Radiolabelled Aptamers for Tumour Imaging and Therapy
By Perkins, A C Missailidis, S
Advances in biotechnology have led to new techniques for the design, selection and production of ligands suitable for molecular targeting. One promising approach is the production of specific receptor binding molecules based on defined nucleic acid sequences that are capable of recognising a wide array of target molecules. These oligonuclide ligands are known as aptamers. The technology that allows production of aptamer molecules is known as systematic evolution of ligands by exponential enrichment (SELEX). Using these techniques, aptamers can now be produced rapidly, inexpensively, and with high homogeneity. Furthermore, they are stable over long term storage at ambient room temperatures. A monomeric aptamer is small in size, with a molecular weight as low as 5 to 10 kDa. However, the aptamer molecule may be used as building block for custom designed targeting agents, offering several advantages. These molecules penetrate tumour readily, reach peak levels quickly and clear from the body rapidly, thus having properties of low toxicity and immunoreactivity. Previous work with radiolabeled aptamers is limited and is currently restricted to preclinical studies, but the body of evidence is steadily growing and aptamers are emerging as valuable clinical products for diagnostic imaging and therapy. We have shown that aptamers directed against the mucin 1 (MUC1) antigen, a tumour marker previously extensively used in tumour imaging and therapy, demonstrated high specificity and uniform penetration in tumour xenografts. The future strategy will be to manipulate the molecular weight of the molecules to achieve an optimum balance between the low immunogenicity and excellent tumour penetration for diagnostic imaging and targeted therapy. In this way, a balance can be achieved between the rapid renal clearance and adequate tumour uptake required for diagnostic imaging and targeted therapy. KEY WORDS: Aptamers, peptides – Radiopharmaceuticals – Neoplasms.
Molecular imaging is the combination of molecular biology and medical imaging, having the aim of targeting specific molecular recognition factors in vivo. In nuclear medicine, the production of custom designed synthetic peptides (e.g. techtides) for diagnostic imaging is a typical example. This approach has now been extended to the concept of molecular targeted therapy and can be described as the design, selection and isolation of ligand-binding molecules that may be used as vectors for receptor mediated therapeutics. Recent advances in this rapidly developing field have employed the use of biotechnological techniques for the production of synthetic targeting molecules that may be used for imaging and therapy. This new generation of molecules is known as aptamers and they are beginning to play an important role in medical diagnosis and treatment.
The word “aptamer” is derived from the Latin word aptus, meaning “to fit”. Aptamers are single or double stranded ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) oligonucleotide ligands usually less than 100 nucleotides in length, that are selected for high affinity and the specific molecular fit with their targets of interest. These molecules can be selected from random pools based on their ability to bind to other molecules, such as nucleic acids, proteins, small organic compounds and even entire cells.1-3 This process uses a chemically synthesised, random sequence library of the order of 1014 sequences of DNA or RNA oligonucleotides, flanked on each side by a known primer sequence, to select a functional oligonucleotide directed against a variety of molecular targets. The process of selection, known as systematic evolution of ligands by exponential enrichment (SELEX), results in the fittest or best binder for target recognition. This methodology involves the exposure of the target molecule to the library in order to allow for interaction of all binding aptamers with this target. The sequences that have not bound are washed away. The bound oligonucleotides are eluted via a chosen elution method, such as by temperature, alterations of pH, salt concentration or use of chaotropic agents. Selected aptamers are amplified using polymerase chain reaction (PCR) and interacted again with their target (Figure 1).
Figure 1.-A schematic representation of systematic evolution of ligands by exponential enrichment (SELEX) process.
TABLE I.-Molecular sizes of targeting molecules.
Various rounds of selection and amplification result in maturation of the ligands through competitive binding, selecting one or few high binding aptamer sequences. After the final round of the aptamer selection process, the PCR products can be cloned into a vector and sequenced to allow for identification of the best binding sequences, which can then be chemically synthesised. The size of a mono-aptamer molecule is between that of a single chain antibody fragment and a small peptide (Table I).
