.

Preiss JR, Wilson C, Stanton M, Diener JL, Epstein D, Grate D, McCauley T

Stabilized aptamers binding to PDGF and their use as oncology therapeutics

ARC127, ARC126, ARC3080 or AX102 [39]

US Pat. App 11/029949 (filed 4 January 2005)


Lupold SE, Lin Y, Hicke BJ, Coffey D

Nucleic acid ligands directed against the PSMA

Aptamer anti-PSMA [40]

US Pat. App 11/518321 (filed 8 September 2006)


Pietras K, Ostman A, Heldin C, Rubin K

Methods and compositions for the treatment of tumors using nucleic acid ligands binding to PDGF

Aptamer anti-PDGF [41]

US Pat. App 09/978,753 (filed 15 October 2001)

Conceptual MindWors, Inc.

Vivekananda J, Kiel JL

Methods and compositions for aptamers against anthrax

Aptamer anti-anthrax [42]

US Pat. App 11 982451 (filed 31 October 2007)

Antisoma Research Ltd

Trent JO, Bates PJ, Miller DM

Antiproliferative activity of G-rich oligonucleotides and method of aptamer binding to nucleolin

Agro100/ AS1411[98]

US Pat. App 11/300662(filed 13 December 2005)


Hicke B, Warren S, Parma D, Gold L

Tenascin-C binding nucleic acid ligands

Aptameranti-tenascin [99]

WO07025049

Archemix Corp.

Diener JL, Wagner-Whyte J, Fontana D

Aptamers that bind thrombin with high affinity

NU172 [100]

WO08028534

Bayer Schering Pharma Aktiengesellschaft

Friebe M, Hecht M, Dinkelborg L, Lehmann L, Noll B

Aptamers labeled with gallium-68

Aptameranti-tenascin [101]

PDGF: Platelet-derived growth factor; PSMA: Prostate-specific membrane antigen.

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cell mitosis [49,50], acting as chemotactic agent for bone marrow derived endothelial cell precursors [51,52], as survival factor against apoptosis [53] and as inducer of blood vessel extravasation [54]. VEGF action involves both upregulation of matrix metalloproteinases and decreased release of metalloproteinase inhibitors [55] as well as elevated expression of intracellular adhesion molecule-1 and adhesion of leukocytes to blood vessels [47,56]. A VEGF isoform (VEGF165) was chosen as a SELEX target due to its well-established role as regulator in pathological angiogenesis, in tumor genesis, ocular neovascular disease and inflammatory processes as well as in rheumatoid arthritis [57,58]. Three different SELEX procedures were performed in order to develop Macugen as a therapeutic VEGFtargeting aptamer molecule. Jellinek and coworkers [57] first showed that it is possible to identify aptamers that block VEGF–receptor interaction. These first aptamers served as a proof of principle but had no therapeutic relevance as they had not been protected against nuclease activity. Therefore, new SELEX enrichments were performed utilizing a RNA library containing 2′-amino and then 2′-fluoropyrimidines [59,23], the latter one originating the aptamer Macugen consisting of a truncated 24 nucleotide minimal sequence. The use of 2′F-substituted nucleotides resulted in a higher affinity of the selected RNAs to their target during SELEX experiments and improved thermal stability when compared to the 2′-amino-modified RNA library. The selected RNA pool was cloned and based on structural characteristics, three aptamer families were identified with potential therapeutic applications. From these families, the t44-OMe aptamer (also known as NX1838 and EYE001 and later as Macugen and Pegaptanib) [45,60-62] was most effective in inhibiting vascular leakage from dermal microvessels following VEGF injection in guinea pigs [46]. The selected aptamer was further modified by the attachment of a 5′-linked 40-kDa PEG moiety. This modification led to an improvement of the inhibition of VEGF-induced vascular leakage to 83% despite the fact that aptamer-binding affinity to VEGF following PEG-coupling was reduced. Preclinical tests in human umbilical microvascular endothelial cells using Pegaptanib resulted in 50% inhibition of 125I labeled VEGF-receptor binding at 0.75 – 1.4 nM receptor concentration. Pharmacokinetics studies showed the stability of Pegaptanib in human plasma for > 18 h when aptamers were intravenously or subcutaneously injected in rhesus monkeys [62]. The half-life time, the plasma clearance and the detection in vitreous humor of the eye were evaluated in further studies including in patients with AMD, diabetic macular edema and ocular diseases. Available treatments for these patients before development of Macugen were restricted to cryotherapy and vitrectomy [63,64]. Aptamer ARC1905, a potent and selective antagonist of the factor C5 of the complement cascade, is currently being tested for its efficiency in inhibiting excessive activation of AMD complement. In the current Phase I clinical trial,