One particularly attractive feature of these synthetic molecules is that they can support site-specific modifications, whilst maintaining their structure and function. Furthermore, they may be linked together, thus enabling the construction of a custom designed targeting molecule of choice. An aptamer can be further modified for in vivo use by simple molecular alterations of such as incorporation of bioconjugates for the addition of biotin, fluorescent tags, radiometal chelators and drugs.4-8 Overall aptamers have the following characteristics: 1) aptamer production techniques are inexpensive, efficient and rapid; 2) aptamers can be manufactured with high reproducibility; 3) aptamers are highly stable: they are suitable for long term storage and can be transported at ambient temperatures; 4) aptamers are versatile molecules that can be easy modified; 5) aptamers are small in size, resulting in low immunogenicity and good tumour penetration when administered; 6) aptamers can be good as inhibitors, antagonists or regulators of pathways (e.g. VEGF, NX1838); 7) aptamers are attractive as high affinity molecular probes and sensor recognition molecules; 8) aptamers can be used as carrier and reporter molecules, for example with fluorophores and radionuclides.
Interest in the production of aptamers for medical application is growing rapidly and many new molecules are emerging. In many ways, aptamers are similar to antibodies, but they have many advantages as listed below. A useful resource of aptamer sequences, that may have diagnostic or therapeutic utility, is held by the Department of Chemistry and Biochemistry, Institute for Cell and Molecular Biology, University of Texas, Austin.9 This contains comprehensive sequence information on aptamers and unnatural ribozymes that have been generated by in vitro selection methods. The database is updated monthly and is publicly available at http://aptamer.icmb. utexas.edu/.
Aptamers in diagnosis and therapy
Aptamers have only recently entered into clinical use. One promising areas of therapeutic potential is with the use of anti- vascular endothelial growth factor (VEGF) aptamers.10 The first pharmaceutical aptamer formulation, Macugen(R) (pegaptanib sodium injection) was approved in the United States in December of 2004. This is an anti-VEGF aptamer formulation used for the treatment of neovascular agerelated macular degeneration. Pegaptanib sodium is a covalent conjugate of an oligonucleotide of 28 nucleotides in length that terminates in a pentylamino linker, to which two 20-kDa monomethoxy polyethylene glycol units are covalently attached via the two amino groups on a lysine residue. It is formulated as a sterile, aqueous solution for intravitreous injection. The regulatory approval of this formulation in the United States and Europe is encouraging, since this will lead the way for the acceptance of other clinical products. There is now a growing body of evidence that this formulation is proving to be of clinical benefit.
Similar developments are taking place in other areas, with the production of aptamers against targets, such as hepatitis B virus core protein, hepatitis C and human immunodeficiency virus (HIV).11- 13 Di Giusto et al.14 have described the design and construction of multivalent circular DNA aptamers for use in anticoagulation. Four aptameric binding motifs directed at blood-borne targets were used, from which larger, multidomain aptamers were constructed. The expectation was that by using circular DNA aptamers, half-lives in serum and plasma would be extended beyond 10 h, making such constructs viable for therapeutic and diagnostic applications. Duplexes and three-way junctions were used as scaffold architectures around which 2, 3, or 4 aptameric motifs were arranged in a structurally defined manner, giving rise to improved binding characteristics through stability and avidity gains. Circular aptamers targeted against thrombin displayed improved anticoagulant potency when compared with those of the canonical GS-522 thrombin DNA aptamer. Development of these anticoagulant aptamers has now reached a point where the principles of DNA nanotechnology are merging with aptameric functions. Aptamers have also been developed against thrombin for thrombus imaging.15 Other developments have resulted in the production of aptamers against the amyloid peptide for use in the diagnosis of Alzheimer’s disease.16 One of the earliest imaging studies was carried out by Charlton et al.17 who used an aptamer inhibitor of human neutrophil elastase to image inflammation. In the area of tumour targeting, aptamers offer enormous potential. In 2000, Hicke et al. used the term “escort aptamers” indicating that aptamers offered a delivery service for diagnosis and therapy.18 Hicke et al. then went on to produce tenascin-C aptamers, generated using both tumour cells and purified protein.19 This has led to one of the most encouraging in vivo pre- clinical tumour targeting studies to date, investigating the preparation of an aptamer (TTA1) directed against the extracellular matrix protein tenascin-C.20 Biodistribution studies were undertaken in nude mice bearing either U251 glioblastoma or MDA-MB-435 breast tumour xenografts. Fluorescence microscopy of rhodamine red-X- labelled aptamer demonstrated rapid tumour uptake within 10 min and diffusion throughout the tumours in the following 3 h. Radiolabelling of TTA1 was carried out with ^sup 99m^Tc using mercapto-acetyl glycene (MAG^sub 2^) and DTPA. Imaging studies demonstrated rapid blood clearance with a half-life of less than 10 min and rapid tumour penetration (6% injected dose per gram) at 10 min. A tumour to blood ratio of 50:1 was achieved after 3 h. This study demonstrates the potential of this type of molecule for tumour imaging and therapy.