ARC1905 is being tested in patients suffering from wet AMD in combination with the anti-VEGF drug ranibizumab (Lucentis®, Genentech). A second clinical trial with dry AMD patients shall be initiated in the year 2009 [65]. E10030 is a PDGF-binding aptamer that leads to regression of neovascularization when used in combination with a VEGF inhibitor. The aptamer is being tested for the safety and tolerability of E10030 in combination with an antiVEGF agent in a Phase I clinical trial for the treatment of wet AMD [65]. 5. The

anti-von Willebrand factor aptamer ARC1779 Coagulation or clotting occurs following injury. Activation of the von Willebrand factor induces platelet adherence to damaged blood vessels. However, excessive activity of von Willebrand factor results in undesired arterial clots, thereby, restricting blood flow, which can lead to heart attack and stroke. An aptamer with antithrombotic activity was developed for binding to the A1 domain of von Willebrand factor, the ligand for the glycoprotein 1b receptor on platelet cell surfaces for blockade of arterial thrombogenesis. Possible therapeutic actions of ARC1779 include prevention of thrombus formation in acute coronary syndromes, von Willebrand’s disease and related disorders [66]. The intended use of this compound was in patients suffering from acute coronary syndromes and undergoing percutaneous coronary intervention (PCI). The first human evaluations (Phase I clinical trials) revealed monophasic binding of ARC1779 in ELISA experiments, an apparent elimination half-life of ∼ 2 h and aptamer distribution in central compartments [67]. Another trial with human volunteers was carried out to assess the safety and tolerability of ARC1779 and to measure the relationship between the administered doses of ARC1779 and the inhibition of plasma von Willebrand activity and platelet function. No serious adverse events were observed in the Phase I trials, however, one volunteer experienced an allergy-like reaction following a rapid bolus administration of ARC1779, resulting in a change of doses from IV bolus to lower dose IV bolus followed by infusion. At present, the aptamer is being evaluated in clinical trials for prevention of excessive blood clot formation resulting from increased von Willebrand activity in thrombotic microangiopathies (TMAs). A Phase IIa clinical trial in patients suffering from microangiopathies with excessive activity of von Willebrand factor was performed in order to verify antagonism of von Willebrand factor activity and to establish conditions of drug administration. Currently, a Phase IIb clinical trial is being planned for evaluation of the efficacy, safety and tolerability of ARC1779 in patients with TMA. Another aptamer, a thrombin inhibitor denominated ARC183 [68], was tested for therapeutic use during coronary


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artery bypass grafting in an open-label dose-escalation trial in 40 healthy volunteers by Archemix Corp. and Nuvelo, Inc. Preliminary results of this trial indicated that ARC183 induced a stable and dose-dependent anticoagulation activity that was reversible upon ceasing aptamer administration. However, further drug development of ARC183 was discontinued due to a poor dosing profile, and NU-172, a second generation molecule with increased pharmacological activity, was developed [69]. Experiments in animals suggested rapid onset and offset of NU-172-induced effects and avoidance of heparin-induced thrombocytopenia [70]. Detailed information on the status of ARC1779 and NU172 is available on the website of Archemix Corp. [65]. 6. REG-1:

an aptamer–antidote system for anticoagulation REG-1, consisting of the factor IXa-binding aptamer RB006 and the RB007 complementary oligonucleotide as antidote, has been developed as potential therapeutic molecules that act on the anticoagulation system. The aptamer RB006 was designed to improve the current therapy used in blood coagulation directed against formation of blood clots and bleeding by interacting with factor IXa [71]. The combination of the two molecules (aptamer and antidote complementary oligonucleotide) significantly reduces the bleeding risk; this is achieved by controlling the activity of the anticoagulation aptamer. The aptamer and its antidote were successfully used to replace heparin and protamine in a porcine cardiopulmonary bypass procedure model [72]. The aptamer pair may be useful in patients diagnosed with heparin-induced thrombocytopenia or in those that have insulin-dependent diabetes in which the insulin activity is modified. The aptamer–antidote pair demonstrated a stable blockade of factor IXa activity in the presence of the aptamer and restoration of factor IXa activity following administration of the antidote oligonucleotide. This aptamer revealed a prolonged duration of effect following chemical stabilization and conjugation to a 40-kDa polyethylene glycol carrier [73]. The median duration of RB006mediated effects was dose-dependent with ≤ 30 h in volunteers receiving 60 mg of the drug. The antidote oligonucleotide RB007 neutralized the pharmacodynamic effects exerted by RB006 within 1 – 5 min [73]. The REG-1 system was evaluated in three Phase I clinical studies regarding its safety and dose evaluation in human safety toleration tests [71,73,74]. No serious adverse effects, such as major bleeding, were observed. Meanwhile Regado started a multicenter, open-label, randomized Phase IIa clinical study that enrolled 26 patients undergoing elective PCI. The aim of the study was to determine whether REG1 can replace standard heparin therapy during a coronary balloon angioplasty dilatation and stenting procedure in patients at low risk for complications associated with therapy-related bleeding or heart attack [75]. 6

7. Other

promising aptamers in clinical trials: the AS1411 or AGRO100 aptamers

AS1411, also known as AGRO100, for treatment of cancer is the furthest advanced aptamer in clinical trials. This aptamer is a member of a novel class of antiproliferative agents known as G-rich oligonucleotides (GRO) as non-antisense, guanosinerich phosphodiester oligodeoxynucleotides forming stable G-quadruplex structures [76]. AS1411, a 26-mer unmodified GRO with in antiproliferation activity in vitro, has shown activity against human tumor xenografts in vivo. The mechanism underlying its activity in cancer growth inhibition seems to involve initial binding to cell surface nucleolin and internalization resulting in inhibition of DNA replication. In contrast to other unmodified oligonucleotides, AS1411 is relatively stable in serum-containing medium, probably as a result of the formation of a G-quartet-mediated dimer. Girvan et al. [76] reported the association of AS1411 with NF-κB, an essential modulator (NEMO) of cell growth. Aptamer binding affects the regulatory subunit of the inhibitor of κB kinase (IKK) complex, called IKK-χ. In the classic NF-κB pathway, the IKK complex is required for phosphorylation of IκB-α and subsequent activation of the transcription factor NF-κB. The treatment of cancer cells with AS1411 inhibited IKK-χ activity and reduced phosphorylation of IκB-α in response to TNF-α stimulation. NEMO is co-precipitated by nucleolin, indicating that both proteins are present in the same complex. Teng et al. [77] demonstrated that protein arginine methyl-transferase 5 (PRMT5), an enzyme that catalyzes the formation of symmetrical dimethylarginine, is a nucleolinassociated protein whose localization and activity are altered by AS1411. Levels of PRMT5 were found to be decreased in the nucleus when treated with AS1411 in DU145 human prostate cancer cells, but increased in the cytoplasm. These changes were dependent on nucleolin and were not observed in cells pretreated with nucleolin-specific small interfering RNA. The clinical Phase I study revealed promising aptamer activity in renal cancer and the absence of any significant adverse effects. In a group of 12 patients with advanced renal cancer, aptamer treatment revealed signs of antitumor activity with tumor regression in two cases. Further trials are ongoing with renal cancer and NSCLC [78]. Antisoma made an announcement to advance the development of AS1411 in acute myeloid leukemia (AML) based on positive data from a Phase II study with patients suffering from relapsed and refractory AML. Co-application of AS1411 with a high dose of cytarabine, a standard chemotherapy treatment, significantly enhanced response rates compared to administration of cytarabine alone. Further information on AS1411 is available on the Archemix and Antisoma websites [65,79]. 8. The

AX102 aptamer

A therapeutic aptamer inhibiting PDGF-B receptor binding is also being studied in preclinical trials [60,78,80]. This growth


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factor is involved in pericyte recruitment and provision of endothelial cell survival signals in cancer [81,82]. The utilization of the novel DNA aptamer AX102 resulted in a progressive reduction of pericytes in Lewis lung carcinoma cells. The inhibition of the PDGF-B receptor by AX102 led to regression of tumor vessels. However, obtained results depended on the respective tumor type [83]. The combination of antiVEGF aptamers and anti-PDGF inhibitors were shown to be more potent in inhibition of tumor growth when compared to effects induced by either of them individually, since neovessels became refractory during continuous absence of VEGF-A signaling [84]. 9. Aptamers in diagnosis: the tenascin

and prostate specific membrane antigen-binding aptamers

As already emphasized, aptamers are also being developed into diagnostic tools. As an example, a tenascin-binding aptamer is under development for cancer imaging by Schering AG. Matrix protein tenascin-C is detected by fluorescent and radiolabeled aptamer forms. The labeled aptamer recognizes solid tumors such as glioblastoma as well as breast, lung and colon cancer and is rapidly cleared from the blood and other nontargeted tissues [85]. In addition to the cited examples, many more aptamers will be optimized for diagnostic and therapeutic applications. Due to their high binding specificities, DNA aptamers have been selected against IgE [86] and IFN-γ [87] and have shown efficient interactions with their targets in cell cultures and animal models [88]. Aptamers recognizing rabbit IgG with binding affinities in the picomolar range may substitute secondary antibodies [89]. Ellington and his research group developed an aptamer database where some published experiments involving aptamers are catalogued [90,91]. Another aptamer with importance for tumor-diagnosis was raised against the prostate-specific membrane antigen (PSMA). Lupold and coworkers identified aptamers binding with affinities of 2 – 12 nM to the extracellular portion of PSMA expressed by prostate cancer cells. Aptamers were confirmed as biologically active since they inhibited PMSA N-acetyl-linked acid dipeptidase (NAALADase) activity. This inhibitory activity was still visible following aptamer truncation at its 5′- and 3′-termini. The highly specific aptamer construct recognizing LNCap human prostate but not PSMA-devoid prostate cancer cells [25] was developed in subsequent studies by Farokhzad to specialized nanoparticle– aptamer conjugates as drug delivery vehicles to prostate cancer cells [92]. As result of this study, these bioconjugates specifically targeted tumor cells and were taken up by LNCAP human epithelial cells expressing PSMA. Furthermore, antiPMSA aptamers were modified for carrying toxins or smallinterfering RNAs to prostate tumor cells [93,94]. As major achievement, in vivo studies revealed the efficiency of such aptamers in prostate tumor reduction in vivo. A single

intra-tumoral injection of docetaxel (Dtxl)-encapsulated nanoparticles containing the aptamer formulation resulted in complete tumor reduction in five of seven LNCaP xenograft nude mice [94]. 10. Aptamers