The authors own experience in this area stems from a collaboration between the Open University at Milton Keynes and The University of Nottingham in the UK. The aim of the collaboration has been to target the well-characterised tumour associated antigen mucin 1 (MUC1) with a radiolabeled aptamer of high affinity and specificity for tumour imaging and therapy. Previous targeting and imaging studies have demonstrated this to be a valuable target for example in imaging ovarian and bladder cancer.21,22 MUC1 mucin is a high molecular weight glycoprotein expressed on glandular epithelial cells in humans. In malignancy, MUC1 is under glycosylated, less “sugar coating”aetherefore, a distinctive tumour marker. It is dominated by a variable-number tandem-repeat sequence of 20 amino acids. Using combinatorial chemistry techniques coupled with PCR aptamers have been selected from degenerate libraries that bind with high affinity and specificity to the protein core of the MUC1 antigen, a tumour marker previously extensively used in tumour imaging and therapy. The aptamer selection process was performed on affinity chromatography matrices. After 10 rounds of selection and amplification, aptamers were cloned and sequenced. Post-SELEX amino modifications have been used to confer nuclease resistance and coupling potential. The aptamers bound to MUC1 antigen with a Kd ranging between 0.3-30 nm and high specificity, demonstrated by fluorescent microscopy on MUC1-expresing tumour cells.23 Using peptide coupling reactions, we have successfully attached chelators for ^sup 99m^Tc radiolabelling. Two of the constructs tested were based on mono-aptamer chelator complexes, one with commercially available MAG3 and one with a novel designed cyclen-based chelator. The other two constructs were based on the use of multi-aptamer complexes, where 4 aptamers were attached to the 4 arms of either DOTA or carboxy-porphyrin (Figure 2).
Figure 2.-Diagrammatic constructs of a mono-aptamer (A) and a multi-aptamer (B) complex as targeted radiopharmaceuticals. The circular portions of the molecule represent the binding site.
The aptamer conjugates demonstrated high coupling efficiency, with >90% labelling of the aptamer. There was no observed digestion of aptamer in gel electrophoresis after 6 h in serum, urine or nuclease solutions. All four complexes demonstrated uptake in tumour, due to their MUC1 specificity. Biodistribution studies of radiolabeled aptamers have been carried out in mice with MCF7 xenografts. The monomelic aptamer complexes had rapid renal clearance from the system, due to their small size (MW of 8 000 Da). In the initial imaging studies, tumour visualisation was poor. However, autoradiographic studies were especially encouraging, demonstrating tumour penetration of the radiolabeled aptamer throughout the volume of the tumour. To increase the retention time, additional constructs based on the design of a tetra-aptamer complex have been prepared. A core chelator, such as DOTA and carboxyporphyrin has been used as a skeleton for the building of multi-aptamer constructs aiming to increase the molecular weight of the complex and potentially its stability of binding due to interactions with more than one MUC1 molecules at the surface of the tumour cell. The increase of the MW to 32 000 Da allowed increased retention times in the system. This work is currently being expanded to develop a higher molecular weight aptamer for radiolabelling with a therapeutic radionuclide, such as ^sup 188^Re.