and patents

The SELEX technique has been constantly optimized to improve the method of selection for the development of aptamers with specific properties. Identified aptamer sequences, especially those with possible biomedical applications, have been protected by patents. In addition to aptamer sequences, improvements in selection procedures as well as chemical modifications such as addition of specific fluorescent tags or incorporation of modified nucleotides to enhance stability of aptamers and their pharmacokinetics have been published. Some of the aptamers are promising candidates in clinical trials and others efficiently bind to diagnostic targets. We have selected some of the patented aptamers that shall be tested or are already evaluated in clinical trials (Table 1) [95]. The use of aptamers has been extended from basic science in cellular processes and gene regulation to applications in therapy and diagnostics. Fomivirsen (Vitravene, Novartis) was the first antisense oligonucleotide for cytomegalovirus retinitis approved by US FDA at the end of the 1990s. Difficulties in the development of these agents into therapeutics due to instability in biological fluids or availability have been bypassed by the development of chemical modifications of oligonucleotides and advances in synthesis. Aptamers are one of the four classes of oligonucleotide reagents that also include antisense oligonucleotides, ribozymes and smallinterfering RNA. Aptamers differ from the other classes of oligonucleotide reagents in the sense that they do not lead to alterations of target gene expression, which could induce undesired changes of expression of related genes. Instead, they interact directly with the products of gene expression involved in disease and can specifically inhibit the activity of an isoform or a product of alternative splicing of a protein without affecting normal function of the remaining gene products. As already discussed throughout this review, aptamers with unique sequences can easily be evolved by in vitro selection and modified according to their respective therapeutic applications. The utilization of defined in vitro selection procedures as well as selected aptamers with their respective biological actions and therapeutic application have been protected by patents. 11. Expert opinion

The approval of Macugen is a breakthrough in the therapeutic use of aptamers, which is encouraging clinical testing of further aptamers directed against targets with implications in disease. While Macugen recognizes the isoform 165, but not the isoform 121 of VEGF A, the new monoclonal antibody


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drugs bevacizumab and ranibizumab that recognize all of the VEGF isoforms have turned out to be more effective in the treatment of AMD disease. The anti-PDGF aptamers ARC126 and ARC127 for treatment of the pathogenesis of proliferative retinopathies showed a significant reduction in epiretinal membrane formation in a transgenic mouse model. This animal model expresses PDGF-B in photoreceptors and develops severe proliferative retinopathy [96]. In this system imatinib mesylate, a competitive inhibitor of a few tyrosine kinases, including PDGF receptors, was inefficient. However, imatinib mesylate showed substantial antiproliferative activity in Philadelphia chromosome-positive acute lymphoblastic leukemia and gastrointestinal stromal tumors. Obviously, the efficiency of the aptamer or traditional drugs depends very much on the tumor type. However, one can imagine that the combination of the conventional therapy using a tyrosine kinase inhibitor and the anti-PDGF aptamer may generally improve therapeutic effects due to the highly specific activity of the aptamer in inhibiting PDGF–receptor interaction and the more general blocking effect of imatinib mesylate on tyrosine kinase receptors involved in disease progression. Without any doubts, an important contribution of the SELEX technique will be in the development of aptamers acting on proteins involved in disease states that so far cannot be successfully treated. The REG-1 aptamer is likely to gain unique importance as an anticoagulation agent due to the

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Declaration of interest The authors state no conflict of interest and have received no payment in preparation of this manuscript. HU Ulrich received grant support from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), Brazil. KG Neves’ doctoral thesis research is supported by a fellowship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

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Affiliation Paromita Majumder1,2 PhD, Katia Gomes Neves1 MSc & Henning Ulrich†1 PhD †Author for correspondence 1Professor, Universidade de São Paulo, Instituto de Quimica, Departamento de Bioquímica, São Paulo, SP, CEP 05508-900, Brazil Tel: +55 11 3091 3810 ext 223; Fax: +55 11 3815 5579; E-mail: [email protected] 2Venetian Institute of Molecular Medicine, Foundation for Advanced Biomedical Research, Via G. Orus 2, 35129 Padova, Italy

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