As could be expected, aptamers are also being used as vectors for non-radioactive cytotoxic agents. Aptamers against prostate- specific membrane antigen (PSMA) have been produced as a means for targeted chemotherapy.24 Chu et al.25 have developed RNA aptamer:gelonin toxin conjugates to target cells overexpressing PSMA as a potential therapeutic for prostate cancer. This work has shown the aptamer: toxin conjugates to have an IC50 of 27 nmol/L, displaying an increased potency of at least 600-fold relative to cells that do not express PSMA. The aptamer was found to promote uptake into target cells and also decreases the toxicity of gelonin in non-target cells.
Conclusions
The use of aptamers in nuclear medicine is still a novel concept and to date experience has been limited to very few pre-clinical studies. There is now a rapidly growing body of evidence to indicate that aptamers are destined to have an important role in diagnostic and therapeutic medicine. As discussed, aptamer molecules can identify a vast range of biological targets with high affinity and high specificity. In cancer therapy, the results of initial pre- clinical studies are encouraging. It has been shown that radiolabeled aptamers can penetrate tumour more readily than whole antibodies, reach peak levels in the tumour more rapidly and clear from the body faster, thereby reducing toxicity to healthy tissues. Our own initial studies are also encouraging. The range of aptamer conjugates is increasing rapidly and, more importantly, they offer a doorway to biotechnological developments in synthetic radiopharmaceutical production avoiding the problems of contamination with viruses or prions of human or animal origin.26 The future strategy will be to manipulate the molecular weight of the molecules to achieve an optimum balance between the low immunogenicity and excellent tumour penetration for diagnostic imaging and targeted therapy. The construction of multi-aptamer complexes will address size issues, resulting in novel “designer” radiopharmaceuticals with excellent properties for tumour imaging and therapy. The concept of “escort aptamers” is now moving closer to becoming a clinical reality.
The authors would like to thank the Breast Cancer Campaign of the United Kingdom for Financial grant support.
References
1. Ellinghton AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature 1990;346:818-22.
2. Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990;249:505-10.
3. Eaton BE, Gold L, Hicke BJ, Janjic N, Jucker FM, Sebesta DP et al. Post-SELEX combinatorial optimization of aptamers. Bioorg Med Chem 1997;5:1087-96.
4. Jayasena SD. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem 1999;45:1628-50.
5. Hilger CS, Willis MC, Wolters M, Wolfgang AP. Synthesis of Tc- 99-labeled, modified RNA. Tetrahedron Lett 1998;39:9403-6.
6. Wagner S, Eisenhut M, Eritja R, Oberdofer F. Synthesis of copper-64 and technetium-99M labelled oligonucleotides with macrocyclic ligands. Nucleosides Nucleotides 1997;16:1789-92.
7. Winnard P, Chang M, Rusckowski G, Mardirossian F, Hnatowich DJ. Preparation and use of NHS-MAGa for technetium-99m labeling of DNA. Nucl Med Biol 1997;24:425-32.
8. Tavitian B. In vivo imaging with oligonucleotides for diagnosis and drug development. Gut 2003;52:iv40-7.
9. Lee JF, Hesselberth JR, Meyers LA, Ellington AD. Aptamer database. Nucleic Acid Res 2004;32:D95-100.
10. Carrasquillo KG, Ricker JA, Rigas IK, Miller JW, Gragoudas ES, Adamis AP. Controlled delivery of the anti-VEGF aptamer EYE001 with poly(lactic-co-glycolic)acid microspheres. Invest Ophthalmol Vis Sci 2003;44:290-9.
11. Butz K, Denk C, Fitscher B, Crnkovic-Mertens I, Ullmann A, Schroder CH et al. Peptide aptamers targeting the hepatitis B virus core protein: a new class of molecules with antiviral activity.Oncogene 2001;20:6579-86.
12. Biroccio A, Hamm J, Incitti I, De Francesco R, Tomei L. Selection of RNA aptamers that are specific and high-affinity ligands of the hepatitis C virus RNA-dependent RNA polymerase. J Virol 2002;76:3688-96.
13. Yamamoto R, Toyoda S, Viljanen P, Machida K, Nishikawa S, Murakami K et al. In vitro selection of RNA aptamers that can bind specifically to Tat protein of HIV-1. Nucleic Acids Symp Ser 1995;34:145-6.
14. Di Giusto DA, King GC. Construction, stability, and activity of multivalent circular anticoagulant aptamers. J Biol Chem 2004;279;46483-9.
15. Dougan H, Weitz JI, Stafford AR, Gillespie KD, Klement P, Hobbs JB et al. Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents. Nucl Med Biol 2003;30:61-72.
16. Ylera F, Lurz R, Erdmann VA, Fuster JP. Selection of RNA aptamers to the Alzheimer’s disease amyloid peptide. Biochem Biophys Res Commun 2002;290:1583-8. 17. Charlton J, Sennello J, Smith D. In vivo imaging of inflammation using an apatamer inhibitor of human neutrophilo elastase. Chem Biol 1997;4:809-16.
18. Hicke BJ, Stephens AW. Escort aptamers: a delivery service for diagnosis and dierapy. J Clin Invest 200; 106:923-8.
19. Hicke BJ, Marion C, Chang YF, Gould T, Lynott CK, Parma D et al. Tenascin-C aptamers are generated using tumor cells and purified protein. J Biol Chem 2001;276:48644-54.
20. Hicke BJ, Stephens AW, Gould T, Chang YF, Lynott CK, Heil J et al. Tumour targeting by an aptamer. J Nucl Med 2006;47:668-78.
21. Perkins AC, Symonds IM, Pimm MV, Price MR, Wastie ML, Symonds EM. Immunoscintigrapny of ovarian carcinoma using a monoclonal antibody (In-111-NCRC48) defining a polymorphic epithelial mucin (PEM) epitope. Nucl Med Commun 1993;14:578-86.
22. Hughes ODM, Bishop MC, Perkins AC, Wastie ML, Denton G, Price MR et al. Targeting superficial bladder cancer by the intravesical administration of Cu-67-labelled anti-MUC1 mucin monoclonal antibody. J Clin Oncol 2000;18:363-70.
23. Ferreira CSM, Matthews CS, Missailidis S. DNA aptamers that bind to MUC1 tumour marker: design and characterisation of MUC1- binding single-stranded DNA aptamers. Tumor Biol 2006;27:289-301.
24. Lupoid SE, Hicke J, Lin Yun C, Donald S. Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res 2002;62:4029-33.
25. Chu TC, Marks JW, Lavery LA, Faulkner S, Rosenblum MG, Ellington AD et al. Aptamer: toxin conjugates that specifically target prostate tumor cells. Cancer Res 2006;66:5989-92.
26. Perkins AC, Frier M. Bad blood and biologicals: the need for new radiopharmaceutical source materials. Nucl Med Commun 1993;20:1- 3.
A. C. PERKINS 1, S. MISSAILIDIS 2
1 Academic Medical Physics, Queen’s Medical Centre
The University of Nottingham, Nottingham, UK
2 Department of Chemistry, The Open University
Walton Hall, Milton Keynes, UK
Address reprint requests to: Prof. A. C. Perkins, Academic Medical Physics, Queen’s Medical Centre, Nottingham, NG7 2UH, UK. E- mail: alan.perkins@nottingham.ac.uk
Copyright Edizioni Minerva Medica Dec 2007
(c) 2007 Quarterly Journal of Nuclear Medicine, The. Provided by ProQuest Information and Learning. All rights Reserved.